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Archive for the ‘Schizophrenia’ Category


Disease related changes in proteomics, protein folding, protein-protein interaction

Curator: Larry H. Bernstein, MD, FCAP

LPBI

 

Frankenstein Proteins Stitched Together by Scientists

http://www.genengnews.com/gen-news-highlights/frankenstein-proteins-stitched-together-by-scientists/81252715/

http://www.genengnews.com/Media/images/GENHighlight/thumb_May11_2016_Wikipedia_1831Frankenstein2192501426.jpg

The Frankenstein monster, stitched together from disparate body parts, proved to be an abomination, but stitched together proteins may fare better. They may, for example, serve specific purposes in medicine, research, and industry. At least, that’s the ambition of scientists based at the University of North Carolina. They have developed a computational protocol called SEWING that builds new proteins from connected or disconnected pieces of existing structures. [Wikipedia]

Unlike Victor Frankenstein, who betrayed Promethean ambition when he sewed together his infamous creature, today’s biochemists are relatively modest. Rather than defy nature, they emulate it. For example, at the University of North Carolina (UNC), researchers have taken inspiration from natural evolutionary mechanisms to develop a technique called SEWING—Structure Extension With Native-substructure Graphs. SEWING is a computational protocol that describes how to stitch together new proteins from connected or disconnected pieces of existing structures.

“We can now begin to think about engineering proteins to do things that nothing else is capable of doing,” said UNC’s Brian Kuhlman, Ph.D. “The structure of a protein determines its function, so if we are going to learn how to design new functions, we have to learn how to design new structures. Our study is a critical step in that direction and provides tools for creating proteins that haven’t been seen before in nature.”

Traditionally, researchers have used computational protein design to recreate in the laboratory what already exists in the natural world. In recent years, their focus has shifted toward inventing novel proteins with new functionality. These design projects all start with a specific structural “blueprint” in mind, and as a result are limited. Dr. Kuhlman and his colleagues, however, believe that by removing the limitations of a predetermined blueprint and taking cues from evolution they can more easily create functional proteins.

Dr. Kuhlman’s UNC team developed a protein design approach that emulates natural mechanisms for shuffling tertiary structures such as pleats, coils, and furrows. Putting the approach into action, the UNC team mapped 50,000 stitched together proteins on the computer, and then it produced 21 promising structures in the laboratory. Details of this work appeared May 6 in the journal Science, in an article entitled, “Design of Structurally Distinct Proteins Using Strategies Inspired by Evolution.”

“Helical proteins designed with SEWING contain structural features absent from other de novo designed proteins and, in some cases, remain folded at more than 100°C,” wrote the authors. “High-resolution structures of the designed proteins CA01 and DA05R1 were solved by x-ray crystallography (2.2 angstrom resolution) and nuclear magnetic resonance, respectively, and there was excellent agreement with the design models.”

Essentially, the UNC scientists confirmed that the proteins they had synthesized contained the unique structural varieties that had been designed on the computer. The UNC scientists also determined that the structures they had created had new surface and pocket features. Such features, they noted, provide potential binding sites for ligands or macromolecules.

“We were excited that some had clefts or grooves on the surface, regions that naturally occurring proteins use for binding other proteins,” said the Science article’s first author, Tim M. Jacobs, Ph.D., a former graduate student in Dr. Kuhlman’s laboratory. “That’s important because if we wanted to create a protein that can act as a biosensor to detect a certain metabolite in the body, either for diagnostic or research purposes, it would need to have these grooves. Likewise, if we wanted to develop novel therapeutics, they would also need to attach to specific proteins.”

Currently, the UNC researchers are using SEWING to create proteins that can bind to several other proteins at a time. Many of the most important proteins are such multitaskers, including the blood protein hemoglobin.

 

Histone Mutation Deranges DNA Methylation to Cause Cancer

http://www.genengnews.com/gen-news-highlights/histone-mutation-deranges-dna-methylation-to-cause-cancer/81252723/

http://www.genengnews.com/Media/images/GENHighlight/thumb_May13_2016_RockefellerUniv_ChildhoodSarcoma1293657114.jpg

In some cancers, including chondroblastoma and a rare form of childhood sarcoma, a mutation in histone H3 reduces global levels of methylation (dark areas) in tumor cells but not in normal cells (arrowhead). The mutation locks the cells in a proliferative state to promote tumor development. [Laboratory of Chromatin Biology and Epigenetics at The Rockefeller University]

They have been called oncohistones, the mutated histones that are known to accompany certain pediatric cancers. Despite their suggestive moniker, oncohistones have kept their oncogenic secrets. For example, it has been unclear whether oncohistones are able to cause cancer on their own, or whether they need to act in concert with additional DNA mutations, that is, mutations other than those affecting histone structures.

While oncohistone mechanisms remain poorly understood, this particular question—the oncogenicity of lone oncohistones—has been resolved, at least in part. According to researchers based at The Rockefeller University, a change to the structure of a histone can trigger a tumor on its own.

This finding appeared May 13 in the journal Science, in an article entitled, “Histone H3K36 Mutations Promote Sarcomagenesis Through Altered Histone Methylation Landscape.” The article describes the Rockefeller team’s study of a histone protein called H3, which has been found in about 95% of samples of chondoblastoma, a benign tumor that arises in cartilage, typically during adolescence.

The Rockefeller scientists found that the H3 lysine 36–to–methionine (H3K36M) mutation impairs the differentiation of mesenchymal progenitor cells and generates undifferentiated sarcoma in vivo.

After the scientists inserted the H3 histone mutation into mouse mesenchymal progenitor cells (MPCs)—which generate cartilage, bone, and fat—they watched these cells lose the ability to differentiate in the lab. Next, the scientists injected the mutant cells into living mice, and the animals developed the tumors rich in MPCs, known as an undifferentiated sarcoma. Finally, the researchers tried to understand how the mutation causes the tumors to develop.

The scientists determined that H3K36M mutant nucleosomes inhibit the enzymatic activities of several H3K36 methyltransferases.

“Depleting H3K36 methyltransferases, or expressing an H3K36I mutant that similarly inhibits H3K36 methylation, is sufficient to phenocopy the H3K36M mutation,” the authors of the Science study wrote. “After the loss of H3K36 methylation, a genome-wide gain in H3K27 methylation leads to a redistribution of polycomb repressive complex 1 and de-repression of its target genes known to block mesenchymal differentiation.”

Essentially, when the H3K36M mutation occurs, the cell becomes locked in a proliferative state—meaning it divides constantly, leading to tumors. Specifically, the mutation inhibits enzymes that normally tag the histone with chemical groups known as methyls, allowing genes to be expressed normally.

In response to this lack of modification, another part of the histone becomes overmodified, or tagged with too many methyl groups. “This leads to an overall resetting of the landscape of chromatin, the complex of DNA and its associated factors, including histones,” explained co-author Peter Lewis, Ph.D., a professor at the University of Wisconsin-Madison and a former postdoctoral fellow in laboratory of C. David Allis, Ph.D., a professor at Rockefeller.

The finding—that a “resetting” of the chromatin landscape can lock the cell into a proliferative state—suggests that researchers should be on the hunt for more mutations in histones that might be driving tumors. For their part, the Rockefeller researchers are trying to learn more about how this specific mutation in histone H3 causes tumors to develop.

“We want to know which pathways cause the mesenchymal progenitor cells that carry the mutation to continue to divide, and not differentiate into the bone, fat, and cartilage cells they are destined to become,” said co-author Chao Lu, Ph.D., a postdoctoral fellow in the Allis lab.

Once researchers understand more about these pathways, added Dr. Lewis, they can consider ways of blocking them with drugs, particularly in tumors such as MPC-rich sarcomas—which, unlike chondroblastoma, can be deadly. In fact, drugs that block these pathways may already exist and may even be in use for other types of cancers.

“One long-term goal of our collaborative team is to better understand fundamental mechanisms that drive these processes, with the hope of providing new therapeutic approaches,” concluded Dr. Allis.

 

Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape

Chao Lu, Siddhant U. Jain, Dominik Hoelper, …, C. David Allis1,, Nada Jabado,, Peter W. Lewis,
Science  13 May 2016; 352(6287):844-849 http://dx.doi.org:/10.1126/science.aac7272  http://science.sciencemag.org/content/352/6287/844

An oncohistone deranges inhibitory chromatin

Missense mutations (that change one amino acid for another) in histone H3 can produce a so-called oncohistone and are found in a number of pediatric cancers. For example, the lysine-36–to-methionine (K36M) mutation is seen in almost all chondroblastomas. Lu et al. show that K36M mutant histones are oncogenic, and they inhibit the normal methylation of this same residue in wild-type H3 histones. The mutant histones also interfere with the normal development of bone-related cells and the deposition of inhibitory chromatin marks.

Science, this issue p. 844

Several types of pediatric cancers reportedly contain high-frequency missense mutations in histone H3, yet the underlying oncogenic mechanism remains poorly characterized. Here we report that the H3 lysine 36–to–methionine (H3K36M) mutation impairs the differentiation of mesenchymal progenitor cells and generates undifferentiated sarcoma in vivo. H3K36M mutant nucleosomes inhibit the enzymatic activities of several H3K36 methyltransferases. Depleting H3K36 methyltransferases, or expressing an H3K36I mutant that similarly inhibits H3K36 methylation, is sufficient to phenocopy the H3K36M mutation. After the loss of H3K36 methylation, a genome-wide gain in H3K27 methylation leads to a redistribution of polycomb repressive complex 1 and de-repression of its target genes known to block mesenchymal differentiation. Our findings are mirrored in human undifferentiated sarcomas in which novel K36M/I mutations in H3.1 are identified.

 

Mitochondria? We Don’t Need No Stinking Mitochondria!

 

http://www.genengnews.com/Media/images/GENHighlight/thumb_fx11801711851.jpg
Diagram comparing typical eukaryotic cell to the newly discovered mitochondria-free organism. [Karnkowska et al., 2016, Current Biology 26, 1–11]
  • The organelle that produces a significant portion of energy for eukaryotic cells would seemingly be indispensable, yet over the years, a number of organisms have been discovered that challenge that biological pretense. However, these so-called amitochondrial species may lack a defined organelle, but they still retain some residual functions of their mitochondria-containing brethren. Even the intestinal eukaryotic parasite Giardia intestinalis, which was for many years considered to be mitochondria-free, was proven recently to contain a considerably shriveled version of the organelle.
  • Now, an international group of scientists has released results from a new study that challenges the notion that mitochondria are essential for eukaryotes—discovering an organism that resides in the gut of chinchillas that contains absolutely no trace of mitochondria at all.
  • “In low-oxygen environments, eukaryotes often possess a reduced form of the mitochondrion, but it was believed that some of the mitochondrial functions are so essential that these organelles are indispensable for their life,” explained lead study author Anna Karnkowska, Ph.D., visiting scientist at the University of British Columbia in Vancouver. “We have characterized a eukaryotic microbe which indeed possesses no mitochondrion at all.”

 

Mysterious Eukaryote Missing Mitochondria

Researchers uncover the first example of a eukaryotic organism that lacks the organelles.

By Anna Azvolinsky | May 12, 2016

http://www.the-scientist.com/?articles.view/articleNo/46077/title/Mysterious-Eukaryote-Missing-Mitochondria

http://www.the-scientist.com/images/News/May2016/620_Monocercomonides-Pa203.jpg

Monocercomonoides sp. PA203VLADIMIR HAMPL, CHARLES UNIVERSITY, PRAGUE, CZECH REPUBLIC

Scientists have long thought that mitochondria—organelles responsible for energy generation—are an essential and defining feature of a eukaryotic cell. Now, researchers from Charles University in Prague and their colleagues are challenging this notion with their discovery of a eukaryotic organism,Monocercomonoides species PA203, which lacks mitochondria. The team’s phylogenetic analysis, published today (May 12) in Current Biology,suggests that Monocercomonoides—which belong to the Oxymonadida group of protozoa and live in low-oxygen environmentsdid have mitochondria at one point, but eventually lost the organelles.

“This is quite a groundbreaking discovery,” said Thijs Ettema, who studies microbial genome evolution at Uppsala University in Sweden and was not involved in the work.

“This study shows that mitochondria are not so central for all lineages of living eukaryotes,” Toni Gabaldonof the Center for Genomic Regulation in Barcelona, Spain, who also was not involved in the work, wrote in an email to The Scientist. “Yet, this mitochondrial-devoid, single-cell eukaryote is as complex as other eukaryotic cells in almost any other aspect of cellular complexity.”

Charles University’s Vladimir Hampl studies the evolution of protists. Along with Anna Karnkowska and colleagues, Hampl decided to sequence the genome of Monocercomonoides, a little-studied protist that lives in the digestive tracts of vertebrates. The 75-megabase genome—the first of an oxymonad—did not contain any conserved genes found on mitochondrial genomes of other eukaryotes, the researchers found. It also did not contain any nuclear genes associated with mitochondrial functions.

“It was surprising and for a long time, we didn’t believe that the [mitochondria-associated genes were really not there]. We thought we were missing something,” Hampl told The Scientist. “But when the data kept accumulating, we switched to the hypothesis that this organism really didn’t have mitochondria.”

Because researchers have previously not found examples of eukaryotes without some form of mitochondria, the current theory of the origin of eukaryotes poses that the appearance of mitochondria was crucial to the identity of these organisms.

“We now view these mitochondria-like organelles as a continuum from full mitochondria to very small . Some anaerobic protists, for example, have only pared down versions of mitochondria, such as hydrogenosomes and mitosomes, which lack a mitochondrial genome. But these mitochondrion-like organelles perform essential functions of the iron-sulfur cluster assembly pathway, which is known to be conserved in virtually all eukaryotic organisms studied to date.

Yet, in their analysis, the researchers found no evidence of the presence of any components of this mitochondrial pathway.

Like the scaling down of mitochondria into mitosomes in some organisms, the ancestors of modernMonocercomonoides once had mitochondria. “Because this organism is phylogenetically nested among relatives that had conventional mitochondria, this is most likely a secondary adaptation,” said Michael Gray, a biochemist who studies mitochondria at Dalhousie University in Nova Scotia and was not involved in the study. According to Gray, the finding of a mitochondria-deficient eukaryote does not mean that the organelles did not play a major role in the evolution of eukaryotic cells.

To be sure they were not missing mitochondrial proteins, Hampl’s team also searched for potential mitochondrial protein homologs of other anaerobic species, and for signature sequences of a range of known mitochondrial proteins. While similar searches with other species uncovered a few mitochondrial proteins, the team’s analysis of Monocercomonoides came up empty.

“The data is very complete,” said Ettema. “It is difficult to prove the absence of something but [these authors] do a convincing job.”

To form the essential iron-sulfur clusters, the team discovered that Monocercomonoides use a sulfur mobilization system found in the cytosol, and that an ancestor of the organism acquired this system by lateral gene transfer from bacteria. This cytosolic, compensating system allowed Monocercomonoides to lose the otherwise essential iron-sulfur cluster-forming pathway in the mitochondrion, the team proposed.

“This work shows the great evolutionary plasticity of the eukaryotic cell,” said Karnkowska, who participated in the study while she was a postdoc at Charles University. Karnkowska, who is now a visiting researcher at the University of British Columbia in Canada, added: “This is a striking example of how far the evolution of a eukaryotic cell can go that was beyond our expectations.”

“The results highlight how many surprises may await us in the poorly studied eukaryotic phyla that live in under-explored environments,” Gabaldon said.

Ettema agreed. “Now that we’ve found one, we need to look at the bigger picture and see if there are other examples of eukaryotes that have lost their mitochondria, to understand how adaptable eukaryotes are.”

  1. Karnkowska et al., “A eukaryote without a mitochondrial organelle,” Current Biology,doi:10.1016/j.cub.2016.03.053, 2016.

organellesmitochondriagenetics & genomics and evolution

 

A Eukaryote without a Mitochondrial Organelle

Anna Karnkowska,  Vojtěch Vacek,  Zuzana Zubáčová,…,  Čestmír Vlček,  Vladimír HamplDOI: http://dx.doi.org/10.1016/j.cub.2016.03.053  Article Info

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Highlights

  • Monocercomonoides sp. is a eukaryotic microorganism with no mitochondria
  • •The complete absence of mitochondria is a secondary loss, not an ancestral feature
  • •The essential mitochondrial ISC pathway was replaced by a bacterial SUF system

The presence of mitochondria and related organelles in every studied eukaryote supports the view that mitochondria are essential cellular components. Here, we report the genome sequence of a microbial eukaryote, the oxymonad Monocercomonoides sp., which revealed that this organism lacks all hallmark mitochondrial proteins. Crucially, the mitochondrial iron-sulfur cluster assembly pathway, thought to be conserved in virtually all eukaryotic cells, has been replaced by a cytosolic sulfur mobilization system (SUF) acquired by lateral gene transfer from bacteria. In the context of eukaryotic phylogeny, our data suggest that Monocercomonoides is not primitively amitochondrial but has lost the mitochondrion secondarily. This is the first example of a eukaryote lacking any form of a mitochondrion, demonstrating that this organelle is not absolutely essential for the viability of a eukaryotic cell.

http://www.cell.com/cms/attachment/2056332410/2061316405/fx1.jpg

 

HIV Particles Used to Trap Intact Mammalian Protein Complexes

Belgian scientists from VIB and UGent developed Virotrap, a viral particle sorting approach for purifying protein complexes under native conditions.

http://www.technologynetworks.com/Proteomics/news.aspx?ID=191122

This method catches a bait protein together with its associated protein partners in virus-like particles that are budded from human cells. Like this, cell lysis is not needed and protein complexes are preserved during purification.

With his feet in both a proteomics lab and an interactomics lab, VIB/UGent professor Sven Eyckerman is well aware of the shortcomings of conventional approaches to analyze protein complexes. The lysis conditions required in mass spectrometry–based strategies to break open cell membranes often affect protein-protein interactions. “The first step in a classical study on protein complexes essentially turns the highly organized cellular structure into a big messy soup”, Eyckerman explains.

Inspired by virus biology, Eyckerman came up with a creative solution. “We used the natural process of HIV particle formation to our benefit by hacking a completely safe form of the virus to abduct intact protein machines from the cell.” It is well known that the HIV virus captures a number of host proteins during its particle formation. By fusing a bait protein to the HIV-1 GAG protein, interaction partners become trapped within virus-like particles that bud from mammalian cells. Standard proteomic approaches are used next to reveal the content of these particles. Fittingly, the team named the method ‘Virotrap’.

The Virotrap approach is exceptional as protein networks can be characterized under natural conditions. By trapping protein complexes in the protective environment of a virus-like shell, the intact complexes are preserved during the purification process. The researchers showed the method was suitable for detection of known binary interactions as well as mass spectrometry-based identification of novel protein partners.

Virotrap is a textbook example of bringing research teams with complementary expertise together. Cross-pollination with the labs of Jan Tavernier (VIB/UGent) and Kris Gevaert (VIB/UGent) enabled the development of this platform.

Jan Tavernier: “Virotrap represents a new concept in co-complex analysis wherein complex stability is physically guaranteed by a protective, physical structure. It is complementary to the arsenal of existing interactomics methods, but also holds potential for other fields, like drug target characterization. We also developed a small molecule-variant of Virotrap that could successfully trap protein partners for small molecule baits.”

Kris Gevaert: “Virotrap can also impact our understanding of disease pathways. We were actually surprised to see that this virus-based system could be used to study antiviral pathways, like Toll-like receptor signaling. Understanding these protein machines in their natural environment is essential if we want to modulate their activity in pathology.“

 

Trapping mammalian protein complexes in viral particles

Sven Eyckerman, Kevin Titeca, …Kris GevaertJan Tavernier
Nature Communications Apr 2016; 7(11416)   http://dx.doi.org:/10.1038/ncomms11416

Cell lysis is an inevitable step in classical mass spectrometry–based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes.

Proteins mostly exert their function within supramolecular complexes. Strategies for detecting protein–protein interactions (PPIs) can be roughly divided into genetic systems1 and co-purification strategies combined with mass spectrometry (MS) analysis (for example, AP–MS)2. The latter approaches typically require cell or tissue homogenization using detergents, followed by capture of the protein complex using affinity tags3 or specific antibodies4. The protein complexes extracted from this ‘soup’ of constituents are then subjected to several washing steps before actual analysis by trypsin digestion and liquid chromatography–MS/MS analysis. Such lysis and purification protocols are typically empirical and have mostly been optimized using model interactions in single labs. In fact, lysis conditions can profoundly affect the number of both specific and nonspecific proteins that are identified in a typical AP–MS set-up. Indeed, recent studies using the nuclear pore complex as a model protein complex describe optimization of purifications for the different proteins in the complex by examining 96 different conditions5. Nevertheless, for new purifications, it remains hard to correctly estimate the loss of factors in a standard AP–MS experiment due to washing and dilution effects during treatments (that is, false negatives). These considerations have pushed the concept of stabilizing PPIs before the actual homogenization step. A classical approach involves cross-linking with simple reagents (for example, formaldehyde) or with more advanced isotope-labelled cross-linkers (reviewed in ref. 2). However, experimental challenges such as cell permeability and reactivity still preclude the widespread use of cross-linking agents. Moreover, MS-generated spectra of cross-linked peptides are notoriously difficult to identify correctly. A recent lysis-independent solution involves the expression of a bait protein fused to a promiscuous biotin ligase, which results in labelling of proteins proximal to the activity of the enzyme-tagged bait protein6. When compared with AP–MS, this BioID approach delivers a complementary set of candidate proteins, including novel interaction partners78. Such particular studies clearly underscore the need for complementary approaches in the co-complex strategies.

The evolutionary stress on viruses promoted highly condensed coding of information and maximal functionality for small genomes. Accordingly, for HIV-1 it is sufficient to express a single protein, the p55 GAG protein, for efficient production of virus-like particles (VLPs) from cells910. This protein is highly mobile before its accumulation in cholesterol-rich regions of the membrane, where multimerization initiates the budding process11. A total of 4,000–5,000 GAG molecules is required to form a single particle of about 145 nm (ref. 12). Both VLPs and mature viruses contain a number of host proteins that are recruited by binding to viral proteins. These proteins can either contribute to the infectivity (for example, Cyclophilin/FKBPA13) or act as antiviral proteins preventing the spreading of the virus (for example, APOBEC proteins14).

We here describe the development and application of Virotrap, an elegant co-purification strategy based on the trapping of a bait protein together with its associated protein partners in VLPs that are budded from the cell. After enrichment, these particles can be analysed by targeted (for example, western blotting) or unbiased approaches (MS-based proteomics). Virotrap allows detection of known binary PPIs, analysis of protein complexes and their dynamics, and readily detects protein binders for small molecules.

Concept of the Virotrap system

Classical AP–MS approaches rely on cell homogenization to access protein complexes, a step that can vary significantly with the lysis conditions (detergents, salt concentrations, pH conditions and so on)5. To eliminate the homogenization step in AP–MS, we reasoned that incorporation of a protein complex inside a secreted VLP traps the interaction partners under native conditions and protects them during further purification. We thus explored the possibility of protein complex packaging by the expression of GAG-bait protein chimeras (Fig. 1) as expression of GAG results in the release of VLPs from the cells910. As a first PPI pair to evaluate this concept, we selected the HRAS protein as a bait combined with the RAF1 prey protein. We were able to specifically detect the HRAS–RAF1 interaction following enrichment of VLPs via ultracentrifugation (Supplementary Fig. 1a). To prevent tedious ultracentrifugation steps, we designed a novel single-step protocol wherein we co-express the vesicular stomatitis virus glycoprotein (VSV-G) together with a tagged version of this glycoprotein in addition to the GAG bait and prey. Both tagged and untagged VSV-G proteins are probably presented as trimers on the surface of the VLPs, allowing efficient antibody-based recovery from large volumes. The HRAS–RAF1 interaction was confirmed using this single-step protocol (Supplementary Fig. 1b). No associations with unrelated bait or prey proteins were observed for both protocols.

Figure 1: Schematic representation of the Virotrap strategy.

http://www.nature.com/ncomms/2016/160428/ncomms11416/images_article/ncomms11416-f1.jpg

 

Expression of a GAG-bait fusion protein (1) results in submembrane multimerization (2) and subsequent budding of VLPs from cells (3). Interaction partners of the bait protein are also trapped within these VLPs and can be identified after purification by western blotting or MS analysis (4).

Virotrap for the detection of binary interactions

We next explored the reciprocal detection of a set of PPI pairs, which were selected based on published evidence and cytosolic localization15. After single-step purification and western blot analysis, we could readily detect reciprocal interactions between CDK2 and CKS1B, LCP2 and GRAP2, and S100A1 and S100B (Fig. 2a). Only for the LCP2 prey we observed nonspecific association with an irrelevant bait construct. However, the particle levels of the GRAP2 bait were substantially lower as compared with those of the GAG control construct (GAG protein levels in VLPs; Fig. 2a, second panel of the LCP2 prey). After quantification of the intensities of bait and prey proteins and normalization of prey levels using bait levels, we observed a strong enrichment for the GAG-GRAP2 bait (Supplementary Fig. 2).

…..

Virotrap for unbiased discovery of novel interactions

For the detection of novel interaction partners, we scaled up VLP production and purification protocols (Supplementary Fig. 5 and Supplementary Note 1 for an overview of the protocol) and investigated protein partners trapped using the following bait proteins: Fas-associated via death domain (FADD), A20 (TNFAIP3), nuclear factor-κB (NF-κB) essential modifier (IKBKG), TRAF family member-associated NF-κB activator (TANK), MYD88 and ring finger protein 41 (RNF41). To obtain specific interactors from the lists of identified proteins, we challenged the data with a combined protein list of 19 unrelated Virotrap experiments (Supplementary Table 1 for an overview). Figure 3 shows the design and the list of candidate interactors obtained after removal of all proteins that were found in the 19 control samples (including removal of proteins from the control list identified with a single peptide). The remaining list of confident protein identifications (identified with at least two peptides in at least two biological repeats) reveals both known and novel candidate interaction partners. All candidate interactors including single peptide protein identifications are given in Supplementary Data 2 and also include recurrent protein identifications of known interactors based on a single peptide; for example, CASP8 for FADD and TANK for NEMO. Using alternative methods, we confirmed the interaction between A20 and FADD, and the associations with transmembrane proteins (insulin receptor and insulin-like growth factor receptor 1) that were captured using RNF41 as a bait (Supplementary Fig. 6). To address the use of Virotrap for the detection of dynamic interactions, we activated the NF-κB pathway via the tumour necrosis factor (TNF) receptor (TNFRSF1A) using TNFα (TNF) and performed Virotrap analysis using A20 as bait (Fig. 3). This resulted in the additional enrichment of receptor-interacting kinase (RIPK1), TNFR1-associated via death domain (TRADD), TNFRSF1A and TNF itself, confirming the expected activated complex20.

Figure 3: Use of Virotrap for unbiased interactome analysis

http://www.nature.com/ncomms/2016/160428/ncomms11416/images_article/ncomms11416-f3.jpg

Figure 4: Use of Virotrap for detection of protein partners of small molecules.

http://www.nature.com/ncomms/2016/160428/ncomms11416/images_article/ncomms11416-f4.jpg

….

Lysis conditions used in AP–MS strategies are critical for the preservation of protein complexes. A multitude of lysis conditions have been described, culminating in a recent report where protein complex stability was assessed under 96 lysis/purification protocols5. Moreover, the authors suggest to optimize the conditions for every complex, implying an important workload for researchers embarking on protein complex analysis using classical AP–MS. As lysis results in a profound change of the subcellular context and significantly alters the concentration of proteins, loss of complex integrity during a classical AP–MS protocol can be expected. A clear evolution towards ‘lysis-independent’ approaches in the co-complex analysis field is evident with the introduction of BioID6 and APEX25 where proximal proteins, including proteins residing in the complex, are labelled with biotin by an enzymatic activity fused to a bait protein. A side-by-side comparison between classical AP–MS and BioID showed overlapping and unique candidate binding proteins for both approaches78, supporting the notion that complementary methods are needed to provide a comprehensive view on protein complexes. This has also been clearly demonstrated for binary approaches15 and is a logical consequence of the heterogenic nature underlying PPIs (binding mechanism, requirement for posttranslational modifications, location, affinity and so on).

In this report, we explore an alternative, yet complementary method to isolate protein complexes without interfering with cellular integrity. By trapping protein complexes in the protective environment of a virus-like shell, the intact complexes are preserved during the purification process. This constitutes a new concept in co-complex analysis wherein complex stability is physically guaranteed by a protective, physical structure. A comparison of our Virotrap approach with AP–MS shows complementary data, with specific false positives and false negatives for both methods (Supplementary Fig. 7).

The current implementation of the Virotrap platform implies the use of a GAG-bait construct resulting in considerable expression of the bait protein. Different strategies are currently pursued to reduce bait expression including co-expression of a native GAG protein together with the GAG-bait protein, not only reducing bait expression but also creating more ‘space’ in the particles potentially accommodating larger bait protein complexes. Nevertheless, the presence of the bait on the forming GAG scaffold creates an intracellular affinity matrix (comparable to the early in vitro affinity columns for purification of interaction partners from lysates26) that has the potential to compete with endogenous complexes by avidity effects. This avidity effect is a powerful mechanism that aids in the recruitment of cyclophilin to GAG27, a well-known weak interaction (Kd=16 μM (ref. 28)) detectable as a background association in the Virotrap system. Although background binding may be increased by elevated bait expression, weaker associations are readily detectable (for example, MAL—MYD88-binding study; Fig. 2c).

The size of Virotrap particles (around 145 nm) suggests limitations in the size of the protein complex that can be accommodated in the particles. Further experimentation is required to define the maximum size of proteins or the number of protein complexes that can be trapped inside the particles.

….

In conclusion, Virotrap captures significant parts of known interactomes and reveals new interactions. This cell lysis-free approach purifies protein complexes under native conditions and thus provides a powerful method to complement AP–MS or other PPI data. Future improvements of the system include strategies to reduce bait expression to more physiological levels and application of advanced data analysis options to filter out background. These developments can further aid in the deployment of Virotrap as a powerful extension of the current co-complex technology arsenal.

 

New Autism Blood Biomarker Identified

Researchers at UT Southwestern Medical Center have identified a blood biomarker that may aid in earlier diagnosis of children with autism spectrum disorder, or ASD

http://www.technologynetworks.com/Proteomics/news.aspx?ID=191268

 

In a recent edition of Scientific Reports, UT Southwestern researchers reported on the identification of a blood biomarker that could distinguish the majority of ASD study participants versus a control group of similar age range. In addition, the biomarker was significantly correlated with the level of communication impairment, suggesting that the blood test may give insight into ASD severity.

“Numerous investigators have long sought a biomarker for ASD,” said Dr. Dwight German, study senior author and Professor of Psychiatry at UT Southwestern. “The blood biomarker reported here along with others we are testing can represent a useful test with over 80 percent accuracy in identifying ASD.”

ASD1 –  was 66 percent accurate in diagnosing ASD. When combined with thyroid stimulating hormone level measurements, the ASD1-binding biomarker was 73 percent accurate at diagnosis

 

A Search for Blood Biomarkers for Autism: Peptoids

Sayed ZamanUmar Yazdani,…, Laura Hewitson & Dwight C. German
Scientific Reports 2016; 6(19164) http://dx.doi.org:/10.1038/srep19164

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social interaction and communication, and restricted, repetitive patterns of behavior. In order to identify individuals with ASD and initiate interventions at the earliest possible age, biomarkers for the disorder are desirable. Research findings have identified widespread changes in the immune system in children with autism, at both systemic and cellular levels. In an attempt to find candidate antibody biomarkers for ASD, highly complex libraries of peptoids (oligo-N-substituted glycines) were screened for compounds that preferentially bind IgG from boys with ASD over typically developing (TD) boys. Unexpectedly, many peptoids were identified that preferentially bound IgG from TD boys. One of these peptoids was studied further and found to bind significantly higher levels (>2-fold) of the IgG1 subtype in serum from TD boys (n = 60) compared to ASD boys (n = 74), as well as compared to older adult males (n = 53). Together these data suggest that ASD boys have reduced levels (>50%) of an IgG1 antibody, which resembles the level found normally with advanced age. In this discovery study, the ASD1 peptoid was 66% accurate in predicting ASD.

….

Peptoid libraries have been used previously to search for autoantibodies for neurodegenerative diseases19 and for systemic lupus erythematosus (SLE)21. In the case of SLE, peptoids were identified that could identify subjects with the disease and related syndromes with moderate sensitivity (70%) and excellent specificity (97.5%). Peptoids were used to measure IgG levels from both healthy subjects and SLE patients. Binding to the SLE-peptoid was significantly higher in SLE patients vs. healthy controls. The IgG bound to the SLE-peptoid was found to react with several autoantigens, suggesting that the peptoids are capable of interacting with multiple, structurally similar molecules. These data indicate that IgG binding to peptoids can identify subjects with high levels of pathogenic autoantibodies vs. a single antibody.

In the present study, the ASD1 peptoid binds significantly lower levels of IgG1 in ASD males vs. TD males. This finding suggests that the ASD1 peptoid recognizes antibody(-ies) of an IgG1 subtype that is (are) significantly lower in abundance in the ASD males vs. TD males. Although a previous study14 has demonstrated lower levels of plasma IgG in ASD vs. TD children, here, we additionally quantified serum IgG levels in our individuals and found no difference in IgG between the two groups (data not shown). Furthermore, our IgG levels did not correlate with ASD1 binding levels, indicating that ASD1 does not bind IgG generically, and that the peptoid’s ability to differentiate between ASD and TD males is related to a specific antibody(-ies).

ASD subjects underwent a diagnostic evaluation using the ADOS and ADI-R, and application of the DSM-IV criteria prior to study inclusion. Only those subjects with a diagnosis of Autistic Disorder were included in the study. The ADOS is a semi-structured observation of a child’s behavior that allows examiners to observe the three core domains of ASD symptoms: reciprocal social interaction, communication, and restricted and repetitive behaviors1. When ADOS subdomain scores were compared with peptoid binding, the only significant relationship was with Social Interaction. However, the positive correlation would suggest that lower peptoid binding is associated with better social interaction, not poorer social interaction as anticipated.

The ADI-R is a structured parental interview that measures the core features of ASD symptoms in the areas of reciprocal social interaction, communication and language, and patterns of behavior. Of the three ADI-R subdomains, only the Communication domain was related to ASD1 peptoid binding, and this correlation was negative suggesting that low peptoid binding is associated with greater communication problems. These latter data are similar to the findings of Heuer et al.14 who found that children with autism with low levels of plasma IgG have high scores on the Aberrant Behavior Checklist (p < 0.0001). Thus, peptoid binding to IgG1 may be useful as a severity marker for ASD allowing for further characterization of individuals, but further research is needed.

It is interesting that in serum samples from older men, the ASD1 binding is similar to that in the ASD boys. This is consistent with the observation that with aging there is a reduction in the strength of the immune system, and the changes are gender-specific25. Recent studies using parabiosis26, in which blood from young mice reverse age-related impairments in cognitive function and synaptic plasticity in old mice, reveal that blood constituents from young subjects may contain important substances for maintaining neuronal functions. Work is in progress to identify the antibody/antibodies that are differentially binding to the ASD1 peptoid, which appear as a single band on the electrophoresis gel (Fig. 4).

……..

The ADI-R is a structured parental interview that measures the core features of ASD symptoms in the areas of reciprocal social interaction, communication and language, and patterns of behavior. Of the three ADI-R subdomains, only the Communication domain was related to ASD1 peptoid binding, and this correlation was negative suggesting that low peptoid binding is associated with greater communication problems. These latter data are similar to the findings of Heuer et al.14 who found that children with autism with low levels of plasma IgG have high scores on the Aberrant Behavior Checklist (p < 0.0001). Thus, peptoid binding to IgG1 may be useful as a severity marker for ASD allowing for further characterization of individuals, but further research is needed.

 

  • Titration of IgG binding to ASD1 using serum pooled from 10 TD males and 10 ASD males demonstrates ASD1’s ability to differentiate between the two groups. (B)Detecting IgG1 subclass instead of total IgG amplifies this differentiation. (C) IgG1 binding of individual ASD (n=74) and TD (n=60) male serum samples (1:100 dilution) to ASD1 significantly differs with TD>ASD. In addition, IgG1 binding of older adult male (AM) serum samples (n=53) to ASD1 is significantly lower than TD males, and not different from ASD males. The three groups were compared with a Kruskal-Wallis ANOVA, H = 10.1781, p<0.006. **p<0.005. Error bars show SEM. (D) Receiver-operating characteristic curve for ASD1’s ability to discriminate between ASD and TD males.

http://www.nature.com/article-assets/npg/srep/2016/160114/srep19164/images_hires/m685/srep19164-f3.jpg

 

Association between peptoid binding and ADOS and ADI-R subdomains

Higher scores in any domain on the ADOS and ADI-R are indicative of more abnormal behaviors and/or symptoms. Among ADOS subdomains, there was no significant relationship between Communication and peptoid binding (z = 0.04, p = 0.966), Communication + Social interaction (z = 1.53, p = 0.127), or Stereotyped Behaviors and Restrictive Interests (SBRI) (z = 0.46, p = 0.647). Higher scores on the Social Interaction domain were significantly associated with higher peptoid binding (z = 2.04, p = 0.041).

Among ADI-R subdomains, higher scores on the Communication domain were associated with lower levels of peptoid binding (z = −2.28, p = 0.023). There was not a significant relationship between Social Interaction (z = 0.07, p = 0.941) or Restrictive/Repetitive Stereotyped Behaviors (z = −1.40, p = 0.162) and peptoid binding.

 

 

Computational Model Finds New Protein-Protein Interactions

Researchers at University of Pittsburgh have discovered 500 new protein-protein interactions (PPIs) associated with genes linked to schizophrenia.

http://www.technologynetworks.com/Proteomics/news.aspx?id=190995

Using a computational model they developed, researchers at the University of Pittsburgh School of Medicine have discovered more than 500 new protein-protein interactions (PPIs) associated with genes linked to schizophrenia. The findings, published online in npj Schizophrenia, a Nature Publishing Group journal, could lead to greater understanding of the biological underpinnings of this mental illness, as well as point the way to treatments.

There have been many genome-wide association studies (GWAS) that have identified gene variants associated with an increased risk for schizophrenia, but in most cases there is little known about the proteins that these genes make, what they do and how they interact, said senior investigator Madhavi Ganapathiraju, Ph.D., assistant professor of biomedical informatics, Pitt School of Medicine.

“GWAS studies and other research efforts have shown us what genes might be relevant in schizophrenia,” she said. “What we have done is the next step. We are trying to understand how these genes relate to each other, which could show us the biological pathways that are important in the disease.”

Each gene makes proteins and proteins typically interact with each other in a biological process. Information about interacting partners can shed light on the role of a gene that has not been studied, revealing pathways and biological processes associated with the disease and also its relation to other complex diseases.

Dr. Ganapathiraju’s team developed a computational model called High-Precision Protein Interaction Prediction (HiPPIP) and applied it to discover PPIs of schizophrenia-linked genes identified through GWAS, as well as historically known risk genes. They found 504 never-before known PPIs, and noted also that while schizophrenia-linked genes identified historically and through GWAS had little overlap, the model showed they shared more than 100 common interactors.

“We can infer what the protein might do by checking out the company it keeps,” Dr. Ganapathiraju explained. “For example, if I know you have many friends who play hockey, it could mean that you are involved in hockey, too. Similarly, if we see that an unknown protein interacts with multiple proteins involved in neural signaling, for example, there is a high likelihood that the unknown entity also is involved in the same.”

Dr. Ganapathiraju and colleagues have drawn such inferences on protein function based on the PPIs of proteins, and made their findings available on a website Schizo-Pi. This information can be used by biologists to explore the schizophrenia interactome with the aim of understanding more about the disease or developing new treatment drugs.

Schizophrenia interactome with 504 novel protein–protein interactions

MK GanapathirajuM Thahir,…,  CE LoscherEM Bauer & S Chaparala
npj Schizophrenia 2016;  2(16012)   http://dx.doi.org:/10.1038/npjschz.2016.12

(GWAS) have revealed the role of rare and common genetic variants, but the functional effects of the risk variants remain to be understood. Protein interactome-based studies can facilitate the study of molecular mechanisms by which the risk genes relate to schizophrenia (SZ) genesis, but protein–protein interactions (PPIs) are unknown for many of the liability genes. We developed a computational model to discover PPIs, which is found to be highly accurate according to computational evaluations and experimental validations of selected PPIs. We present here, 365 novel PPIs of liability genes identified by the SZ Working Group of the Psychiatric Genomics Consortium (PGC). Seventeen genes that had no previously known interactions have 57 novel interactions by our method. Among the new interactors are 19 drug targets that are targeted by 130 drugs. In addition, we computed 147 novel PPIs of 25 candidate genes investigated in the pre-GWAS era. While there is little overlap between the GWAS genes and the pre-GWAS genes, the interactomes reveal that they largely belong to the same pathways, thus reconciling the apparent disparities between the GWAS and prior gene association studies. The interactome including 504 novel PPIs overall, could motivate other systems biology studies and trials with repurposed drugs. The PPIs are made available on a webserver, called Schizo-Pi at http://severus.dbmi.pitt.edu/schizo-pi with advanced search capabilities.

Schizophrenia (SZ) is a common, potentially severe psychiatric disorder that afflicts all populations.1 Gene mapping studies suggest that SZ is a complex disorder, with a cumulative impact of variable genetic effects coupled with environmental factors.2 As many as 38 genome-wide association studies (GWAS) have been reported on SZ out of a total of 1,750 GWAS publications on 1,087 traits or diseases reported in the GWAS catalog maintained by the National Human Genome Research Institute of USA3 (as of April 2015), revealing the common variants associated with SZ.4 The SZ Working Group of the Psychiatric Genomics Consortium (PGC) identified 108 genetic loci that likely confer risk for SZ.5 While the role of genetics has been clearly validated by this study, the functional impact of the risk variants is not well-understood.6,7 Several of the genes implicated by the GWAS have unknown functions and could participate in possibly hitherto unknown pathways.8 Further, there is little or no overlap between the genes identified through GWAS and ‘candidate genes’ proposed in the pre-GWAS era.9

Interactome-based studies can be useful in discovering the functional associations of genes. For example,disrupted in schizophrenia 1 (DISC1), an SZ related candidate gene originally had no known homolog in humans. Although it had well-characterized protein domains such as coiled-coil domains and leucine-zipper domains, its function was unknown.10,11 Once its protein–protein interactions (PPIs) were determined using yeast 2-hybrid technology,12 investigators successfully linked DISC1 to cAMP signaling, axon elongation, and neuronal migration, and accelerated the research pertaining to SZ in general, and DISC1 in particular.13 Typically such studies are carried out on known protein–protein interaction (PPI) networks, or as in the case of DISC1, when there is a specific gene of interest, its PPIs are determined by methods such as yeast 2-hybrid technology.

Knowledge of human PPI networks is thus valuable for accelerating discovery of protein function, and indeed, biomedical research in general. However, of the hundreds of thousands of biophysical PPIs thought to exist in the human interactome,14,15 <100,000 are known today (Human Protein Reference Database, HPRD16 and BioGRID17 databases). Gold standard experimental methods for the determination of all the PPIs in human interactome are time-consuming, expensive and may not even be feasible, as about 250 million pairs of proteins would need to be tested overall; high-throughput methods such as yeast 2-hybrid have important limitations for whole interactome determination as they have a low recall of 23% (i.e., remaining 77% of true interactions need to be determined by other means), and a low precision (i.e., the screens have to be repeated multiple times to achieve high selectivity).18,19Computational methods are therefore necessary to complete the interactome expeditiously. Algorithms have begun emerging to predict PPIs using statistical machine learning on the characteristics of the proteins, but these algorithms are employed predominantly to study yeast. Two significant computational predictions have been reported for human interactome; although they have had high false positive rates, these methods have laid the foundation for computational prediction of human PPIs.20,21

We have created a new PPI prediction model called High-Confidence Protein–Protein Interaction Prediction (HiPPIP) model. Novel interactions predicted with this model are making translational impact. For example, we discovered a PPI between OASL and DDX58, which on validation showed that an increased expression of OASL could boost innate immunity to combat influenza by activating the RIG-I pathway.22 Also, the interactome of the genes associated with congenital heart disease showed that the disease morphogenesis has a close connection with the structure and function of cilia.23Here, we describe the HiPPIP model and its application to SZ genes to construct the SZ interactome. After computational evaluations and experimental validations of selected novel PPIs, we present here 504 highly confident novel PPIs in the SZ interactome, shedding new light onto several uncharacterized genes that are associated with SZ.

We developed a computational model called HiPPIP to predict PPIs (see Methods and Supplementary File 1). The model has been evaluated by computational methods and experimental validations and is found to be highly accurate. Evaluations on a held-out test data showed a precision of 97.5% and a recall of 5%. 5% recall out of 150,000 to 600,000 estimated number of interactions in the human interactome corresponds to 7,500–30,000 novel PPIs in the whole interactome. Note that, it is likely that the real precision would be higher than 97.5% because in this test data, randomly paired proteins are treated as non-interacting protein pairs, whereas some of them may actually be interacting pairs with a small probability; thus, some of the pairs that are treated as false positives in test set are likely to be true but hitherto unknown interactions. In Figure 1a, we show the precision versus recall of our method on ‘hub proteins’ where we considered all pairs that received a score >0.5 by HiPPIP to be novel interactions. In Figure 1b, we show the number of true positives versus false positives observed in hub proteins. Both these figures also show our method to be superior in comparison to the prediction of membrane-receptor interactome by Qi et al’s.24 True positives versus false positives are also shown for individual hub proteins by our method in Figure 1cand by Qi et al’s.23 in Figure 1d. These evaluations showed that our predictions contain mostly true positives. Unlike in other domains where ranked lists are commonly used such as information retrieval, in PPI prediction the ‘false positives’ may actually be unlabeled instances that are indeed true interactions that are not yet discovered. In fact, such unlabeled pairs predicted as interactors of the hub gene HMGB1 (namely, the pairs HMGB1-KL and HMGB1-FLT1) were validated by experimental methods and found to be true PPIs (See the Figures e–g inSupplementary File 3). Thus, we concluded that the protein pairs that received a score of ⩾0.5 are highly confident to be true interactions. The pairs that receive a score less than but close to 0.5 (i.e., in the range of 0.4–0.5) may also contain several true PPIs; however, we cannot confidently say that all in this range are true PPIs. Only the PPIs predicted with a score >0.5 are included in the interactome.

Figure 1

http://www.nature.com/article-assets/npg/npjschz/2016/npjschz201612/images_hires/w582/npjschz201612-f1.jpg

Computational evaluation of predicted protein–protein interactions on hub proteins: (a) precision recall curve. (b) True positive versus false positives in ranked lists of hub type membrane receptors for our method and that by Qi et al. True positives versus false positives are shown for individual membrane receptors by our method in (c) and by Qi et al. in (d). Thick line is the average, which is also the same as shown in (b). Note:x-axis is recall in (a), whereas it is number of false positives in (bd). The range of y-axis is observed by varying the threshold from 1.0–0 in (a), and to 0.5 in (bd).

SZ interactome

By applying HiPPIP to the GWAS genes and Historic (pre-GWAS) genes, we predicted over 500 high confidence new PPIs adding to about 1400 previously known PPIs.

Schizophrenia interactome: network view of the schizophrenia interactome is shown as a graph, where genes are shown as nodes and PPIs as edges connecting the nodes. Schizophrenia-associated genes are shown as dark blue nodes, novel interactors as red color nodes and known interactors as blue color nodes. The source of the schizophrenia genes is indicated by its label font, where Historic genes are shown italicized, GWAS genes are shown in bold, and the one gene that is common to both is shown in italicized and bold. For clarity, the source is also indicated by the shape of the node (triangular for GWAS and square for Historic and hexagonal for both). Symbols are shown only for the schizophrenia-associated genes; actual interactions may be accessed on the web. Red edges are the novel interactions, whereas blue edges are known interactions. GWAS, genome-wide association studies of schizophrenia; PPI, protein–protein interaction.

http://www.nature.com/article-assets/npg/npjschz/2016/npjschz201612/images_hires/m685/npjschz201612-f2.jpg

 

Webserver of SZ interactome

We have made the known and novel interactions of all SZ-associated genes available on a webserver called Schizo-Pi, at the addresshttp://severus.dbmi.pitt.edu/schizo-pi. This webserver is similar to Wiki-Pi33 which presents comprehensive annotations of both participating proteins of a PPI side-by-side. The difference between Wiki-Pi which we developed earlier, and Schizo-Pi, is the inclusion of novel predicted interactions of the SZ genes into the latter.

Despite the many advances in biomedical research, identifying the molecular mechanisms underlying the disease is still challenging. Studies based on protein interactions were proven to be valuable in identifying novel gene associations that could shed new light on disease pathology.35 The interactome including more than 500 novel PPIs will help to identify pathways and biological processes associated with the disease and also its relation to other complex diseases. It also helps identify potential drugs that could be repurposed to use for SZ treatment.

Functional and pathway enrichment in SZ interactome

When a gene of interest has little known information, functions of its interacting partners serve as a starting point to hypothesize its own function. We computed statistically significant enrichment of GO biological process terms among the interacting partners of each of the genes using BinGO36 (see online at http://severus.dbmi.pitt.edu/schizo-pi).

 

Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution

Massimo Stefani · Christopher M. Dobson

Abstract The deposition of proteins in the form of amyloid fibrils and plaques is the characteristic feature of more than 20 degenerative conditions affecting either the central nervous system or a variety of peripheral tissues. As these conditions include Alzheimer’s, Parkinson’s and the prion diseases, several forms of fatal systemic amyloidosis, and at least one condition associated with medical intervention (haemodialysis), they are of enormous importance in the context of present-day human health and welfare. Much remains to be learned about the mechanism by which the proteins associated with these diseases aggregate and form amyloid structures, and how the latter affect the functions of the organs with which they are associated. A great deal of information concerning these diseases has emerged, however, during the past 5 years, much of it causing a number of fundamental assumptions about the amyloid diseases to be reexamined. For example, it is now apparent that the ability to form amyloid structures is not an unusual feature of the small number of proteins associated with these diseases but is instead a general property of polypeptide chains. It has also been found recently that aggregates of proteins not associated with amyloid diseases can impair the ability of cells to function to a similar extent as aggregates of proteins linked with specific neurodegenerative conditions. Moreover, the mature amyloid fibrils or plaques appear to be substantially less toxic than the prefibrillar aggregates that are their precursors. The toxicity of these early aggregates appears to result from an intrinsic ability to impair fundamental cellular processes by interacting with cellular membranes, causing oxidative stress and increases in free Ca2+ that eventually lead to apoptotic or necrotic cell death. The ‘new view’ of these diseases also suggests that other degenerative conditions could have similar underlying origins to those of the amyloidoses. In addition, cellular protection mechanisms, such as molecular chaperones and the protein degradation machinery, appear to be crucial in the prevention of disease in normally functioning living organisms. It also suggests some intriguing new factors that could be of great significance in the evolution of biological molecules and the mechanisms that regulate their behaviour.

The genetic information within a cell encodes not only the specific structures and functions of proteins but also the way these structures are attained through the process known as protein folding. In recent years many of the underlying features of the fundamental mechanism of this complex process and the manner in which it is regulated in living systems have emerged from a combination of experimental and theoretical studies [1]. The knowledge gained from these studies has also raised a host of interesting issues. It has become apparent, for example, that the folding and unfolding of proteins is associated with a whole range of cellular processes from the trafficking of molecules to specific organelles to the regulation of the cell cycle and the immune response. Such observations led to the inevitable conclusion that the failure to fold correctly, or to remain correctly folded, gives rise to many different types of biological malfunctions and hence to many different forms of disease [2]. In addition, it has been recognised recently that a large number of eukaryotic genes code for proteins that appear to be ‘natively unfolded’, and that proteins can adopt, under certain circumstances, highly organised multi-molecular assemblies whose structures are not specifically encoded in the amino acid sequence. Both these observations have raised challenging questions about one of the most fundamental principles of biology: the close relationship between the sequence, structure and function of proteins, as we discuss below [3].

It is well established that proteins that are ‘misfolded’, i.e. that are not in their functionally relevant conformation, are devoid of normal biological activity. In addition, they often aggregate and/or interact inappropriately with other cellular components leading to impairment of cell viability and eventually to cell death. Many diseases, often known as misfolding or conformational diseases, ultimately result from the presence in a living system of protein molecules with structures that are ‘incorrect’, i.e. that differ from those in normally functioning organisms [4]. Such diseases include conditions in which a specific protein, or protein complex, fails to fold correctly (e.g. cystic fibrosis, Marfan syndrome, amyotonic lateral sclerosis) or is not sufficiently stable to perform its normal function (e.g. many forms of cancer). They also include conditions in which aberrant folding behaviour results in the failure of a protein to be correctly trafficked (e.g. familial hypercholesterolaemia, α1-antitrypsin deficiency, and some forms of retinitis pigmentosa) [4]. The tendency of proteins to aggregate, often to give species extremely intractable to dissolution and refolding, is of course also well known in other circumstances. Examples include the formation of inclusion bodies during overexpression of heterologous proteins in bacteria and the precipitation of proteins during laboratory purification procedures. Indeed, protein aggregation is well established as one of the major difficulties associated with the production and handling of proteins in the biotechnology and pharmaceutical industries [5].

Considerable attention is presently focused on a group of protein folding diseases known as amyloidoses. In these diseases specific peptides or proteins fail to fold or to remain correctly folded and then aggregate (often with other components) so as to give rise to ‘amyloid’ deposits in tissue. Amyloid structures can be recognised because they possess a series of specific tinctorial and biophysical characteristics that reflect a common core structure based on the presence of highly organised βsheets [6]. The deposits in strictly defined amyloidoses are extracellular and can often be observed as thread-like fibrillar structures, sometimes assembled further into larger aggregates or plaques. These diseases include a range of sporadic, familial or transmissible degenerative diseases, some of which affect the brain and the central nervous system (e.g. Alzheimer’s and Creutzfeldt-Jakob diseases), while others involve peripheral tissues and organs such as the liver, heart and spleen (e.g. systemic amyloidoses and type II diabetes) [7, 8]. In other forms of amyloidosis, such as primary or secondary systemic amyloidoses, proteinaceous deposits are found in skeletal tissue and joints (e.g. haemodialysis-related amyloidosis) as well as in several organs (e.g. heart and kidney). Yet other components such as collagen, glycosaminoglycans and proteins (e.g. serum amyloid protein) are often present in the deposits protecting them against degradation [9, 10, 11]. Similar deposits to those in the amyloidoses are, however, found intracellularly in other diseases; these can be localised either in the cytoplasm, in the form of specialised aggregates known as aggresomes or as Lewy or Russell bodies or in the nucleus (see below).

The presence in tissue of proteinaceous deposits is a hallmark of all these diseases, suggesting a causative link between aggregate formation and pathological symptoms (often known as the amyloid hypothesis) [7, 8, 12]. At the present time the link between amyloid formation and disease is widely accepted on the basis of a large number of biochemical and genetic studies. The specific nature of the pathogenic species, and the molecular basis of their ability to damage cells, are however, the subject of intense debate [13, 14, 15, 16, 17, 18, 19, 20]. In neurodegenerative disorders it is very likely that the impairment of cellular function follows directly from the interactions of the aggregated proteins with cellular components [21, 22]. In the systemic non-neurological diseases, however, it is widely believed that the accumulation in vital organs of large amounts of amyloid deposits can by itself cause at least some of the clinical symptoms [23]. It is quite possible, however, that there are other more specific effects of aggregates on biochemical processes even in these diseases. The presence of extracellular or intracellular aggregates of a specific polypeptide molecule is a characteristic of all the 20 or so recognised amyloid diseases. The polypeptides involved include full length proteins (e.g. lysozyme or immunoglobulin light chains), biological peptides (amylin, atrial natriuretic factor) and fragments of larger proteins produced as a result of specific processing (e.g. the Alzheimer βpeptide) or of more general degradation [e.g. poly(Q) stretches cleaved from proteins with poly(Q) extensions such as huntingtin, ataxins and the androgen receptor]. The peptides and proteins associated with known amyloid diseases are listed in Table 1. In some cases the proteins involved have wild type sequences, as in sporadic forms of the diseases, but in other cases these are variants resulting from genetic mutations associated with familial forms of the diseases. In some cases both sporadic and familial diseases are associated with a given protein; in this case the mutational variants are usually associated with early-onset forms of the disease. In the case of the neurodegenerative diseases associated with the prion protein some forms of the diseases are transmissible. The existence of familial forms of a number of amyloid diseases has provided significant clues to the origins of the pathologies. For example, there are increasingly strong links between the age at onset of familial forms of disease and the effects of the mutations involved on the propensity of the affected proteins to aggregate in vitro. Such findings also support the link between the process of aggregation and the clinical manifestations of disease [24, 25].

The presence in cells of misfolded or aggregated proteins triggers a complex biological response. In the cytosol, this is referred to as the ‘heat shock response’ and in the endoplasmic reticulum (ER) it is known as the ‘unfolded protein response’. These responses lead to the expression, among others, of the genes for heat shock proteins (Hsp, or molecular chaperone proteins) and proteins involved in the ubiquitin-proteasome pathway [26]. The evolution of such complex biochemical machinery testifies to the fact that it is necessary for cells to isolate and clear rapidly and efficiently any unfolded or incorrectly folded protein as soon as it appears. In itself this fact suggests that these species could have a generally adverse effect on cellular components and cell viability. Indeed, it was a major step forward in understanding many aspects of cell biology when it was recognised that proteins previously associated only with stress, such as heat shock, are in fact crucial in the normal functioning of living systems. This advance, for example, led to the discovery of the role of molecular chaperones in protein folding and in the normal ‘housekeeping’ processes that are inherent in healthy cells [27, 28]. More recently a number of degenerative diseases, both neurological and systemic, have been linked to, or shown to be affected by, impairment of the ubiquitin-proteasome pathway (Table 2). The diseases are primarily associated with a reduction in either the expression or the biological activity of Hsps, ubiquitin, ubiquitinating or deubiquitinating enzymes and the proteasome itself, as we show below [29, 30, 31, 32], or even to the failure of the quality control mechanisms that ensure proper maturation of proteins in the ER. The latter normally leads to degradation of a significant proportion of polypeptide chains before they have attained their native conformations through retrograde translocation to the cytosol [33, 34].

….

It is now well established that the molecular basis of protein aggregation into amyloid structures involves the existence of ‘misfolded’ forms of proteins, i.e. proteins that are not in the structures in which they normally function in vivo or of fragments of proteins resulting from degradation processes that are inherently unable to fold [4, 7, 8, 36]. Aggregation is one of the common consequences of a polypeptide chain failing to reach or maintain its functional three-dimensional structure. Such events can be associated with specific mutations, misprocessing phenomena, aberrant interactions with metal ions, changes in environmental conditions, such as pH or temperature, or chemical modification (oxidation, proteolysis). Perturbations in the conformational properties of the polypeptide chain resulting from such phenomena may affect equilibrium 1 in Fig. 1 increasing the population of partially unfolded, or misfolded, species that are much more aggregation-prone than the native state.

Fig. 1 Overview of the possible fates of a newly synthesised polypeptide chain. The equilibrium ① between the partially folded molecules and the natively folded ones is usually strongly in favour of the latter except as a result of specific mutations, chemical modifications or partially destabilising solution conditions. The increased equilibrium populations of molecules in the partially or completely unfolded ensemble of structures are usually degraded by the proteasome; when this clearance mechanism is impaired, such species often form disordered aggregates or shift equilibrium ② towards the nucleation of pre-fibrillar assemblies that eventually grow into mature fibrils (equilibrium ③). DANGER! indicates that pre-fibrillar aggregates in most cases display much higher toxicity than mature fibrils. Heat shock proteins (Hsp) can suppress the appearance of pre-fibrillar assemblies by minimising the population of the partially folded molecules by assisting in the correct folding of the nascent chain and the unfolded protein response target incorrectly folded proteins for degradation.

……

Little is known at present about the detailed arrangement of the polypeptide chains themselves within amyloid fibrils, either those parts involved in the core βstrands or in regions that connect the various β-strands. Recent data suggest that the sheets are relatively untwisted and may in some cases at least exist in quite specific supersecondary structure motifs such as β-helices [6, 40] or the recently proposed µ-helix [41]. It seems possible that there may be significant differences in the way the strands are assembled depending on characteristics of the polypeptide chain involved [6, 42]. Factors including length, sequence (and in some cases the presence of disulphide bonds or post-translational modifications such as glycosylation) may be important in determining details of the structures. Several recent papers report structural models for amyloid fibrils containing different polypeptide chains, including the Aβ40 peptide, insulin and fragments of the prion protein, based on data from such techniques as cryo-electron microscopy and solid-state magnetic resonance spectroscopy [43, 44]. These models have much in common and do indeed appear to reflect the fact that the structures of different fibrils are likely to be variations on a common theme [40]. It is also emerging that there may be some common and highly organised assemblies of amyloid protofilaments that are not simply extended threads or ribbons. It is clear, for example, that in some cases large closed loops can be formed [45, 46, 47], and there may be specific types of relatively small spherical or ‘doughnut’ shaped structures that can result in at least some circumstances (see below).

…..

The similarity of some early amyloid aggregates with the pores resulting from oligomerisation of bacterial toxins and pore-forming eukaryotic proteins (see below) also suggest that the basic mechanism of protein aggregation into amyloid structures may not only be associated with diseases but in some cases could result in species with functional significance. Recent evidence indicates that a variety of micro-organisms may exploit the controlled aggregation of specific proteins (or their precursors) to generate functional structures. Examples include bacterial curli [52] and proteins of the interior fibre cells of mammalian ocular lenses, whose β-sheet arrays seem to be organised in an amyloid-like supramolecular order [53]. In this case the inherent stability of amyloid-like protein structure may contribute to the long-term structural integrity and transparency of the lens. Recently it has been hypothesised that amyloid-like aggregates of serum amyloid A found in secondary amyloidoses following chronic inflammatory diseases protect the host against bacterial infections by inducing lysis of bacterial cells [54]. One particularly interesting example is a ‘misfolded’ form of the milk protein α-lactalbumin that is formed at low pH and trapped by the presence of specific lipid molecules [55]. This form of the protein has been reported to trigger apoptosis selectively in tumour cells providing evidence for its importance in protecting infants from certain types of cancer [55]. ….

Amyloid formation is a generic property of polypeptide chains ….

It is clear that the presence of different side chains can influence the details of amyloid structures, particularly the assembly of protofibrils, and that they give rise to the variations on the common structural theme discussed above. More fundamentally, the composition and sequence of a peptide or protein affects profoundly its propensity to form amyloid structures under given conditions (see below).

Because the formation of stable protein aggregates of amyloid type does not normally occur in vivo under physiological conditions, it is likely that the proteins encoded in the genomes of living organisms are endowed with structural adaptations that mitigate against aggregation under these conditions. A recent survey involving a large number of structures of β-proteins highlights several strategies through which natural proteins avoid intermolecular association of β-strands in their native states [65].  Other surveys of protein databases indicate that nature disfavours sequences of alternating polar and nonpolar residues, as well as clusters of several consecutive hydrophobic residues, both of which enhance the tendency of a protein to aggregate prior to becoming completely folded [66, 67].

……

Precursors of amyloid fibrils can be toxic to cells

It was generally assumed until recently that the proteinaceous aggregates most toxic to cells are likely to be mature amyloid fibrils, the form of aggregates that have been commonly detected in pathological deposits. It therefore appeared probable that the pathogenic features underlying amyloid diseases are a consequence of the interaction with cells of extracellular deposits of aggregated material. As well as forming the basis for understanding the fundamental causes of these diseases, this scenario stimulated the exploration of therapeutic approaches to amyloidoses that focused mainly on the search for molecules able to impair the growth and deposition of fibrillar forms of aggregated proteins. ….

Structural basis and molecular features of amyloid toxicity

The presence of toxic aggregates inside or outside cells can impair a number of cell functions that ultimately lead to cell death by an apoptotic mechanism [95, 96]. Recent research suggests, however, that in most cases initial perturbations to fundamental cellular processes underlie the impairment of cell function induced by aggregates of disease-associated polypeptides. Many pieces of data point to a central role of modifications to the intracellular redox status and free Ca2+ levels in cells exposed to toxic aggregates [45, 89, 97, 98, 99, 100, 101]. A modification of the intracellular redox status in such cells is associated with a sharp increase in the quantity of reactive oxygen species (ROS) that is reminiscent of the oxidative burst by which leukocytes destroy invading foreign cells after phagocytosis. In addition, changes have been observed in reactive nitrogen species, lipid peroxidation, deregulation of NO metabolism [97], protein nitrosylation [102] and upregulation of heme oxygenase-1, a specific marker of oxidative stress [103]. ….

Results have recently been reported concerning the toxicity towards cultured cells of aggregates of poly(Q) peptides which argues against a disease mechanism based on specific toxic features of the aggregates. These results indicate that there is a close relationship between the toxicity of proteins with poly(Q) extensions and their nuclear localisation. In addition they support the hypotheses that the toxicity of poly(Q) aggregates can be a consequence of altered interactions with nuclear coactivator or corepressor molecules including p53, CBP, Sp1 and TAF130 or of the interaction with transcription factors and nuclear coactivators, such as CBP, endowed with short poly(Q) stretches ([95] and references therein)…..

Concluding remarks
The data reported in the past few years strongly suggest that the conversion of normally soluble proteins into amyloid fibrils and the toxicity of small aggregates appearing during the early stages of the formation of the latter are common or generic features of polypeptide chains. Moreover, the molecular basis of this toxicity also appears to display common features between the different systems that have so far been studied. The ability of many, perhaps all, natural polypeptides to ‘misfold’ and convert into toxic aggregates under suitable conditions suggests that one of the most important driving forces in the evolution of proteins must have been the negative selection against sequence changes that increase the tendency of a polypeptide chain to aggregate. Nevertheless, as protein folding is a stochastic process, and no such process can be completely infallible, misfolded proteins or protein folding intermediates in equilibrium with the natively folded molecules must continuously form within cells. Thus mechanisms to deal with such species must have co-evolved with proteins. Indeed, it is clear that misfolding, and the associated tendency to aggregate, is kept under control by molecular chaperones, which render the resulting species harmless assisting in their refolding, or triggering their degradation by the cellular clearance machinery [166, 167, 168, 169, 170, 171, 172, 173, 175, 177, 178].

Misfolded and aggregated species are likely to owe their toxicity to the exposure on their surfaces of regions of proteins that are buried in the interior of the structures of the correctly folded native states. The exposure of large patches of hydrophobic groups is likely to be particularly significant as such patches favour the interaction of the misfolded species with cell membranes [44, 83, 89, 90, 91, 93]. Interactions of this type are likely to lead to the impairment of the function and integrity of the membranes involved, giving rise to a loss of regulation of the intracellular ion balance and redox status and eventually to cell death. In addition, misfolded proteins undoubtedly interact inappropriately with other cellular components, potentially giving rise to the impairment of a range of other biological processes. Under some conditions the intracellular content of aggregated species may increase directly, due to an enhanced propensity of incompletely folded or misfolded species to aggregate within the cell itself. This could occur as the result of the expression of mutational variants of proteins with decreased stability or cooperativity or with an intrinsically higher propensity to aggregate. It could also occur as a result of the overproduction of some types of protein, for example, because of other genetic factors or other disease conditions, or because of perturbations to the cellular environment that generate conditions favouring aggregation, such as heat shock or oxidative stress. Finally, the accumulation of misfolded or aggregated proteins could arise from the chaperone and clearance mechanisms becoming overwhelmed as a result of specific mutant phenotypes or of the general effects of ageing [173, 174].

The topics discussed in this review not only provide a great deal of evidence for the ‘new view’ that proteins have an intrinsic capability of misfolding and forming structures such as amyloid fibrils but also suggest that the role of molecular chaperones is even more important than was thought in the past. The role of these ubiquitous proteins in enhancing the efficiency of protein folding is well established [185]. It could well be that they are at least as important in controlling the harmful effects of misfolded or aggregated proteins as in enhancing the yield of functional molecules.

 

Nutritional Status is Associated with Faster Cognitive Decline and Worse Functional Impairment in the Progression of Dementia: The Cache County Dementia Progression Study1

Sanders, Chelseaa | Behrens, Stephaniea | Schwartz, Sarahb | Wengreen, Heidic | Corcoran, Chris D.b; d | Lyketsos, Constantine G.e | Tschanz, JoAnn T.a; d;
Journal of Alzheimer’s Disease 2016; 52(1):33-42,     http://content.iospress.com/articles/journal-of-alzheimers-disease/jad150528   http://dx.doi.org:/10.3233/JAD-150528

Nutritional status may be a modifiable factor in the progression of dementia. We examined the association of nutritional status and rate of cognitive and functional decline in a U.S. population-based sample. Study design was an observational longitudinal study with annual follow-ups up to 6 years of 292 persons with dementia (72% Alzheimer’s disease, 56% female) in Cache County, UT using the Mini-Mental State Exam (MMSE), Clinical Dementia Rating Sum of Boxes (CDR-sb), and modified Mini Nutritional Assessment (mMNA). mMNA scores declined by approximately 0.50 points/year, suggesting increasing risk for malnutrition. Lower mMNA score predicted faster rate of decline on the MMSE at earlier follow-up times, but slower decline at later follow-up times, whereas higher mMNA scores had the opposite pattern (mMNA by time β= 0.22, p = 0.017; mMNA by time2 β= –0.04, p = 0.04). Lower mMNA score was associated with greater impairment on the CDR-sb over the course of dementia (β= 0.35, p <  0.001). Assessment of malnutrition may be useful in predicting rates of progression in dementia and may provide a target for clinical intervention.

 

Shared Genetic Risk Factors for Late-Life Depression and Alzheimer’s Disease

Ye, Qing | Bai, Feng* | Zhang, Zhijun
Journal of Alzheimer’s Disease 2016; 52(1): 1-15.                                      http://dx.doi.org:/10.3233/JAD-151129

Background: Considerable evidence has been reported for the comorbidity between late-life depression (LLD) and Alzheimer’s disease (AD), both of which are very common in the general elderly population and represent a large burden on the health of the elderly. The pathophysiological mechanisms underlying the link between LLD and AD are poorly understood. Because both LLD and AD can be heritable and are influenced by multiple risk genes, shared genetic risk factors between LLD and AD may exist. Objective: The objective is to review the existing evidence for genetic risk factors that are common to LLD and AD and to outline the biological substrates proposed to mediate this association. Methods: A literature review was performed. Results: Genetic polymorphisms of brain-derived neurotrophic factor, apolipoprotein E, interleukin 1-beta, and methylenetetrahydrofolate reductase have been demonstrated to confer increased risk to both LLD and AD by studies examining either LLD or AD patients. These results contribute to the understanding of pathophysiological mechanisms that are common to both of these disorders, including deficits in nerve growth factors, inflammatory changes, and dysregulation mechanisms involving lipoprotein and folate. Other conflicting results have also been reviewed, and few studies have investigated the effects of the described polymorphisms on both LLD and AD. Conclusion: The findings suggest that common genetic pathways may underlie LLD and AD comorbidity. Studies to evaluate the genetic relationship between LLD and AD may provide insights into the molecular mechanisms that trigger disease progression as the population ages.

 

Association of Vitamin B12, Folate, and Sulfur Amino Acids With Brain Magnetic Resonance Imaging Measures in Older Adults: A Longitudinal Population-Based Study

B Hooshmand, F Mangialasche, G Kalpouzos…, et al.
AMA Psychiatry. Published online April 27, 2016.    http://dx.doi.org:/10.1001/jamapsychiatry.2016.0274

Importance  Vitamin B12, folate, and sulfur amino acids may be modifiable risk factors for structural brain changes that precede clinical dementia.

Objective  To investigate the association of circulating levels of vitamin B12, red blood cell folate, and sulfur amino acids with the rate of total brain volume loss and the change in white matter hyperintensity volume as measured by fluid-attenuated inversion recovery in older adults.

Design, Setting, and Participants  The magnetic resonance imaging subsample of the Swedish National Study on Aging and Care in Kungsholmen, a population-based longitudinal study in Stockholm, Sweden, was conducted in 501 participants aged 60 years or older who were free of dementia at baseline. A total of 299 participants underwent repeated structural brain magnetic resonance imaging scans from September 17, 2001, to December 17, 2009.

Main Outcomes and Measures  The rate of brain tissue volume loss and the progression of total white matter hyperintensity volume.

Results  In the multi-adjusted linear mixed models, among 501 participants (300 women [59.9%]; mean [SD] age, 70.9 [9.1] years), higher baseline vitamin B12 and holotranscobalamin levels were associated with a decreased rate of total brain volume loss during the study period: for each increase of 1 SD, β (SE) was 0.048 (0.013) for vitamin B12 (P < .001) and 0.040 (0.013) for holotranscobalamin (P = .002). Increased total homocysteine levels were associated with faster rates of total brain volume loss in the whole sample (β [SE] per 1-SD increase, –0.035 [0.015]; P = .02) and with the progression of white matter hyperintensity among participants with systolic blood pressure greater than 140 mm Hg (β [SE] per 1-SD increase, 0.000019 [0.00001]; P = .047). No longitudinal associations were found for red blood cell folate and other sulfur amino acids.

Conclusions and Relevance  This study suggests that both vitamin B12 and total homocysteine concentrations may be related to accelerated aging of the brain. Randomized clinical trials are needed to determine the importance of vitamin B12supplementation on slowing brain aging in older adults.

 

 

Notes from Kurzweill

This vitamin stops the aging process in organs, say Swiss researchers

A potential breakthrough for regenerative medicine, pending further studies

http://www.kurzweilai.net/this-vitamin-stops-the-aging-process-in-organs-say-swiss-researchers

Improved muscle stem cell numbers and muscle function in NR-treated aged mice: Newly regenerated muscle fibers 7 days after muscle damage in aged mice (left: control group; right: fed NR). (Scale bar = 50 μm). (credit: Hongbo Zhang et al./Science) http://www.kurzweilai.net/images/improved-muscle-fibers.png

EPFL researchers have restored the ability of mice organs to regenerate and extend life by simply administering nicotinamide riboside (NR) to them.

NR has been shown in previous studies to be effective in boosting metabolism and treating a number of degenerative diseases. Now, an article by PhD student Hongbo Zhang published in Science also describes the restorative effects of NR on the functioning of stem cells for regenerating organs.

As in all mammals, as mice age, the regenerative capacity of certain organs (such as the liver and kidneys) and muscles (including the heart) diminishes. Their ability to repair them following an injury is also affected. This leads to many of the disorders typical of aging.

Mitochondria —> stem cells —> organs

To understand how the regeneration process deteriorates with age, Zhang teamed up with colleagues from ETH Zurich, the University of Zurich, and universities in Canada and Brazil. By using several biomarkers, they were able to identify the molecular chain that regulates how mitochondria — the “powerhouse” of the cell — function and how they change with age. “We were able to show for the first time that their ability to function properly was important for stem cells,” said Auwerx.

Under normal conditions, these stem cells, reacting to signals sent by the body, regenerate damaged organs by producing new specific cells. At least in young bodies. “We demonstrated that fatigue in stem cells was one of the main causes of poor regeneration or even degeneration in certain tissues or organs,” said Zhang.

How to revitalize stem cells

Which is why the researchers wanted to “revitalize” stem cells in the muscles of elderly mice. And they did so by precisely targeting the molecules that help the mitochondria to function properly. “We gave nicotinamide riboside to 2-year-old mice, which is an advanced age for them,” said Zhang.

“This substance, which is close to vitamin B3, is a precursor of NAD+, a molecule that plays a key role in mitochondrial activity. And our results are extremely promising: muscular regeneration is much better in mice that received NR, and they lived longer than the mice that didn’t get it.”

Parallel studies have revealed a comparable effect on stem cells of the brain and skin. “This work could have very important implications in the field of regenerative medicine,” said Auwerx. This work on the aging process also has potential for treating diseases that can affect — and be fatal — in young people, like muscular dystrophy (myopathy).

So far, no negative side effects have been observed following the use of NR, even at high doses. But while it appears to boost the functioning of all cells, it could include pathological ones, so further in-depth studies are required.

Abstract of NAD+ repletion improves mitochondrial and stem cell function and enhances life span in mice

Adult stem cells (SCs) are essential for tissue maintenance and regeneration yet are susceptible to senescence during aging. We demonstrate the importance of the amount of the oxidized form of cellular nicotinamide adenine dinucleotide (NAD+) and its impact on mitochondrial activity as a pivotal switch to modulate muscle SC (MuSC) senescence. Treatment with the NAD+ precursor nicotinamide riboside (NR) induced the mitochondrial unfolded protein response (UPRmt) and synthesis of prohibitin proteins, and this rejuvenated MuSCs in aged mice. NR also prevented MuSC senescence in the Mdx mouse model of muscular dystrophy. We furthermore demonstrate that NR delays senescence of neural SCs (NSCs) and melanocyte SCs (McSCs), and increased mouse lifespan. Strategies that conserve cellular NAD+ may reprogram dysfunctional SCs and improve lifespan in mammals.

references:

Hongbo Zhang, Dongryeol Ryu, Yibo Wu, Karim Gariani, Xu Wang, Peiling Luan, Davide D’amico, Eduardo R. Ropelle, Matthias P. Lutolf, Ruedi Aebersold, Kristina Schoonjans, Keir J. Menzies, Johan Auwerx. NAD repletion improves mitochondrial and stem cell function and enhances lifespan in mice. Science, 2016 DOI: 10.1126/science.aaf2693

 

Enhancer–promoter interactions are encoded by complex genomic signatures on looping chromatin

Sean WhalenRebecca M Truty & Katherine S Pollard
Nature Genetics 2016; 48:488–496
    
    doi:10.1038/ng.3539

Discriminating the gene target of a distal regulatory element from other nearby transcribed genes is a challenging problem with the potential to illuminate the causal underpinnings of complex diseases. We present TargetFinder, a computational method that reconstructs regulatory landscapes from diverse features along the genome. The resulting models accurately predict individual enhancer–promoter interactions across multiple cell lines with a false discovery rate up to 15 times smaller than that obtained using the closest gene. By evaluating the genomic features driving this accuracy, we uncover interactions between structural proteins, transcription factors, epigenetic modifications, and transcription that together distinguish interacting from non-interacting enhancer–promoter pairs. Most of this signature is not proximal to the enhancers and promoters but instead decorates the looping DNA. We conclude that complex but consistent combinations of marks on the one-dimensional genome encode the three-dimensional structure of fine-scale regulatory interactions.

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Schizophrenia genomics

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Histone Methylation at H3K9; Evidence for a Restrictive Epigenome in Schizophrenia

Schizophr Res. 2013 Sep; 149(0): 15–20.      doi:  10.1016/j.schres.2013.06.021

Epigenetic changes are stable and long-lasting chromatin modifications that regulate genomewide and local gene activity. The addition of two methyl groups to the 9th lysine of histone 3 (H3K9me2) by histone methyltransferases (HMT) leads to a restrictive chromatin state, and thus reduced levels of gene transcription. Given the numerous reports of transcriptional down-regulation of candidate genes in schizophrenia, we tested the hypothesis that this illness can be characterized by a restrictive epigenome.

METHODS   We obtained parietal cortical samples from the Stanley Foundation Neuropathology Consortium and lymphocyte samples from the University of Illinois at Chicago (UIC). In both tissues we measured mRNA expression of HMTs GLP, SETDB1 and G9a via real-time RT-PCR and H3K9me2 levels via western blot. Clinical rating scales were obtained from the UIC cohort.

RESULTS   A diagnosis of schizophrenia is a significant predictor for increased GLP, SETDB1 mRNA expression and H3K9me2 levels in both postmortem brain and lymphocyte samples. G9a mRNA is significantly increased in the UIC lymphocyte samples as well. Increased HMT mRNA expression is associated with worsening of specific symptoms, longer durations of illness and a family history of schizophrenia.

CONCLUSIONS   These data support the hypothesis of a restrictive epigenome in schizophrenia, and may associate with symptoms that are notoriously treatment resistant. The histone methyltransferases measured here are potential future therapeutic targets for small molecule pharmacology, and better patient prognosis.

Schizophrenia is conceptualized as a disorder of gene transcription and regulation. Consequently, chromatin is the ideal scaffold to examine this manifested pathophysiology of schizophrenia, as it constitutes the interface between the underlying genetic code and its surrounding biochemical environment. Through post-transcriptional modifications of histone proteins, gene expression can be either transcriptionally active in a ‘euchromatic’ environment, temporarily quieted in ‘facultative heterochromatin,’ or completely silenced in ‘constitutive heterochromatin’ (Zhang and Reinberg 2001). Post-translational modifications to lysine 9 of the H3 protein (H3K9) are uniquely able to reflect these three levels of transcriptional regulation. H3K9 modifications located in the promoter regions of actively transcribed genes are often acetylated (H3K9acetyl). Conversely, quieted transcription in gene-rich areas of the genome are often associated with mono- or dimethyl H3K9 (H3K9me2), while completely silenced areas of the genome are associated with trimethylated H3K9 (H3K9me3). In particular, the formation of H3K9me2 is catalyzed by histone methyltransferases (HMTs), including Eu-HMTase2 (G9a), Eu-HMTase1 (GLP), and SETDB1 (Krishnan et al. 2011) The different degrees of lysine methylation are possible due to the cooperation of these HMTs, which are able to form large heteromeric complexes (Fritsch et al. 2010).

H3K9 methylation has not been extensively studied in the brain, and until recently the regulation and role of the enzymes responsible for its formation were not known. Postnatal, neuronal-specific GLP/G9a knockdown produces a significant decrease in global H3K9me2 levels and inappropriate gene expression, leading to deficits in learning, reduction in exploratory behaviors and motivation in mice (Schaefer et al. 2009; Shinkai and Tachibana 2011;Tachibana et al. 2005;Tzeng et al. 2007). In humans, deletions or loss-of-function mutations of G9a results in Kleefstra Syndrome, characterized by a severe learning disability and developmental delay (Nillesen et al. 2011; Kleefstra et al. 2005). In humans, increased SETDB1 mRNA expression and resultant elevated H3K9me3 levels have been documented in Huntington Disease (HD) (Ryu et al. 2006; Fox et al. 2004).

A hallmark of schizophrenia is aberrant gene regulation, with the vast majority of studies reporting a down-regulation of gene transcription, suggesting that the epigenome of patients with schizophrenia is restrictive (Akbarian et al. 1995;Guidotti et al. 2000;Fatemi et al. 2005; Impagnatiello et al. 1998; Jindal et al. 2010). Postmortem brain studies indicate a reduction of an open histone modification, H3K4me3, and elevated expression of the histone deacetylase HDAC1 mRNA expression (Cheung et al. 2010; Sharma et al. 2008). The use of peripheral blood mononuclear cells as a reflection of overall chromatin state or at particular gene promoters has been successfully implemented in clinical studies of subjects afflicted depression, alcoholism, and schizophrenia. Peripheral blood cell studies have indicated that schizophrenia is associated with an abnormally condensed chromatin structure; (Issidorides et al. 1975; Kosower et al. 1995) specifically increased restrictive H3K9me2 and reduced H3K9 acetylation (Gavin et al. 2009b). Additionally, H3K9 acetylation in schizophrenia patients is less responsive to in vivo treatment with HDAC inhibitors when compared to both patients with bipolar disorder and nonpsychiatric controls (Sharma et al. 2006;Gavin et al. 2008). Finally, a correlation exists between age of onset of psychiatric symptoms of schizophrenia and baseline levels of H3K9me2 (Gavin et al. 2009b). It is the hypothesis of this paper that schizophrenia can be characterized by a restrictive epigenome, which is observable in both brain and peripheral blood, and has specific and observable effects on psychopathology. We have focused on levels of H3K9me2, indicative of facultative heterochromatin, and the enzymes that catalyze this modification, in patients with schizophrenia to examine their role in this illness.

3.1. mRNA Levels of HMT Gene Expression

We performed a multiple linear regression with each HMT gene of interest as the dependent variable. For postmortem brain tissue we examined sex, age, pH, RIN and diagnosis, whereas for lymphocytes we examined sex, age, and diagnosis as explanatory variables. In these two cohorts, we found that a diagnosis of schizophrenia is a significant predictor for GLP mRNA expression in both postmortem brain samples (β=0.44, F(1,24)=5.80, p<0.05), and in lymphocytes (β=−0.41, F(1,40)=7.91, p<0.01), indicating that patients with schizophrenia demonstrated increased levels compared to nonpsychiatric controls (Fig. 1a). Similarly, a diagnosis of schizophrenia is also a significant predictor for increased SETDB1 mRNA levels in both postmortem brain samples (β=0.39, F(1, 24)=4.33,p<0.05), and in lymphocytes (β=0.37, F(1,40)=6.19, p<0.05; Fig. 1b). A diagnosis of schizophrenia is not a significant predictor for elevated G9a mRNA levels in postmortem brain samples (β=0.22, F(1,24)=1.22, p=ns), but is for lymphocytes (β=−0.317, F(1,40)=4.46, p<0.05; Fig. 1c).

Interestingly, in both postmortem tissue (r=0.79, p<0.001) and lymphocytes (r=0.54, p<0.001), GLP and SETDB1 mRNA expression are positively correlated (data not shown).

Fig. 1

mRNA expression in both postmortem parietal cortical samples from the Stanley Foundation Neuropathology Consortium (on the left) and lymphocyte samples from University of Illinois at Chicago (on the right) and a. GLP mRNA levels, b. G9a mRNA levels and

To establish whether there exist differences in HMT mRNA among schizophrenic patients taking psychotropic medication, and those who were not, we performed a second multiple linear regression analysis on each individual cohort. The overall or type-specific use of antipsychotic, antidepressant or mood stabilizing medication are not significant predictors of HMT mRNA levels in either the postmortem or the lymphocyte cohorts.

3.2. H3K9me2 levels in the Postmortem Brain

In a previously published study we documented elevated global H3K9me2 levels in lymphocytes obtained from schizophrenia patients compared to nonpsychiatric controls (Gavin et al. 2009b). In the current study we attempted to discern whether this abnormality in a restrictive histone modification is present in brain tissue from the SFNC cohort as well. We performed a multiple linear regression with H3K9me2 levels as the dependent variable, with sex, age, and diagnosis as explanatory variables. We found that diagnosis of schizophrenia is a significant predictor of H3K9me2 levels extracted from postmortem brain tissue (β=0.40, F(1,24)=4.58, p<0.05; Fig. 2). GLP (r=0.65, p<0.001) and SETDB1 (r=0.44,p<0.05) are positively correlated with H3K9me2 levels, as discovered through a Pearson Correlation (data not shown).

Fig. 2

H3K9me2 levels are significantly increased parietal cortical samples from patients with schizophrenia when compared to nonpsychiatric controls. Below graph, a representative western blot image is shown. All data is shown as a ratio of optical density …     
3.3. Clinical Correlates with Lymphocyte HMT mRNA Levels

Lymphocyte levels of G9a mRNA demonstrated a positive correlation with the PANSS negative subscale total (r=0.61, p<0.05; Fig. 3a), GLP mRNA is positively correlated with the PANSS general subscale total, (r=0.64, p<0.01; Fig. 3b), and SETDB1 mRNA is more highly expressed in patients with longer durations of illness compared to both normal controls and patients in the ‘first episode psychosis’ group (ANOVA, F(2,30)=3.66, p<0.01; Fig. 3c). Patients with a family history of schizophrenia also had significantly increased levels of lymphocyte SETDB1 mRNA (t18=2.52, p<0.05; Fig. 3d).

Fig. 3

Clinical Correlates with Lymphocyte HMT mRNA Levels a. A rise in G9a mRNA is significantly correlated with increasing PANSS negative subscale totals; p<0.05. b. GLP mRNA is significantly increased upon worsening of PANSS general subscale scores;
4. Discussion

The current paper demonstrates an increase in GLP and SETDB1 mRNA in both postmortem parietal cortex and lymphocyte samples from patients with schizophrenia, as well as an increase in G9a mRNA in lymphocytes. G9a and GLP are responsible for the bulk of H3K9me2 modifications across the genome (Shinkai and Tachibana 2011; Tachibana et al. 2005), and SETDB1 is the only euchromatic HMT to specifically di- and tri-methylate H3K9 (Zee et al. 2010;Wang et al. 2003), but all three of these HMTs are able to form large heteromeric complexes, thus allowing for the sequential degrees of lysine methylation (Fritsch et al. 2010). Further, we demonstrate that the ultimate outcome of their catalytic activity, H3K9me2, is significantly increased in patients with schizophrenia as compared to nonpsychiatric controls. Moreover, GLP and SETDB1 mRNA are positively correlated with H3K9me2 levels. These findings add gravity to our previous demonstration of increased H3K9me2 levels in lymphocytes from schizophrenic patients (Gavin et al. 2009b).

Our investigations into the role of H3K9me2 in schizophrenia pathophysiology, as opposed to other H3K9 modifications, were motivated by the hypothesis that initial inactivation of gene promoter activity at various schizophrenia candidate genes can result in gradual entrenchment of the heterochromatin state as a result of disease chronicity and disuse (Sharma et al. 2012). Areas of H3K9me2 can then act as a platform for additional restrictive adaptors, thus resulting in the spreading of heterochromatin across previously unmodified gene rich areas. As such, the gene altering effects of medications are unable to overcome this restrictive burden, leading to repeated medication failures (Sharma et al. 2012). Support for this hypothesis has been previously demonstrated, (Sharma et al. 2008; Benes et al. 2007) including the finding that schizophrenia patients clinically treated for four weeks with the HDAC inhibitor, valproic acid, displayed no increase in peripheral blood cell acetylated histones 3 or 4 as compared to bipolar patients (Sharma et al. 2006). Here, we find an increase in both H3K9me2 levels and the enzymes which catalyze this modification, providing additional evidence supporting an increased heterochromatin state in schizophrenia.

The major role of the parietal cortex is to integrate and evaluate sensory information (Andersen & Buneo, 2003; Cohen & Andersen, 2002). It is one of the last areas of the human brain to fully mature, (Geschwind, 1965) thus early life environmental insults could have a profound effect. Disordered thought, a common symptom in schizophrenia, is most likely explainable through disruption of this system (Torrey, 2007). Patients with schizophrenia report either acute (McGhie & Chapman, 1961) or blunted (Freedman, 1974) sensitivity to sensory stimuli, and demonstrate overall impairment of sensory integration (Manschreck & Ames, 1984; Torrey, 1980). Similar patterns of transcriptional regulation are observed across the cortex, consequently, results from the parietal cortex likely reflect patterns of gene transcription in other brain regions (Hawrylycz et al., 2012).

Due to its heterogeneity, examining schizophrenia as a binary measurement of illness when examining biological relevancy can be limiting (Arango et al. 2000;Buchanan and Carpenter 1994). Through utilizing the PANSS, biological underpinnings that do not demarcate cleanly with diagnostic categories, can be correlated directly with specific symptomatology. Correlations between methyltransferase enzymes and clinical symptomatology indicate that these restrictive enzymes could contribute to specific facets of the illness, particularly negative and general symptoms, which are particularly resistant to improvement. Increased severity of negative symptoms are correlated with poorer disease prognosis, (Wieselgren et al. 1996) and are not alleviated through our current regimen of psychotropics.

Additionally, SETDB1 mRNA levels are also correlated with other markers of a worse disease prognosis, including a more chronic form of the illness, and a history of schizophrenia in the family. Pharmacological targeting of increased levels of SETDB1improves motor performance and extends survival in HD mice, indicating the promise of treatments that modulate gene silencing mechanisms in neuropsychiatric disorders (Ryu et al. 2006).

The main weakness of this current study was that clinical symptoms were correlated with mRNA extracted from peripheral tissue. Enzymes relating specifically to synaptic function were not examined, but rather overall mechanisms of epigenetic regulation that are not tissue specific. While postmortem investigations are able to serve as a useful snapshot at the time of death, the ability to measure and monitor histone marks over time as marker of disease progression, improvement, or as a predictor of pharmacological response are only possible using peripheral blood cells. A strong rationale for the use of blood chromatin ‘levels’ as a type of biosensor that registers the epigenetic milieu has been proposed elsewhere (Sharma 2012). Furthermore, previous studies have indicated the mRNA patterns of expression patterns in lymphocytes are capable of distinguishing between psychiatric diagnostic groups (Middleton et al. 2005).

The present study hypothesized that schizophrenia may be due to abnormal regulation of fundamental epigenetic mechanisms, thus, we chose to measure overall levels of H3K9me2 opposed to specific gene promoters, based on the assumption that while the individual genes silenced in the brain and blood may not be the same, similar global pathogenic processes may be occurring in both tissues.

The results of this paper indicate that chromatin is more restrictive in patients with schizophrenia, and may be significantly contributing to disease pathology. If, through pharmacological interventions, a reduction in this histone hyper-restrictive insult in schizophrenia can be relaxed, inducing a type of “genome softening,” then neuronal gene expression can be enhanced, thus allowing for increased plasticity and improved therapeutic response (Sharma 2005).

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Balancing Histone Methylation Activities in Psychiatric Disorders

Alterations in histone lysine methylation and other epigenetic regulators of gene expression contribute to changes in brain transcriptomes in mood and psychosis spectrum disorders, including depression and schizophrenia. Genetic association studies and animal models implicate multiple lysine methyltransferases (KMTs) and demethylases (KDMs) in the neurobiology of emotion and cognition. Here, we review the role of histone lysine methylation and transcriptional regulation in normal and diseased neurodevelopment and discuss various KMTs and KDMs as potential therapeutic targets in the treatment of neuropsychiatric disease.

Schizophrenia and depression are major psychiatric disorders that lack consensus neuropathology and, in a large majority of cases, a straightforward genetic risk architecture. Furthermore, many patients on the mood and psychosis spectrum show an incomplete response to conventional pharmacological treatments which are mainly aimed at monoamine signaling pathways in the brain (Box 1).

Box 1  Schizophrenia and Depression

Schizophrenia affects 1% of the general population and typically begins during young-adult years, although cognitive disturbances could be evident much earlier. The disease is, in terms of genetics and etiology, highly heterogeneous, and increasingly defined as different and partially independent symptom complexes: (i) psychosis with delusions, hallucinations and disorganized thought; (ii) cognitive dysfunction including deficits in attention, memory and executive function; and (iii) depressed mood and negative symptoms including inability to experience pleasure (anhedonia), social withdrawal and poor thought and speech output [42]. Currently prescribed antipsychotics, which are mainly aimed at dopaminergic and/or serotonergic receptor systems, exert therapeutic effects on psychosis in approximately 75% of patients. However, it is the cognitive impairment which is often the more disabling and persistent feature of schizophrenia [42]. Currently there are no established pharmacological treatments for this symptom complex. However, given that cognitive dysfunction is an important predictor for long-term outcome, this area is considered a high priority in schizophrenia research, as reflected by initiatives combining efforts from government agencies, academia and industry, including MATRICS (the Measurement and Treatment Research to Improve Cognition in Schizophrenia) [42].

Affective disorders as a group show, in terms of genetic risk architecture, some overlap with schizophrenia. For example, rare structural variants, including the balanced translocation at the Disrupted-in-Schizophrenia 1 (DISC-1) locus (1q42) or the 22q11 deletion are, in different individuals, associated with either mood disorder or schizophrenia [81, 82].

Depression, including its more severe manifestation, major depressive disorder which has a lifetime risk of 10–15% for the U.S. general population, is associated with excessive sadness, anhedonia, negative thoughts, and neurovegetative symptoms including changes in sleep pattern and appetite [1]. The disorder, which in more severe cases is accompanied by delusions, hallucinations and other symptoms of psychosis, often takes a chronic and recurrent course. Conventional antidepressant therapies primarily target monoamine metabolism and reuptake mechanisms at the terminals of serotonergic, noradrenergic and dopaminergic neurons. Unfortunately, up to 40% of cases show an insufficient response to these pharmacological treatments [1]. In addition, many antipsychotic and antidepressant drugs have significant side effect burden, including weight gain, diabetes and metabolic defects, extrapyramidal symptoms and sexual dysfunction [83, 84].

However, there is evidence that dysregulated gene transcription, indicative of compromised neural circuitry, contributes to disordered brain function in psychosis and mood spectrum disorder [1, 2]. While no single gene transcript is consistently affected, alterations in RNA levels contribute to defects in GABAergic inhibitory neurotransmission and more generally, synapse organization and function, metabolism and mitochondrial functions, and oligodendrocyte pathology [35]. While a number of transcriptional and post-transcriptional mechanisms may contribute to these changes, chromatin-associated proteins and epigenetic regulators invoked in sustained alterations of gene expression and function (Box 2) could play a critical role in the pathophysiology, or treatment of mental illness [6,7]. Indeed, there is evidence that changes in acetylation of histone lysine residues, which are broadly associated with active gene expression [8] and considered a potential therapeutic target for cancer and other medical conditions [9], also impact gene expression patterns in the brain and thereby influence emotional and cognitive functions. For example, mice or rats exposed to systemic treatment, or localized intracranial injections of class I/II histone deacetylase inhibitors (HDACi) exhibit behavioral changes reminiscent of those elicited by conventional antidepressant drugs [1013]. The short chain fatty acid derivative valproic acid, widely prescribed for its mood-stabilizing and anticonvulsant effects, induces brain histone hyperacetylation at a select set of gene promoters when administered to animals at comparatively high doses [14]. Conversely, overexpression of selected HDACs in neuronal structures implicated in the neurobiology of depression, including the hippocampus, elicit a pro-depressant behavioral phenotype [12]. Similarly, animals treated with class I/II HDACi often show improved performance in learning and memory paradigms and furthermore, drug-induced inhibition or activation of class III HDAC (also known as sirtuins) elicits changes in motivational and reward-related behaviors [15]. Therefore, the orderly balance between histone acetyl-transferase and deacetylase activities is critical for cognitive performance and synaptic and behavioral plasticity [16]. Likewise, However, HDACs interfere with acetylation of many non-histone proteins in the nucleus and cytoplasm [16], and moreover, some of these drugs carry a significant side effect burden [9]. Therefore, in light of the emerging role of epigenetic mechanisms in the neurobiology of these and other psychiatric conditions [6], the therapeutic potential of chromatin modifying drugs, other than the HDACi, warrants further investigations. This review will focus on histone lysine methylation, one of the most highly regulated chromatin markings in brain and other tissues. Multiple methyltransferases (KMTs) and demethylases (KDMs) were recently implicated in emotional and cognitive disorders (Fig. 1), and these types of chromatin modifying enzymes could emerge as novel targets in the treatment of mood and psychosis spectrum disorders.

Box 2  Epigenetic regulators and chromatin structure and function

Epigenetics, in the broader sense, applies both to dividing and postmitotic cells, and refers to a type of cellular memory that involves sustained changes in chromatin structure and function, including gene expression, in the absence of DNA sequence alterations (For in depth discussion, see [85]). Chromatin is essentially a repeating chain of nucleosomes comprised of genomic DNA wrapped around an octamer of core histones H2A/H2B/H3/H4. The histone proteins are intensely decorated with epigenetic information, with more than 70 (amino acid) residue-specific sites subject to various types of post-translational modifications (PTM). These include lysine (K) acetylation, methylation and poly ADP-ribosylation, arginine (R) methylation, and serine (S), threonine (T), tyrosine (Y) and histidine (H) phosphorylation [86]. In addition, a subset of the histone H2A, H2B and H4 lysines are covalently linked to the small protein modifiers ubiquitin and SUMO [87, 88]. Finally, epigenetic markings in genomic DNA include 5-methyl-cytosine and the related form, 5-hydroxy-methyl-cytosine [85]. These DNA and nucleosomal histone markings define the functional architecture of chromatin (see main text).

Proteins associated with methylation and other histone PTM are typically defined either as ‘writers’, ‘erasers’ or ‘readers’, essentially differentiating between the process of establishing or removing a mark as opposed to providing a docking site for chromatin remodeling complexes that regulate transcription, or induce and maintain chromatin condensation [18, 86, 89]. As it pertains to the brain, especially in the context of neuropsychiatric disease, a substantial body of knowledge has been generated for a select set of site-specific (K) methyltransferases and demethylases (Fig. 1A). In contrast, many PTMs are recognized by large numbers of reader proteins [90], but to date only very few of these readers have been explored in the brain. To mention just two examples, there are approximately 75 reader proteins specifically associated with histone H3-trimethyl-lysine 4 (H3K4me3), including several components of the SAGA complex ascribed with a key role for transcriptional initiation at RNA polymerase II target genes [90]. In contrast, H3K9me3, generally considered a repressive mark, provides a central hub for heterochromatin (associated) proteins including several members of the HP1 family and zinc finger domain containing molecules [90]. There is additional complexity because pluripotent stem cells and additional cell types decorate many of their promoters with ‘bivalent domains’ which include both open chromatin-associated (methylated H3K4 and H3/H4 acetylation) and repressive (methylated H3K27) marks [91, 92].

An external file that holds a picture, illustration, etc. Object name is nihms275106f1.jpg

Regulation of histone (K) methylation. (A) Listings of residue-specific KMTs and KDMs for H3K4/9/27/36/79 and H4K20. The majority of KMT and KDM are highly specific for a single histone residue, while a few enzymes target multiple residues, as indicated. Red marked KMT/KDM are implicated in neurodevelopment or psychiatric disease as discussed in main text. The non-catalytic JARID2 regulates activity and function of related KMTs. (B) Simplified scheme for selected mono- and trimethylated histone lysine markings implicated in transcriptional regulation, silencing and enhancer function.

The methylation of lysine and arginine residues, like other histone PTM, define chromatin states and function [8, 17]. To date, more than 20 methyl-marks on K and R residues have been described [18, 19]. As it pertains to the lysines, the majority of studies focused on the regulation and methylation-related functions of six specific sites: H3K4, H3K9, H3K27, H3K36, H3K79 and H4K20 [18]. For H3K4 and H3K9/K27, there is additional complexity because specific information is also conveyed (i) for H3K4, the unmethylated lysine effectively serving as a DNA methylation signal [20, 21], and (ii) for H3K9/K27 acetylation as an alternative PTM [22, 23] (Fig. 1A). For the aforementioned H3/H4 residues, specific biological functions and their interrelations with functional chromatin states, including transcriptional initiation and elongation, heterochromatic silencing and other mechanisms, have been described for the trimethyl-, and for some of the mono- and dimethyl-modifications (Representative examples are provided in Fig. 1B. See ref [17] for a detailed description of the histone methylation code and its relation to other types of histone PTM).

The following examples further illustrate the complex regulation of histone lysine methylation. Monomethylation of histone H3-lysine 4 (H3K4me1) plays an important role in neuronal activity-induced transcription at enhancer sequences [24], but the related forms, H3K4me3/2 are primarily found at the 5′ end of genes, with H3K4me3 mostly arranged as distinct and sharp peaks within 1–2Kb of transcription start sites. The H3K4me3 mark provides a docking site at the 5′ end of genes for chromatin remodeling complexes that either facilitate or repress transcription [25]. Furthermore, mono-methyl-H4-K20 shows strong positive correlation with gene expression at promoters enriched with CpGs, which contrasts to the trimethylated form of the same residue which generally is associated with repressed chromatin [23]. Taken together, these examples illustrate that even closely related histone lysine methylation markings are potentially associated with very different chromatin states.

To date, H3K4, H4K9, H3K27 and H4K20 methylation signals were measured at specific loci and genome-wide in human brain, essentially confirming that each of these epigenetic markings defines the same type of chromatin as in the peripheral tissues or animal brain [2630]. Interestingly, a subset of psychotherapeutic drugs including the mood-stabilizer valproate, the atypical antipsychotic clozapine and some monoamine oxidase inhibitors and stimulant drugs interfere with brain histone methylation (Table 1).

Molecular mechanisms of histone (lysine) methylation

A complex system of site-specific methyltransferases, which transfer the methyl-group of S-Adenosyl-Methionine (SAM) to lysine residues, has evolved in the vertebrate cell. There are an estimated 70 human genes harboring the Su(var)3–9,Enhancer of Zeste,Trithorax (SET) domain, which spans approximately 130 amino acids essential for KMT enzymatic activity [31]. The only known exception is the H3K79-specific methyltransferase, KMT4/DOT1L [31, 32], which lacks a SET. Each of the histone K residues discussed above is the preferential target of a distinct set of methyltransferase proteins (Fig. 1A)[19]. Of note, these histone-modifying enzymes are thought not to access histone substrates directly unless recruited by DNA-bound activators and repressors, a mechanism which could target each methyltransferase to a highly specific set of genomic loci [19].

An equally complex system exists for the site-specific lysine demethylases (Fig. 1A). There are at least two different mechanisms for active histone demethylation. The first enzyme type, represented by lysine-specific demethylase 1 (LSD1/KDM1A), contains an amine oxidase domain and requires flavin adenine dinucleotide (FAD) as a cofactor to demethylate di- and mono-methylated lysines. LSD1 and its homologue, LSD2, are primarily H3K4 demethylases, albeit depending on species and context, and activity against H3K9 also has been described [18]. Interestingly, monoamine oxidase inhibitors (MAOi) such as tranylcypromine or phenelzine — powerful antidepressants that exert their therapeutic effects mainly by elevating brain monoamine levels through inhibition of MAO-A/B — also block LSD1 type histone demethylases [18]. While LSD1 is thought to regulate histone methylation at promoters, LSD2 is bound to transcriptional elongation complexes and removes H3K4 methyl markings in gene bodies, thereby facilitating gene expression by reducing spurious transcriptional initiation outside of promoters [33]. The second type of demethylase, which in contrast to LSD1/LSD2 is capable of demethylating trimethyl markings, involves Fe2+-dependent dioxygenation by Jumonji-C (JmJC) domain-mediated catalysis [18]. Given that each of the KMTs and KDMs described has a different combinatorial set of functional domains and (protein) binding partners [18, 34], it is likely that the various site-specific methyltransferases and demethylases are largely non-redundant in function.

KMTs and KDMs with a role in cognition and neuropsychiatric disease

An increasing number of KMTs and KDMs are implicated in neurodevelopment and major psychiatric diseases (marked in red in Fig. 1A).

H3K4

The first histone lysine methyltransferase explored in the nervous system was KMT2A/MLL1, a member of the mixed-lineage leukemia (MLL) family of molecules. Mice heterozygous for an insertional (lacZ) loss-of-function Mll1mutation show distinct abnormalities in hippocampal plasticity and signaling [35], in conjunction with defects of learning and memory [36]. Of note, the hippocampus, and other portions of the forebrain including prefrontal cortex and ventral striatum, are frequently implicated in the neural circuitry of mood and psychosis spectrum disorders [1]. Furthermore, conditional deletion of Mll1resulted in defective neurogenesis during the early postnatal period [37]. While the full spectrum of MLL1 target genes in neurons and glia awaits further investigation, dysregulated expression of certain transcription factors such as DLX2, a key regulator for the differentiation of forebrain GABAergic neurons (which are essential for inhibitory neurotransmission and orderly synchronization of neural networks) [38], may contribute to the cognitive phenotype of the Mll1mutant mice. These observations may be relevant for the pathophysiology of schizophrenia, because some patients show in the prefrontal cortex a deficit in H3K4-trimethylation and gene expression at a subset of GABAergic promoters, including GAD1 encoding a GABA synthesis enzyme [28]. While the timing and age-of-onset for this ‘molecular lesion’ remains unknown, it is of interest that in the normal PFC, H3K4 methylation at the site of GABAergic genes progressively increases during the transition from fetal period to childhood to adulthood [28]. The epigenetic vulnerability of the Gad1 promoter during such prolonged developmental periods is further emphasized by recent animal studies demonstrating that Gad1-DNA methylation and histone acetylation are heavily influenced by the level of maternal care in the neonatal period/pre-weanling period [39].

There is additional evidence that epigenetic fine-tuning of the brain’s H3K4 methyl-markings is critical for orderly neurodevelopment. Of note, loss-of-function mutations in KDM5C/JARID1C/SMCX, an X-linked gene encoding a H3K4 demethylase, have been linked to mental retardation [40] and autism spectrum disorders [41]. The KDM5C gene product operates in a chromatin remodeling complex together with HDAC1/2 histone deacetylases and the transcriptional repressor REST, thereby poising neuron-restrictive silencer elements for H3K4 demethylation and decreased expression of target genes including synaptic proteins and sodium channels [40]. However, because this study was conducted with the HeLa cell line, it remains to be determined whether similar mechanisms operate in the nervous system.

In addition to its role in neurodevelopment, MLL-mediated H3K4 methylation could play a potential role for the treatment of psychosis. The atypical antipsychotic clozapine, which has a somewhat higher therapeutic efficacy when compared to conventional antipsychotics that function primarily as dopamine D2 receptor antagonists [42], upregulates H3K4 tri-methylation at the Gad1/GAD1GABA synthesis enzyme gene promoter. These effects were not mimicked in dopamine receptors D2/D3 (Drd2/3) compound null mutant mice, suggesting that blockade of dopamine D2-like receptors is not sufficient for clozapine-induced H3K4 methylation [28]. In the human PFC, GAD1-associated H3K4 methylation was increased in subjects exposed to clozapine, as compared to subjects treated with conventional antipsychotics. Conversely, mice heterozygous for the H3K4-specific KMT, mixed-lineage leukemia 1 (MLL1), exhibited decreased H3K4 methylation at brain Gad1 [28]. Therefore, it is possible that MLL1, which is highly expressed in GABAergic and other neurons of the adult cerebral cortex [28], will in the future emerge as a novel target for the treatment of psychosis. Questions that remain to be resolved include (i) the molecular pathways linking clozapine — a drug that impacts dopaminergic, serotonergic, muscarinic and other signaling pathways — to MLL1-mediated histone methylation, and (ii) whether or not the clozapine-induced changes in H3K4 methylation are restricted to GABAergic gene promoters or, alternatively, the reflection of more widespread epigenetic changes throughout the genome. Of note, clozapine’s effects on H3K4 methylation require intact brain circuitry and cannot be mimicked in cultured neurons differentiated from forebrain progenitor cells [43]. This finding is in good agreement with the recent observation that some of clozapine’s therapeutic effects require an intact serotonergic system, particularly its presynaptic components [44].

H3K9

The 9q34 subtelomeric deletion syndrome, which includes mental retardation and other developmental defects, is caused by deleterious mutations and haploinsufficiency of euchromatin histone methyltransferase 1 (EHMT1, also known as GLP and KMT1D) [45]. This gene encodes a H3K9-specific methyltransferase that operates in a multimeric complex that includes its closest homologue, G9a/KMT1C, and additional H3K9-specific HMTs [46]. Studies in mutant mice suggest that the GLP/G9a complex is important for suppression of non-neuronal and progenitor genes in mature neurons, and loss of this complex has deleterious effects on cognition and other higher brain functions [47]. Furthermore, G9a-mediated H3K9 methylation events within the reward circuitry, including the ventral striatum, are critical intermediates for the long-term effects of cocaine on reward behavior and neuronal morphology [48]. This would suggest that GLP/G9a, and proper regulation of H3K9 levels, is important for orderly brain function both in developing and mature brain.

Furthermore, changes in motivational and affective behaviors could be elicited by overexpression of the H3K9-HMT, SET domain bifurcated 1 (KMT1C/SETDB1/ESET), in adult forebrain neurons [49]. Interestingly, SETDB1 occupancy in neuronal chromatin is highly restricted, and may be confined to less than 0.75% of annotated genes [49]. However, among these are several NMDA and other ionotropic glutamate receptor subunit genes, including Grin2a/b (Nr2a/b)[49]. Mild to moderate inhibition of NMDA receptor-mediated (including Grin2b) neurotransmission elicits a robust improvement of depressive symptoms in some mood disorder patients [50], and, indeed, SETDB1-mediated H3K9 methylation and repressive chromatin remodeling at the Grin2b locus was associated with antidepressant-like behavioral phenotypes in the Setdb1 transgenic mice [49]. Of note, NMDA receptor antagonists, including GRIN2B-specific drugs, elicit significant therapeutic benefits even in subjects who failed multiple trials of selective serotonin reuptake inhibitors (SSRI) and other conventional antidepressants [50]. However, drugs directly acting at the NMDA receptor site have an unfavorable side effect profile, and therapeutic strategies aimed at SETDB1 expression and activity may therefore provide an alternative strategy.

Interestingly, mice with a genetic ablation of Kap1, encoding the SETDB1 binding partner KRAB-associated protein 1, also known as TRIM28/TIF1b/KRIP1)[51], show increased anxiety and deficits in cognition and memory [52], which are phenotypes that are broadly opposite from those observed in mice with increasedSetdb1 expression in brain [49]. These findings further speak to the therapeutic potential of the Kap1-Setdb1 repressor complex in the context of neuropsychiatric disease.

Finally, the H3K9-specific demethylase, KDM3A/Jmjd1A, showed increased H3K9-methylation at its own promoter in the ventral striatum of mice exposed to social defeat (a type of stressor associated with a depression-like syndrome in these animals), while mice that were treated with a conventional antidepressant or that were resilient to this type of stress did not show changes in KDM3A promoter methylation [53]. While it is unclear whether KDM3A or some other demethylase acitivity is altered in the depressed animals, the same study [53] reported widespread repressive histone methylation changes, including increased dimethyl-H3K9 and methylated H3K27 at hundreds of gene promoters in stress susceptible animals, which further emphasizes the importance of these PTMs for the epigenetics of mood disorder.

H3K27

The H3K27-selective methyltransferase, KMT6A, also known as Enhancer of zeste homolog2 (EZH2), is associated with the polycomb repressive chromatin remodeling complex 2 (PRC2) [54], and essential for cortical progenitor cell and neuron production. Consequently, loss of EZH2 function is associated with severe thinning of the cerebral cortex and a disproportionate loss of neurons residing in upper cortical layers I–IV [55]. Likewise, the H3K27-specific demethylase, JMJD3, is important for neurogenesis and neuronal lineage commitment [56]. Furthermore, H3K27 methylation is dynamically regulated in mature brain and involved in the neurobiology of major psychiatric disease. For example, changes in expression of brain-derived neurotrophic factor (Bdnf) in hippocampus of mice exposed to environmental enrichment or chronic stress are associated with opposite changes in the H3K27me3 mark at a subset of Bdnf gene promoters [12,57]. In addition, acute stress leads to an overall decrease in hippocampal H3K27me3 and H3K9me3 [58]. Furthermore, in the orbitofrontal cortex of suicide completers, alterations in H3K27 methylation were described at the TRKB gene, encoding the high affinity receptor for the nerve growth factor molecule, BDNF [27]. Changes in the balance between histone H3K4 and H3K27 methylation, or DNA cytosine and H3K27 methylation may also contribute to GABAergic gene expression deficits in schizophrenia [28, 43]. To date it remains unclear which of the various H3K27-specific KMTs and KDMs (Fig. 1) are involved in these disease-related alterations in postmortem brain tissue. Of note, the Jumonji and Arid containing protein 2 (JARID2), which by itself lacks catalytic activity but is crucial for subsequent H3K27 or H3K9 methylation by recruiting the polycomb PRC2 complex to its target genes [59, 60], is located within the schizophrenia susceptibility locus on chromosome 6p22 and confers genetic risk in multiple populations of different ethnic origin [61, 62]. While the biological functions of JARID2 have been studied primarily in the context of transcriptional regulation in stem cells [63, 64], this gene shows widespread expression in the mature nervous system [65], implying JARID2-mediated control over polycomb repressive chromatin remodeling in the adult brain.

H3K36 and H4K20

Epigenetic dysregulation of nuclear receptor-binding SET domain containing protein 1/KMT3B could play a role in some neuro- and glioblastomas [66], but like for other H3K36 and H4K20 regulating enzymes (Fig. 1), to date little is known about their role in neurodevelopment, cognition and psychiatric disease. Strikingly, however, KMT3A/HYPB/SETD2, a member of the SET2 family of KMTs mediating H3K36 methylation [67], is also known as huntingtin-interacting protein 1 (HIP-1) or huntingtin(yeast)-interacting protein B (HYPB) [68]. Huntington’s is a triplet repeat disorder and chronic neurodegenerative condition with motor symptoms and cognitive defects, and significant changes in mood and affect [69]. Whether or not there is altered H3K36 methylation in the neuronal populations that are at risk for degeneration is unclear. Furthermore, the huntingtin/KMT3A interaction has been documented for yeast [68] but not brain. Of note, wildtype huntingtin is a facilitator of polycomb complex PCR2-mediated H3K27 methylation [70], and furthermore, H3K4 and H3K9 methylation changes have been reported in preclinical model systems and postmortem brains with Huntington’s disease [71, 72]. Therefore, it is possible that transcriptional dysregulation in this condition is associated with aberrant methylation patterns of multiple lysine residues.

KMTs and KDMs as Novel Drug Targets

Given the emerging role of histone methylation in the neurobiology of psychiatric disease, the next obvious question is whether this type of PTM could provide a target for a new generation of psychotropic therapeutics. In principle, KMTs and KDMs should provide fertile ground for the development of novel drugs, because these enzymes are considered more specific than, for example, HDACs, because each HDAC enzyme is likely to affect a much larger number of histone residues as compared to KMTs/KDMs [73]. However, like for other histone modifying enzymes, the specificity of KMTs and KDMs is not limited to histones but includes the (de)methylation of lysines of non-histone proteins, including the p53 tumor suppressor protein and the VEGF growth factor [74]. Druggable domains within the KMTs and KDMs could involve not only their catalytic sites, such as the SET domain for the KMTs or the amino oxidase and JmjC domains for the LSD1 and JMJD subtypes of KDMs, respectively, but also some of the many other functional domains that are specific to subsets of these proteins [75]. One potential candidate would be the bromodomain of the MLLs and other H3K4-specific methyltransferases [75]. Bromodomains, which are present in many different types of nuclear proteins, bind to acetylated histones and small molecules interfering with some of these interactions recently emerged as powerful modulators of systemic inflammation [76].

The catalytic activity of the SET domain containing KMTs requires the universal methyl donor, S-adenosyl-methionine (also known as AdoMET). Crystallographic and functional studies revealed that the SAM binding pocket of KMTs is different from the SAM pockets of other proteins, which may increase the chance to develop compounds which specifically target histone methyltransferases but not other enzymes and proteins [31]. Currently, however, no KMT or KDM related drug is in clinical trials. However, several of these compounds show therapeutic promise in preclinical studies. For example, the S-adenosylhomocysteine hydrolase inhibitor, 3-deazaneplanocin A (DZNep) induces apoptosis in breast cancer cells [77]. This drug alters H3K27 and H4K20 trimethylation via interference with polycomb PRC2 repressive chromatin remodeling [73]. Antioncogenic effects were also observed with BIX-01294, a drug that downregulates H3K9 methylation levels by binding to the SET domain of the G9a/GLP(EHMT1) methyltransferases [73]. The same drug was shown to alter addictive behaviors and H3K9 methylation when infused locally into the brain of cocaine-exposed mice [48]. As discussed above, while tranylcypromine and other monoamine oxidase inhibitors used for the treatment of depression are weak inhibitors of the LSD1 type of KDM, recently several compounds emerged with much stronger activity against LSD1/LSD2 [18]. It will be extremely interesting to explore these drugs in preclinical models for mood and psychosis spectrum disorders. Finally, microRNA-based therapeutic strategies, aimed at decreasing levels and expression of chromatin remodeling complexes, including some of the histone modifying enzymes discussed here, are gaining increasing prominence in the field of cancer therapy [73] and may in the future emerge as a novel therapeutic option in the context of neuropsychiatric disease.

Emerging Concept in DNA Methylation: Role of Transcription Factors in Shaping DNA Methylation Patterns
CLAIRE MARCHAL AND BENOIT MIOTTO*   Journal of Cellular Physiology Volume 230, Issue 4,  http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1097-4652

DNA methylation in mammals is a key epigenetic modification essential to normal genome regulation and development. DNA methylation patterns are established during early embryonic development, and subsequently maintained during cell divisions. Yet, discrete site-specific de novo DNA methylation or DNA demethylation events play a fundamental role in a number of physiological and pathological contexts, leading to critical changes in the transcriptional status of genes such as differentiation, tumor suppressor or imprinted genes. How the DNA methylation machinery targets specific regions of the genome during early embryogenesis and in adult tissues remains poorly understood. Here, we report advances being made in the field with a particular emphasis on the implication of transcription factors in establishing and in editing DNA methylation profiles. J. Cell. Physiol. 230: 743–751, 2015.

DNA methylation is a well-studied epigenetic modification in mammalian genomes, discovered in 1948. It is involved in a number of essential cellular processes such as transcription regulation, cellular differentiation, cellular identity maintenance, X inactivation, gene imprinting, and the cellular response to environmental changes (Klose and Bird, 2006; Guibert and Weber, 2013; Smith and Meissner, 2013; Subramaniam et al., 2014). DNA methylation has proved to be a dynamic process, requiring continuous regulation and potentially having an important regulatory role for tissuespecific differentiation or cellular signaling. Indeed, the analysis of the distribution of DNA methylation at the genome scale, and nowadays at the single-base resolution, in different physiological and pathological states, unraveled that local changes in DNA methylation contribute to cell-type specific variation in gene expression. Furthermore, aberrant DNA methylation patterns are documented in a number of human diseases from Immunodeficiency, Centromere instability, and Facial anomalies (ICF) syndrome to cancer, and contribute to the onset or development of these diseases (Smith and Meissner, 2013; Weng et al., 2013; Subramaniam et al., 2014). Needless to say, these discoveries also fuel the promising idea that therapeutic strategies targeting DNA methylation can be used in the prevention and the treatment of cancer and other human diseases, including neuro-developmental disorders (Weng et al., 2013; Subramaniam et al., 2014). As an example, antipsychotic drugs clozapine and sulpiride, combined with histone deacetylase inhibitor valproate, have a beneficial action in schizophrenia and bipolar patients, maybe because they revert the aberrant DNA methylation status at GABAergic gene promoters (Dong et al., 2008). In 2004, 5-azacytidine (VidazaTM, Celgene Corporation, Summit, NJ). A drug blocking DNA methylation, received approval by the Food and Drug Administration for the treatment of myelodysplastic syndromes (Kaminskas et al., 2005).

Figure 1. Overview of the DNA methylation and demethylation pathway. (A) DNMT1 is responsible for the maintenance of DNA methylation during DNA replication. It recognizes hemi-methylated CpG, thanks to its interaction with co-factor UHRF1, and it adds methylation on the un-methylated strand. Black bubbles: methylated CpG. Empty bubbles: un-methylated CpG. (B) DNMT3A/B are responsible for de novo DNA methylation. They establish new patterns of methylation directly from unmethylated CpG-containing sequences. In the embryo, their activity is modulated by a catalytically inactive family member DNMT3L. (C) Passive demethylation occurs through loss of DNMT1/3 activity in actively dividing cells. Loss can be attributed to post-translational modifications, gene mutations, gene silencing or any other mechanism that will eventually lead to DNMT activity inhibition. (D) Active DNA demethylation is catalyzed by the TET family of enzymes. TET1, 2 and 3 can oxydate 5mC into 5hmC (represented in grey bubbles), and eventually oxidate 5hmC into 5-formylcytosine and 5-carboxy-cytosine. None of these bases is recognized by DNMTs causing loss of DNA methylation during DNA replication. In addition, these oxidated bases are recognized by the base-excision repair (BER) pathway and catalytically removed.

Figure 2. Summary of the nuclear factors and epigenetic marks involved in the maintenance of DNA methylation status in different regions of the genome. The table recapitulates our current knowledge on transcription factors, chromatin remodellers and histone marks contributing to the establishment of DNA methylation and its erasure. The information is presented according to genomic features, sharing common regulators, such as promoters/enhancers, tumor suppressor genes, germline gene promoters, imprinted regions, DNA repeats, and retroviral elements and peri-centromeric regions.

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t KAP1/DNMTs control the maintenance of DNA methylation, independently of DNA replication, on a number of genomic targets. Yet, DNMTs have been shown to be recruited onto the chromatin by other chromatin remodelers, such as SETDB1 or G9a, or secondary to gene silencing (Gibbons et al., 2000; Dennis et al., 2001; Guibert and Weber, 2013; Pacaud et al., 2014). Thus, only the identification of the full-spectrum of transcription factors involved in the regulation of DNA methylation will tell whether this function is predominantly confer to KRAB-ZNF factors. This systematic analysis might help understand why only a limited number of factors per family are involved in the shaping of DNA methylation. In the case of ZNF factors several explanations have been postulated. The resolution of the structure of the ZNF fingers of Zfp57 bound onto methylated DNA indicated that a specific amino-acid sequence in the DNA binding ZNF fingers might be required for the recognition and binding of methylated CpG sequences (Liu et al., 2012; BuckKoehntop and Defossez, 2013). Using this knowledge, researchers have postulated that ZNF factors containing this motif might likely contribute to shape DNA methylation profile (Liu et al., 2013). An alternative hypothesis rely on the observation that KRAB-ZNF factors are present uniquely in vertebrate genomes and have expanded quite dramatically in mammalian genomes. As DNA repeats sequences also quickly evolved in mammalian genomes, it is suggested that humanspecific KRAB-ZNF factors might primarily contribute in DNA repeats silencing (Lukic et al., 2014).

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DNA methylation plays an important role in the control of gene expression and cell fate in mammals. Its regulation and function has been upon intense scrutiny since its discovery in mid-1900s. Yet, how DNA methylation patterns are established during embryogenesis, and edited in adult tissue, remains a matter of intense debate. Profiling of DNA methylation in many cell type, species and environmental set up indicates that the DNA methylation profile is thighly correlated with the cell type and its environment. As a consequence, de novo methylation and DNA demethylation events are not randomly distributed but are actually targeted to particular regulatory DNA elements in the genome, including promoters, enhancers or repeated DNAs. For this latter reason researchers have focused on the role of transcription factor in these DNA methylation events. Yet, it is also recognized that non-coding RNAs, short and long, contribute to the establishment and editing of DNA methylation profiles in mammals. Non-coding RNAs may directly interact and control methylation and demethylation activities and, as a consequence, the pattern of DNA methylation in the genome (Di Ruscio et al., 2013; Arab et al., 2014; Castro-Diaz et al., 2014; Molaro et al., 2014; Turelli et al., 2014). For instance, antisense long non-coding RNA TARID (TCF21 antisense RNA inducing demethylation), activates TCF21 expression by inducing promoter demethylation. TARID sequence is complementary to the sequence of the TCF21 promoter. Its transcription causes the anchoring of GADD45A (growth arrest and DNA-damageinducible, alpha), a regulator of DNA demethylation, at the TCF21 promoter and its subsequent chromatin remodelling (Arab et al., 2014). Understanding the interplay between noncoding RNAs and transcription factors in the establishment and the maintenance of DNA methylation is therefore an important challenge for the future.

The Expanding Role of MBD Genes in Autism: Identification of a MECP2 Duplication and Novel Alterations in MBD5, MBD6, and SETDB1

The methyl-CpG-binding domain (MBD) gene family was first linked to autism over a decade ago when Rett syndrome, which falls under the umbrella of autism spectrum disorders (ASDs), was revealed to be predominantly caused by MECP2mutations. Since that time, MECP2 alterations have been recognized in idiopathic ASD patients by us and others. Individuals with deletions across the MBD5 gene also present with ASDs, impaired speech, intellectual difficulties, repetitive behaviors, and epilepsy. These findings suggest that further investigations of the MBD gene family may reveal additional associations related to autism. We now describe the first study evaluating individuals with ASD for rare variants in four autosomal MBD family members, MBD5, MBD6, SETDB1, and SETDB2, and expand our initial screening in the MECP2 gene. Each gene was sequenced over all coding exons and evaluated for copy number variations in 287 patients with ASD and an equal number of ethnically matched control individuals. We identified 186 alterations through sequencing, approximately half of which were novel (96 variants, 51.6%). We identified seventeen ASD specific, nonsynonymous variants, four of which were concordant in multiplex families: MBD5 Tyr1269Cys, MBD6 Arg883Trp, MECP2 Thr240Ser, and SETDB1 Pro1067del. Furthermore, a complex duplication spanning the MECP2 gene was identified in two brothers who presented with developmental delay and intellectual disability. From our studies, we provide the first examples of autistic patients carrying potentially detrimental alterations in MBD6 and SETDB1, thereby demonstrating that the MBD gene family potentially plays a significant role in rare and private genetic causes of autism.

There is growing evidence of the involvement of the methyl-CpG-binding domain (MBD) genes in neurological disorders. To date, pathogenic mutations have been found in patients with clinical features along the autism continuum for two genes in this family, methyl-CpG-binding domain protein 5 (MBD5) and methyl-CpG-binding protein 2 (MECP2). Both genes carry an MBD domain, the unifying feature for the family that includes nine additional genes; BAZ1A, BAZ1B, MBD1,MBD2, MBD3, MBD4, MBD6, SETDB1 and SETDB2 (Roloff et al., 2003). The MBD genes are involved in a variety of functions, including chromatin remodeling (BAZ1A, BAZ1B, MBD1, MBD2, MBD3, and MECP2), DNA damage repair (BAZ1A and MBD4), histone methylation (SETBD1 and SETDB2), and X chromosome inactivation (MBD2, Roloff et al., 2003, Bogdanovic & Veenstra, 2009). There is also functional interplay among members of this family as they have been found to bind at the same promoter regions (MBD1, MBD2, MBD3, andMECP2), partner with each other in complexes (MBD1 and SETBD1), or act in the same complexes in a mutually exclusive manner (MBD2 and MBD3, Sarraf & I. Stancheva 2004; Ballestar et al., 2005; Le Guezennec et al., 2006; Matarazzo et al., 2007). Little is known thus far about the functions of MBD5 and MBD6; they each encode proteins that localize to chromatin but fail to bind methylated DNA (Laget et al., 2010).

One specific disorder in the autism spectrum, Rett syndrome, is caused almost exclusively by alterations in MECP2 (Amir et al., 1999). Due to the location ofMECP2 on the X chromosome, mutations in females can lead to Rett syndrome while males with the same genetic changes typically present with neonatal encephalopathy (Moretti & Zoghbi 2006). Further investigations have demonstrated that MECP2 misregulation can lead to a wide range of clinical features including autism, Angelman-like symptoms, mental retardation with or without infantile seizures, mild learning disabilities, and schizophrenia (Watson et al., 2001; Klauck et al., 2002; Carney et al., 2003; Shibayama et al., 2004;Coutinho et al., 2007; Harvey et al., 2007; Lugtenberg et al., 2009). Our group previously evaluated the MECP2 gene in a dataset of female ASD patients and identified two mutations reported in classic Rett syndrome patients; an Arg294X mutation and a 41 base pair deletion (Leu386fs) predicted to generate a truncated protein (Carney et al., 2003). Furthermore, while point mutations in MECP2 were first recognized to result in abnormal clinical phenotypes, increased expression of the wild type protein due to gene duplication also results in neurodevelopmental disorders (Meins et al., 2005; Van Esch et al., 2005; del Gaudio et al., 2006;Ramocki et al., 2009).

A second gene in the MBD family, MBD5, was tied to neurodevelopmental disorders following the identification of microdeletions on chromosome 2q22–2q23 (Vissers et al., 2003; Koolen et al., 2004; de Vries et al., 2005; Wagenstaller et al., 2007; Jaillard et al., 2008; van Bon et al., 2009; Williams et al., 2009; Chung et al., 2011; Talkowski et al., 2011; Noh & J. M. Graham Jr 2012). The minimal region for these nonrecurrent deletions covers only a single gene, MBD5 (van Bon et al., 2009; Williams et al., 2009; Talkowski et al., 2011). This suggests that the common features of ASDs, delayed or impaired speech, intellectual disability, epilepsy, and stereotypic hand movements found across microdeletion patients manifest due to a decreased expression of this critical gene (van Bon et al., 2009;Williams et al., 2009; Talkowski et al., 2011). Notably, two cases of individuals with duplications across the critical MBD5 region also present with autistic features and developmental delay (Chung et al., 2012). This demonstrates that precise regulation of both MBD5 and MECP2 must be maintained as either increased or decreased expression of each gene can result in a range of neurodevelopmental disorders.

Supplementing clinical evidence, mouse models have reiterated the potential significance of the MBD family in autism etiology. Mbd1 and Mecp2 null models have abnormal neurobehavioral phenotypes including increased anxiety, and impaired social interactions and synaptic plasticity (Guy et al., 2001; Shahbazian et al., 2002b; Zhao et al., 2003; Allan et al., 2008). Furthermore, a transgenicSetdb1 model established a link between this gene and behavior (Jiang et al., 2010a). Additionally, Setdb1 plays a role in the repression of Grin2b, a gene linked to autism, bipolar disorder, intellectual disability, and schizophrenia (Avramopoulos et al., 2007; Allen et al., 2008; Endele et al., 2010; Jiang et al., 2010a; Myers et al., 2011; O’Roak et al., 2011).

Studies have demonstrated that each of the MBD genes are expressed in the brain, while their specific functions having only been determined for a subset of genes (Shahbazian et al., 2002a, Bogdanovic & Veenstra, 2009, Jiang et al., 2010b, Laget et al., 2010, Safran et al., 2010). MeCP2 is a transcriptional regulator believed to act in neuronal maturation as levels increase over time (Shahbazian et al., 2002a,Chahrour et al., 2008). Stable levels of MeCP2 are required through adulthood, as elimination of this protein in adult mice mimics features seen in knockout Mecp2mice (McGraw et al., 2011). The H3K9 methyltransferase SETDB1 acts both in early development as well as later stages of life (Jiang et al., 2010a, Cho et al., 2012). Removal of Setdb1 in mice results in peri-implantation lethality (Dodge et al., 2004). Studies in the forebrain of transgenic Setdb1 mice demonstrate that it targets the NMDA receptors Grin2a and Grin2b as well as the glutamate receptorGrid2 (Yang et al., 2002, Jiang et al., 2010a).

While there is clinical evidence of MECP2 and MBD5 playing a role in autism, only two studies to date have evaluated patients with ASD for mutations in additional MBD family members (Li et al., 2005; Cukier et al., 2010). Previous work in our laboratory analyzed the coding regions of MBD1, MBD2, MBD3, andMBD4 in over 200 individuals with ASD of African and European ancestry and identified multiple variants that altered the amino acid sequence, were unique to patients with autism, and concordant with disease in multiplex families (Cukier et al., 2010). In contrast, a study by Li and colleagues was restricted to a dataset of 65 Japanese autistic patients and reported only a single variation that might be related to autism (Li et al., 2005). We now expand our initial study of MECP2 to a larger dataset that includes male patients and perform the first study evaluating patients with ASD for alterations in four additional MBD family members: MBD5MBD6, SETDB1 and SETDB2.

Sequencing across the five MBD genes in 287 patients with ASD and 288 ethnically matched control individuals identified a total of 186 unique variations (Table 1, Supplemental Tables 37). These variants included 177 single nucleotide polymorphisms (SNPs), five deletions and four insertions. Ninety (48.4%) of the variations have been previously reported in either the dbSNP 134 database (http://www.ncbi.nlm.nih.gov/projects/SNP/) or RettBASE (http://mecp2.chw.edu.au/), while the remaining 96 variants (51.6%) are novel. Fifty-six variations are predicted to alter the amino acid sequence. Fifty-three of the changes were found solely in patients with ASD and absent from controls. To determine variants most likely to contribute to ASD susceptibility, we prioritized changes that were either unique to affected individuals or that had an increased frequency in cases when compared to controls. The 17 most interesting variants were nonsynonymous and unique to our ASD population (Table 1). We utilized four distinct programs to characterize the variants; GERP (Cooper et al., 2005) and PhastCons (Siepel et al., 2005) to measure the level of amino acid conservation across species and PolyPhen (Adzhubei et al., 2010) and SIFT (Kumar et al., 2009) to predict which alterations might have the damaging consequences to protein function.

ASD Unique, Nonsynonymous Variations

The mutational burden between cases and controls of African or European ancestry for each gene was not statistically significant by the chi-squared test (Supplemental Table 8). This was determined for the overall load of all variants as well as nonsynonymous alterations (Supplemental Table 9).

MBD5

Thirty-two changes were identified in MBD5, 18 of which have been previously reported (Supplemental Table 3). A distinct set of 11 alterations were nonsynonymous, four of which were only identified in patients with ASD (Val443Met, Ile1247Thr, Tyr1269Cys, and Arg1299Gln, Figure 1A–D, Table 1). Three of these four alterations (75%) are predicted to be damaging by SIFT, as compared to only two of seven nonsynonymous variants (28.6%) identified solely in control individuals (Supplemental Table 3). One alteration of high interest, MBD5 Tyr1269Cys, was inherited paternally in all three ASD children in multiplex family 7763 (Figure 1C). Two of the affected individuals (0001 and 0100) were intellectually impaired with measured IQ in the moderate to severe range (Full Scale IQ: 40 and 50, respectively), while the remaining brother with autism (0101) had borderline intellectual functioning (Full Scale IQ=78). Furthermore, all three siblings had a delay in language and displayed self-injurious behaviors. Two individuals presented with macrocephaly (0100 and 0101), and individual 0100 has a history of epilepsy (recurrent non-febrile seizures).

Pedigrees of ASD families carrying alterations in MBD5 and MBD6

MBD6

A total of 44 alterations were detected in MBD6, two being single base pair insertions and the remainder of which were SNPs (Supplemental Table 4). Sixteen of the single nucleotide changes have been previously reported and 28 are novel. A subset of 17 alterations was identified only in individuals with ASD, seven of which are predicted to cause missense changes (Table 1, Figure 1E–K). While each of these changes was only identified in a single proband, three of the alterations have high PolyPhen and SIFT scores and are novel (Arg883Trp, Pro943Arg and Arg967Cys), suggesting a strong functional consequence. Furthermore, one of these alterations, Arg883Trp, was identified in multiplex family 7979 and passed maternally to both affected children (Figure 1I). Individual 0001 has a diagnosis of autism and is nonverbal with moderate intellectual disability. His sister (0100) has a diagnosis of Pervasive Developmental Disorder-Not Otherwise Specified and mild intellectual disability, displaying some phrase speech. Both siblings have a history self-injurious behavior. Their mother (1001), who also carries the alteration, was diagnosed with anxiety/panic disorders, depression, obsessive compulsive disorder, and has a history of epilepsy (adolescent onset seizures).

Along with novel variations of interest in MBD6, we found that two known SNPs occur at a higher frequency within our affected population compared to our control population. The first variation, rs61741508 (c.-2C>A), was recognized in sixteen patients with ASD and five controls and is located just upstream of the ATG start site in the Kozak consensus sequence. This variation also has high conservation scores (Supplemental Table 4). The second SNP, rs117084250 (c.2407-64C>T), falls within intron nine and was found in twelve individuals with ASD but only four controls. However, the conservation scores were relatively low, thereby making this a variant of lesser interest (Supplemental Table 4).

MECP2

Twenty-eight alterations were identified in MECP2 (Supplemental Table 5). Sixteen of these are currently in the dbSNP database and another one has been previously reported in RettBASE, leaving 11 novel variations. While none of the frequently recurring, classic Rett syndrome variations were identified in this study, there are two previously reported MeCP2 alterations of undetermined pathogenicity (Thr240Ser and Ala370Thr) that may cause clinical phenotypes. This first variation, MeCP2 Thr240Ser (rs61749738), was identified in two families of African ancestry (1072 and 17130) and absent in control individuals (Figure 2A,B). Further investigation into additional family members showed that the variation was inherited maternally in both cases and concordant with disease in multiplex family 1072. The second alteration, Ala370Thr (rs147017239), was also inherited maternally in a single proband of African ancestry (family18024, Figure 2C).

Pedigrees of ASD families carrying alterations in MeCP2

SETDB1

A total of 44 changes were found in SETDB1, comprised of 19 known and 25 novel alterations (Supplemental Table 6). Eight changes are predicted to be nonsynonymous, but only one of these, Pro1067del, was found solely in patients with ASD. This change is also the only ASD specific, nonsynonymous deletion identified in the entire study. The variant removes three nucleotides and predicts an in-frame deletion of a single amino acid. This deletion falls within the SET domain of the protein and was inherited maternally in both affected sons in family 17187 (Figure 3A).

Figure 3

Pedigrees of ASD families carrying alterations in SETDB1 and SETDB2

Another novel variation of interest in SETDB1 that we identified in a high proportion of cases versus controls, Pro529Leu, was identified in five ASD families of European ancestry and only a single control (Figure 3B–F). This variant was inherited paternally in one family and maternally in the remaining four families. In family 37265, the variation was passed from the father, who has dyslexia, to both the female proband with autism (0001) who was diagnosed with developmental and language delays as well as her brother (0100) who presented with ADHD, anxiety/panic disorder, language delay and macrocephaly (Figure 3E). In two of the families with maternal inheritance (17663 and 37673), the mothers presented with anxiety/panic disorder. In family 17663, the mother also presented with a history of seizures, sleep disorder and self-reported depression, while the mother in family 37673 reported history of adolescent onset Anorexia Nervosa. The increased incidence of this alteration in cases versus controls, along with neuropsychiatric and neurodevelopmental disorders in parents carrying the alteration, suggests that this variation may confer a variety of clinical consequences.

SETDB2

Thirty-eight single base pair alterations were identified in the SETDB2gene, 21 of which have been previously reported and the remaining 17 are novel (Supplemental Table 7). Eight SNPs are predicted to alter amino acids and three of these were unique to affected individuals: Ile425Thr, Thr475Met and Pro536Arg (Table 1, Figure 3C,G,H). However, these alterations are not predicted to have a highly detrimental effect on the protein and occur within singleton families, making it difficult to determine whether they may play a pathogenic role in ASD.

Along with isolating additional variations in MBD5 and MECP2 that may contribute to neuropsychiatric disease, this study is the first to report prospective pathogenic variations in MBD6 and SETDB1. These include two novel, nonsynonymous alterations in MBD6 (Arg883Trp and Pro943Arg) and one more in SETDB1 (Pro1067del). Furthermore, the MBD6 Arg883Trp and SetDB1 Pro1067del variations each segregated with ASD in the multiplex families. Potential for SETDB1 to play a role in neurobehavioral phenotypes is supported by results from transgenic Setdb1 mice demonstrating a role in mood behaviors (Jiang et al., 2010a).

To date, MBD5 mutations have been identified in individuals presenting a range of clinical phenotypes including ASD, developmental delay, intellectual disability, epilepsy, repetitive movements, and language impairments (Vissers et al., 2003;Koolen et al., 2004; de Vries et al., 2005; Wagenstaller et al., 2007; Jaillard et al., 2008; van Bon et al., 2009; Williams et al., 2009; Chung et al., 2011; Talkowski et al., 2011; Noh & Graham Jr 2012). These results suggest a significant role for theMBD5 isoform 1, which presents with increased expression in the brain (Laget et al., 2010). It has been estimated that between microdeletions and point mutations of MBD5, this gene may play a contributing genetic role in up to 1% of individuals with ASD (Talkowski et al., 2011). Of the nonsynonymous alterations identified in this study, ASD specific changes were more likely to be predicted to be damaging as compared to those variations found in control individuals (Supplemental Table 3). MBD5 Tyr1269Cys is a strong potentially pathogenic change due to its co-segregation with ASD in a multiplex family of three affected children, high conservation of this amino acid across species and altered function in the luciferase transcriptional activation assay. While this alteration does not fall in a known protein domain, it is specific to isoform 1, the isoform predominately expressed in brain (Laget et al., 2010). It seems likely that most alterations inMBD5 related to disease will be rare and unique, as the one alteration previously reported to have an increased frequency in patients with ASD, Gly79Glu, was only identified in a single control in the current study (Talkowski et al., 2011).

The role of MECP2 in developmental disorders is undisputed (Samaco & Neul 2011). Our study supports the possible pathogenicity of two specific MeCP2 alterations: Thr240Ser and Ala370Thr. The first variant, Thr240Ser was identified in two male probands from families of African ancestry, including the multiplex family 1072 where the variant segregated with ASD (Figure 2A,B). The maternal inheritance in family 17130 and presence of an unaffected carrier sister suggests that the variation may only present with a clinical phenotype in a hemizygous state. This variant falls within the transcriptional repression domain and has been previously reported in four studies; three cases of males with intellectual disability and one female with Rett syndrome (Yntema et al., 2002; Bourdon et al., 2003;Bienvenu & J. Chelly 2006; Campos et al., 2007; Bunyan & D. O. Robinson 2008). The second alteration, Ala370Thr, was identified in a singleton family of African ancestry and previously reported in three Chinese individuals: one female with Rett syndrome, her unaffected mother and a male presenting with epileptic encephalopathy (Figure 2C, Li et al., 2007; Wong & Li 2007). Both of these alterations must be further evaluated to isolate their potential functional consequences.

Finally, while we did identify variants of interest in four of the genes studied,SETDB2 alterations did not appear to be related to the occurrence of ASDs.

This is the first study to evaluate the coding regions of MBD5, MBD6, SETDB1, and SETDB2 for rare alterations in individuals with ASD. We identified novel point mutations predicted to be damaging and concordant with disease in multiplex families, as well as a complex duplication encompassing MECP2. Additional studies, ideally both in patients and animal models, are required to determine the precise consequences of these alterations. The results described here compound the evidence of MECP2 and MBD5’s involvement in ASDs and neurodevelopmental disorders and provide the first examples of autistic patients carrying potentially detrimental alterations in MBD6 and SETDB1. This study demonstrates the expanding role MBD genes play in autism etiology.

PI3K/Akt: getting it right matters

T F Franke1          Oncogene (2008) 27, 6473–6488;      http://dx.doi.org:/10.1038/onc.2008.313

The Akt serine/threonine kinase (also called protein kinase B) has emerged as a critical signaling molecule within eukaryotic cells. Significant progress has been made in clarifying its regulation by upstream kinases and identifying downstream mechanisms that mediate its effects in cells and contribute to signaling specificity. Here, we provide an overview of present advances in the field regarding the function of Akt in physiological and pathological cell function within a more generalized framework of Akt signal transduction. An emphasis is placed on the involvement of Akt in human diseases ranging from cancer to metabolic dysfunction and mental disease.

The molecular mechanisms of Akt regulation are summarized in Figure 1.

Figure 1.

Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorCanonical schematic depicting the present state of our understanding of Akt activation and regulation of downstream biological responses. Autophosphorylation of RTKs induces the recruitment of p85 regulatory subunits leading to PI3K activation. Once activated, p110 catalytic subunits phosphorylate plasma membrane-bound phosphoinositides (PI-4-P and PI-4,5-P2) on the D3-position of their inositol rings. The second messengers resulting from this PI3K-dependent reaction are PI-3,4-P2and PI-3,4,5-P3 (also called PIP3). PIP3, in turn, is the substrate for the phosphoinositide 3-phosphatase PTEN, an endogenous inhibitor of PI3K signaling in cells. The phosphoinositide products of PI3K form high-affinity binding sites for the PH domains of intracellular molecules. PDK1 and Akt are two of the many targets of PI3K products in cells. Following binding of the Akt PH domain to PI3K products, Akt is phosphorylated by PDK1 on a critical threonine residue in its kinase domain. mTORC2 is the main kinase activity that through phosphorylation of a C-terminal HM serine residue locks Akt enzyme into an active conformation. Other kinases such as DNA-PK and ILK1 are also capable of phosphorylating Akt at the HM site but may do so in a cell- or context-dependent manner. Akt activation is blunted by phosphatases including PP2A and PHLPP that inhibit Akt activity by dephosphorylation. Studies examining Akt-interacting proteins such as CTMP or second messengers such as Ins(1,3,4,5)P4 suggest that this common pathway of Akt regulation may be further specified within certain functional contexts or during development. Once activated, Akt activation is channeled into a plethora of downstream biological responses reaching from angiogenesis, cell survival, proliferation, translation to metabolism.

Full figure and legend (367K)

 …….

Consequences of Akt activation include diverse biological responses, ranging from primarily metabolic functions such as glucose transport, glycolysis, glycogen synthesis and the suppression of gluconeogenesis to protein synthesis, increased cell size, cell-cycle progression and apoptosis suppression.

Insights into the molecular consequences of increased Akt activation were derived from seminal studies that ultimately identified the ‘orphan’ proto-oncogene as an obligate intermediate downstream of PI3K in the insulin-dependent metabolic control of glycogen synthesis. When searching for kinases that could regulate GSK3, the groups of Brian Hemmings and Phil Cohen realized that Akt inhibited GSK3 activity in an insulin-stimulated and PI3K-dependent manner by direct phosphorylation of an N-terminal regulatory serine residue (Cross et al., 1995). By systematically permutating the amino-acid sequence surrounding the Akt phosphorylation site in GSK3, Alessi et al. (1996b) derived an optimal peptide sequence for Akt phosphorylation (R-X-R-X-X-S/T; where R is an arginine residue, S is serine, T is threonine and X is any amino acid). This Akt consensus motif is a common feature of known substrates of Akt, and its presence predicts reasonably well whether a given protein may be phosphorylated by Akt enzyme in vitro (for review, see Manning and Cantley, 2007). Experiments using randomized permutations on the basis of the motif to optimize substrate peptides have defined the requirement for optimal phosphorylation by Akt further (Obata et al., 2000). The preferred phosphoacceptor for Akt-dependent phosphorylation is a serine residue, but a synthetic substrate peptide with a threonine residue as the phosphoacceptor instead (R-P-R-A-A-T-F; P=proline, A=alanine, F=phenylalanine) is also easily phosphorylated. For achieving optimal phosphorylation efficiency, the phosphoacceptor is best followed by a hydrophobic residue with a large side-chain in the p+1 position, and preceded by a serine or threonine at the p−2 position.

One of the first targets of Akt to be identified that has direct implications for regulating cell survival is the pro-apoptotic BCL2-antagonist of death (BAD) protein. BAD regulation by Akt has exemplified the molecular pathways linking survival factor signaling to apoptosis suppression (for review, see Franke and Cantley, 1997). When BAD is not phosphorylated, it will inhibit Bcl-xL and other anti-apoptotic Bcl-2 family members by direct binding of its Bcl-2 homology domain to their hydrophobic grooves (Gajewski and Thompson, 1996). Once phosphorylated, these phospho-serine residues of BAD form high-affinity binding sites for cytoplasmic 14-3-3 molecules. As a result, phosphorylated BAD is retained in the cytosol where its pro-apoptotic activity is effectively neutralized (Zha et al., 1996). The importance of BAD as an integration point of survival signaling is underscored by the fact that it is a substrate for multiple independent kinase pathways in cells, not all of which phosphorylate BAD at the same site(s) as Akt (Datta et al., 2000). The mechanisms of 14-3-3-dependent regulation of BAD function hereby resemble the Akt-dependent inhibition of FoxO transcription factors that regulate the transcription of pro-apoptotic genes (Brunet et al., 1999).

The function of Akt extends beyond maintaining mitochondrial integrity to keep cytochrome c and other apoptogenic factors in the mitochondria (Kennedy et al., 1999). Akt activity also mitigates the response of cells to the release of cytochrome c into the cytoplasm. Although caspase-9 is an Akt substrate in human cells, where it may explain cytochrome c resistance (Cardone et al., 1998), it may not be the only, or even the most important, target because Akt-dependent cytochrome c resistance can be observed in animal species where caspase-9 lacks a potential Akt phosphorylation site (Fujita et al., 1999; Zhou et al., 2000). Not surprisingly, other components of the post-mitochondrial machinery such as the X-linked inhibitor of apoptotic proteins (XIAP) have been suggested as potential Akt substrates (Dan et al., 2004).

Another important class of Akt targets are proteins involved in the stress-activated/mitogen activated protein kinase (SAPK/MAPK) cascades. Growing experimental evidence points to a close functional relationship between the Akt survival pathway and SAPK/MAPK cascades that are activated by various cellular stresses and are linked to apoptosis. Increased Akt activity has been shown to suppress the JNK and p38 pathways (Berra et al., 1998; Cerezoet al., 1998; Okubo et al., 1998). It has been shown that apoptosis signal-regulating kinase 1 (ASK1) is regulated by Akt and contains an Akt-specific phosphorylation site (Kim et al., 2001). These findings have been confirmed independently by other groups (Yuan et al., 2003; Mabuchi et al., 2004). Thus, ASK1 is likely to be one of the points of convergence between PI3K/Akt signaling and stress-activated kinase cascades, although probably not the only one. Akt also phosphorylates the small G protein Rac1 (Kwon et al., 2000), the MAP2K stress-activated protein kinase kinase-1 (SEK1; also known as JNKK1 or MKK4) (Park et al., 2002) and the MAP3K mixed lineage kinase 3 (MLK3) (Barthwal et al., 2003; Figueroa et al., 2003). Using yeast-2-hybrid screens to identify interacting partners for Akt kinases, Figueroa et al. (2003) found binding of Akt2 to the JNK adaptor POSH. These authors showed that the binding of Akt2 to POSH results in an inhibition of JNK activity, and that this inhibition is mediated by phosphorylation of the upstream kinase MLK3 and leads to the disassembly of the JNK signaling complex. In turn, POSH is also an Akt substrate (Lyons et al., 2007). Taken together, these findings point to an intriguing model for the regulation of the JNK pathway by Akt, in which the Akt-dependent phosphorylation of specific components can block signal transduction through the stress-regulated kinase cascade. In spite of this, it has been reported that Akt also blocks the pro-apoptotic activity of other MAP3Ks such as MLK1 and MLK2 that act in parallel to MLK3 but do not contain a typical Akt consensus phosphorylation motif (Xu et al., 2001). Thus, phosphorylation-based mechanisms may be limited in explaining the role of Akt in blocking JNK signaling.

Although many of its substrates are involved in clearly defined biological functions within a circumscribed context such as cell proliferation, a more thorough analysis of Akt signaling has suggested that the boundaries between metabolic processes and apoptosis suppression may be artificial. For example, the Akt target GSK3 has been implicated both in the regulation of glucose metabolism and cell survival (Pap and Cooper, 1998). These findings suggest that the distinctions between cell growth, survival, metabolism and apoptosis regulation do not properly reflect functional interactions between concurrent biological processes in cells. This shift in perception has been fueled by studies from the Korsmeyer laboratory that have demonstrated a canonical function for the pro-apoptotic Bcl-2 family member BAD in the regulation of glucokinase activity (Danial et al., 2003). It is conceivable that findings of PKA-dependent regulation of BAD in glucose metabolism can be extrapolated to BAD inhibition by Akt. Still, a formal confirmation for a role of Akt in this process has yet to be presented (for review, see Downward, 2003).

The critical importance of Akt signaling for neuronal function is implied from several lines of in vitro evidence using neuronal cell lines and dispersed primary neuronal cultures that have demonstrated a requirement for Akt in the protection against trophic factor deprivation, oxidative stress and ischemic injury (Dudek et al., 1997; Salinas et al., 2001; Noshita et al., 2002). Dysregulation of Akt activity is observed in neurodegenerative diseases including Alzheimer’s disease (Rickle et al., 2004; Ryderet al., 2004), Parkinson’s disease (Hashimoto et al., 2004) and Huntington’s disease (Humbert et al., 2002), and it is also associated with the pathobiological mechanisms underlying spinocerebellar ataxia (Chen et al., 2003). A mechanistic involvement of impaired Akt signaling in neurodegeneration is further supported by the Akt-dependent phosphorylation of the disease-related proteins huntingtin (Humbert et al., 2002) and ataxin (Chen et al., 2003).

Other studies suggest that the involvement of Akt in brain function extends beyond the protection of neuronal cells against apoptotic insults. Indeed, pathological changes in Akt signal transduction have been described that are associated with mental diseases. Significantly decreased Akt1 expression has been reported in patients suffering from familial schizophrenia (Emamian et al., 2004). Decreased Akt1 levels are correlated with increased GSK3 activity, presumably because of the lack of the Akt-dependent inhibitory input on GSK3. In support of AKT1 being a susceptibility gene for schizophrenia, Akt1(−/−) mice exhibit increased sensitivity to the sensorimotor disruptive effect of amphetamine, which is partly reversed by the treatment of mutant mice with the antipsychotic drug haloperidol (Emamian et al., 2004). Additional support for a contribution of impaired Akt signaling in the pathogenesis of schizophrenia derives from the finding of mutant PI3K signaling in schizophrenia (for review, see Arnold et al., 2005). A direct involvement of Akt in dopaminergic action is indicated by the observation that Akt1(−/−) mutant mice exhibit a behavioral phenotype resembling enhanced dopaminergic transmitter function (Emamian et al., 2004). By interacting with the GSK3 pathway, Akt modulates the suppression of dopamine (DA)-associated behaviors after treatment with the mood stabilizer lithium (Beaulieu et al., 2004). Furthermore, a β-arrestin 2-mediated kinase/phosphatase scaffold of Akt and protein phosphatase A (PP2A) is required for the regulation of Akt downstream of DA receptors (Beaulieu et al., 2005). Still, the role of Akt in dopaminergic responses by far exceeds actions downstream of DA receptors: the insulin-dependent regulation of DA transporter also depends on Akt activity (Garcia et al., 2005).

Since the field of Akt signaling in psychiatric disorders is still emerging, it may be too early to speculate about the molecular involvement of Akt in regulating higher brain function. Possible functional outlets of Akt include some of the substrates mentioned above, including mTORC1 and GSK3. In addition, substrates of Akt related to synaptic plasticity and transmission have been described. One such novel substrate of Akt related to neuronal excitability is the β2-subunit of the type A γ-aminobutyric acid receptor (GABAA-R) (Lin et al., 2001). In support of a direct involvement of Akt in synaptic function, studies directed at working memory performance performed in Akt1(−/−) mice (Lai et al., 2006) and in healthy individuals carrying the AKT1 coding variation observed in familial schizophrenia (Tan et al., 2008) find a strong correlation with cognitive performance. Additional roles for Akt in higher brain function are suggested by studies from the Nestler laboratory that have explored the IRS2-Akt pathway during the development of tolerance to opiate reward (Russo et al., 2007). By using viral-mediated gene transfer to express mutant Akt in midbrain neurons, these authors demonstrate that downregulation of the IRS2-Akt pathway mediates morphine-induced decreases in cell size of DA neurons in brain regions that are critically involved in the reward circuitry and affected in individuals addicted to drugs of abuse.

Finally, TSC patients show an increased incidence of autism spectrum disorders (ASD) ranging from 25 to 50% (for review, seeWiznitzer, 2004). Individuals with macrocephaly due to Lhermitte–Duclos disease are prone to ASD and show a pronounced incidence of mutations in the PTEN tumor suppressor gene (Butler et al., 2005). Additional experimental support for a possible involvement of PTEN/Akt in ASD is provided by data from the Parada laboratory examining the morphology and behavior of mutant mice with neuron-specific knockout of PTEN (Kwon et al., 2006). Future studies will be required to clarify the function of Akt in cognition and characterize the underlying molecular mechanisms. In spite of this, these initial studies suggest a complex function of Akt in conditions affecting brain function and mental health.

The emerging involvement of Akt in higher brain function is summarized in Figure 2.

Figure 2.

Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Akt kinase regulates diverse aspects of neuronal cell function. Akt activation in neuronal cells follows similar mechanisms to those outlined in Figure 1, including activation of PI3K by RTKs including IGF-1/insulin and nerve growth factor (BDNF/NT-3) receptors. Other mechanisms governing Akt activity in neuronal cells include G-coupled receptors for the monoamine neurotransmitters serotonin (5-HT) (for review, see Raymond et al., 2001) and dopamine (DA) (for review, see Beaulieu et al., 2007). Depending on receptor type (D2-DA and 5-HT1A receptors vs D1-DA receptors), binding of 5-HT or DA decreases or increases the activity of adenylyl cyclase (AC), respectively. Changes in cAMP second messenger levels, in turn, alter PKA and PP2A activity. PP2A is inhibited by increased PKA activity, thus maintaining Akt in an activated state after 5-HT1Areceptor simulation (Hsiung et al., 2008). After binding of DA to D2-DA receptor, following initial inhibition of AC, a secondary internalization complex is formed between β-arrestin 2, PP2A and Akt leading to the inhibition of Akt. In neuronal cells, activated Akt regulates diverse targets that have been implicated in the regulation of protein translation and cell size (mTORC1), axonal outgrowth (GSK3), apoptosis suppression (BAD) and synaptic plasticity (GABAA-R). Details regarding functional consequences of Akt regulation for higher brain function are discussed in the text.

Full figure and legend (306K)

 

When considering the present understanding of all the signals leading to and from Akt, we face a growing complexity that is in part compounded by the intersection of multiple signaling cascades. Many substrates of Akt are shared with other kinases that have similar specificities. Moreover, signals originating from activated Akt do not simply lead to changes in the biological activity of specific downstream substrates, but affect entire signaling networks. In spite of this, there is hope that there is order to the far-reaching physiological involvement of Akt. One possibility is that differential regulation of the binding partners of Akt may determine cell- and context-specific signaling by Akt. Studies are now needed to elucidate the physiological functions of the binding partners of Akt in mammalian physiology.

A second challenge that the field is facing arises from the involvement of Akt in multiple areas of physiology. These now exceed cancer and diabetes and, as briefly outlined above, include higher brain functions related to cognition.

SETDB1 in Early Embryos and Embryonic Stem Cells

Yong-Kook Kang

The histone methyltransferase SETDB1 contributes to the silencing of local chromatin and the target specificity appears to be determined through various proteins that SETDB1 interacts with. This fundamental function endows SETDB1 with specialized roles in embryonic cells. Keeping the genomic and transcriptomic integrity via proviral silencing and maintaining the pluripotency by repressing the differentiation-associated genes have been demonstrated as the roles of SETDB1 in embryonic stem cells. In early developing embryos, SETDB1 exhibits characteristic nuclear mobilizations that might account for its pleiotropic roles in these rapidly changing cells as well. Early lethality of SETDB1-null embryos, along with other immunolocalization findings, suggests that SETDB1 is necessary for reprogramming and preparing the genomes of zygotes and pluripotent cells for the post-implantation developmental program.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706629/

Exome sequencing of probands with autism have revealed broadly similar results:de novo mutations in a large set of genes occur in a significant fraction of patients, with relatively high OR’s for damaging mutations in genes expressed in the brain9,1921. Most interestingly, CHD8, which like CHD7 reads H3K4me marks, is frequently mutated in autism22, raising the question of whether the H3K4me pathway may play a role in many congenital diseases. Among 249 protein-alteringde novo mutations in CHD (Supplementary Table 4) and 570 such mutations in autism9,19,20,23, there were two genes, CUL3 and NCKAP1, with damaging mutations in both CHD and autism and none in controls (P = 0.001, Monte Carlo simulation), and several others with mutations in both (e.g., SUV40H1 and CHD7). Similarly, rare copy number variants at 22q11.2, 1q21, and 16p11 are found in patients with autism, CHD or both diseases2426. These observations suggest variable expressivity of mutations in key developmental genes. Identification of the complete set of these developmental genes and the full spectrum of the resulting phenotypes will likely be important for patient care and genetic counseling.

 

Context-specific microRNA function in developmental complexity

Adam P. Carroll1,2Paul A. Tooney1,2 and Murray J. Cairns1,2,*      http://jmcb.oxfordjournals.org/content/early/2013/03/01/jmcb.mjt004.full     J Mol Cell Biol (2013)    doi: 10.1093/jmcb/mjt004

Since their discovery, microRNAs (miRNA) have been implicated in a vast array of biological processes in animals, from fundamental developmental functions including cellular proliferation and differentiation, to more complex and specialized roles such as longterm potentiation and synapse-specific modifications in neurons. This review recounts the history behind this paradigm shift, which has seen small non-coding RNA molecules coming to the forefront of molecular biology, and introduces their role in establishing developmental complexity in animals. The fundamental mechanisms of miRNA biogenesis and function are then considered, leading into a discussion of recent discoveries transforming our understanding of how these molecules regulate gene network behaviour throughout developmental and pathophysiological processes. The emerging complexity of this mechanism is also examined with respect to the influence of cellular context on miRNA function. This discussion highlights the absolute imperative for experimental designs to appreciate the significance of context-specific factors when determining what genes are regulated by a particular miRNA. Moreover, by establishing the timing, location, and mechanism of these regulatory events, we may ultimately understand the true biological function of a specific miRNA in a given cellular environment.

It was once considered the central dogma of molecular biology that gene expression was regulated in a unidirectional manner whereby cellular instructions were encoded in DNA to be transcribed to produce RNA, which simply acted as a messenger molecule to produce the protein end-products that executed these cellular instructions. In fact, signs of a biological phenomenon whereby non-protein-coding RNA molecules could interfere with this very process were not even realized until the 1970s and early 1980s, when exogenous oligonucleotides complementary to ribosomal RNA were found to interfere with ribosome function (Taniguchi and Weissmann, 1978; Eckhardt and Luhrmann, 1979;Jayaraman et al., 1981). A number of experiments in both prokaryotes and eukaryotes further supported the notion of antisense RNA as an antagonist to RNA function (Chang and Stoltzfus, 1985; Ellison et al., 1985; Harland and Weintraub, 1985; Izant and Weintraub, 1985; Melton, 1985), and one such experiment elegantly demonstrated that the introduction of synthetic oligonucleotides complementary to 3′- and 5′-terminal repeats of Rous sarcoma virus 35S RNA not only attenuated viral replication and cell transformation, but also inhibited viral RNA translation in vitro (Stephenson and Zamecnik, 1978; Zamecnik and Stephenson, 1978).

In addition to this, the successful inhibition of thymidine kinase gene expression by antisense RNA in eukaryotic cells precipitated the concept of antisense RNA not only as an experimental tool, but also as a therapeutic design (Izant and Weintraub, 1984). Determining the functionality of a previously identified gene sequence without identifying, isolating, or characterizing the protein product; interfering with RNAs that are never translated; and silencing the expression of disease-associated transcripts in a sequence-specific manner: these were some very appealing prospects. By the late 1980s and early 1990s, a variety of techniques had evolved in the field of molecular and applied genetics whereby various antisense DNA and RNA construct designs were employed to efficiently downregulate target gene expression (Fire et al., 1991).

Meanwhile, the scientific community was also beginning to appreciate a role for endogenous antisense RNA. Short antisense transcripts were found to form an RNA–RNA duplex with the 5′ end of the replication primer of the ColE1 plasmid (Tomizawa et al., 1981; Tomizawa and Itoh, 1982). Endogenous antisense RNA control in prokaryotes was also linked with various biological processes such as plasmid replication, transposition, temporal bacteriophage development, and catabolite repression in bacteria (Light and Molin, 1983; Simons and Kleckner, 1983; Kumar and Novick, 1985). Evidence was also beginning to mount to implicate antisense control mechanisms in eukaryotic organisms (Adeniyi-Jones and Zasloff, 1985; Farnham et al., 1985; Heywood, 1986; Spencer et al., 1986; Williams and Fried, 1986; Stevens et al., 1987), including the demonstration that antisense transcripts in the bovine papillomavirus type 1 (BPV-1) genome prevented episomal replication (Bergman et al., 1986).

It was only a matter of time before phenomena of gene silencing began to unfold in animals. Previous work in the 1980s with Caenorhabditis elegans had established that mutations in the genes for lin-4, lin-14, lin-28, lin-29, and lin-41 altered the heterochronic lineage of developing larvae, resulting in a failure to control temporal aspects of post-embryonic development (Chalfie et al., 1981; Ambros and Horvitz, 1984; 1987; Ambros, 1989); thus, these genes were referred to as being ‘heterochronic’. However, in 1993 it was discovered that lin-4 was located within an intron and was thus unlikely to encode a protein. More significantly, two lin-4 transcripts ∼22 and 61 nucleotides in length were identified that exhibited complementarity to a repeat sequence element in the 3′ untranslated region (UTR) of lin-14 mRNA (Lee et al., 1993). With another report soon replicating this finding in C. elegans andCaenorhabditis briggsae (Wightman et al., 1993), the notion was set forth that the 22-nucloetide lin-4 transcript represented an active mature form of the 61-nucelotide transcript and functioned to control worm larval development by binding to the 3′-UTR of lin-14, thereby negatively regulating its function via an antisense RNA–RNA interaction. Furthermore, lin-4 exhibited complementarity to seven regions within the 3′-UTR of lin-14, demonstrating that gene expression was more potently inhibited as more of these non-coding transcripts bound to the mRNA (He and Hannon, 2004). Retrospectively, we can identify the lin-4 gene in C. elegans as the pioneer of a new class of small, non-coding RNAs called microRNA (miRNA) (Lee et al., 1993), which utilize the RNA interference (RNAi) pathway to regulate the expression of protein-encoding genes at post-transcriptional level (He and Hannon, 2004).

The following few years were somewhat quiet at the forefront of miRNA research, with lin-4 mechanism assumed to be a unique event. Meanwhile, RNAi was coming to prominence in 1998 with Fire and Mello (along with their colleagues) reporting double-stranded RNA (dsRNA) to be far more potent at mediating gene suppression in C. elegans than single-stranded antisense RNA (Fire et al., 1998). Interestingly, only small quantities of dsRNA were required to induce post-transcriptional gene silencing (PTGS), and it was hypothesized that an endogenous catalytic or amplification component was mediating mRNA degradation prior to translation (Montgomery et al., 1998). RNAi was soon thereafter reported as an ATP-dependent process in an in vitroDrosophila embryo lysate system where dsRNA was processed into 21–23-nucleotide species that appeared to guide sequence-specific mRNA cleavage (Zamore et al., 2000). When dsRNA was shown by the Tuschl laboratory to be processed into 21–22-nucleotide short interfering RNA (siRNA) by a ribonuclease III enzyme to mediate sequence-specific RNAi in human embryonic kidney HEK-293 cells, the prospect was set forth for exogenous 21–22-nucleotide siRNA to be developed as gene-specific therapeutic molecules (Elbashir et al., 2001a).

With incredible excitement surrounding the implications of RNAi, Ruvkun and colleagues discovered a second miRNA inC. elegans in 2000. Like lin-4, the newly discovered let-7 exhibited complementarity to the 3′-UTR of heterochronic genes, in this case lin-14, lin-28, lin-41, lin-42, and daf-12 (Reinhart et al., 2000). Moreover, they discovered that let-7 was highly conserved in its temporal regulation across phylogeny (Pasquinelli et al., 2000), refuting the widely believed concept that lin-4 and let-7 were a worm-specific oddity and propelling miRNA to significance as native endogenous clients of the RNAi machinery. This catalysed intense genome-wide searches for the discovery of more endogenous small regulatory RNAs in numerous species, to the point that miRBase Release 19 currently contains sequence data for 25141 mature miRNA products in 193 organism species (Kozomara and Griffiths-Jones, 2011).

The significance of non-coding RNA was further illuminated in 2001 when the completion of the human genome project revealed that <2% of the human genome encoded proteins (Lander et al., 2001). It has been realized that the ratio of non-coding to protein-coding DNA in the genome correlates with developmental complexity (Mattick, 2004), and a recent publication has reported on the exponential correlation of miRNA gene number and 3′-UTR length—but not 5′-UTR or coding sequence length—with morphological complexity in animals (Chen et al., 2012). This was measured according to the number of cell types within each organism, and also confirmed earlier observations that 3′-UTR length in housekeeping genes has remained short across organisms, thereby minimizing miRNA-binding site potential and reducing the complexity with which these constitutively expressed genes are regulated (Stark et al., 2005). Today we certainly have a stronger appreciation for RNA molecules to function not only as messengers of protein production, but also as complex regulatory molecules facilitating the intricate control of gene expression required for developmental complexity (Kosik, 2009).

Mechanisms of miRNA function

When considering non-coding RNA function, miRNAs constitute one of the largest classes of endogenous, non-coding regulatory RNA molecules in animals. In their mature form they are ∼19–22 nucleotides in length, and they interact via Watson–Crick binding with regions of complementarity primarily within the 3′-UTR of mRNA transcripts. In doing so, miRNAs act as sequence-specificity guides for the RNAi machinery to mediate repression of target gene expression at post-transcriptional level by negatively regulating mRNA stability and/or protein translation.

miRNA biogenesis

miRNAs are typically transcribed by RNA polymerase II (pol II) as long primary miRNA (pri-miRNA) transcripts, which undergo sequential cleavage into a precursor miRNA (pre-miRNA) transcript before being cleaved again into the mature miRNA duplex (Figure 1). These pri-miRNA transcripts range in length from several hundred nucleotides to several kilobases, can contain either a single miRNA or clusters of several miRNAs, and originate from intronic regions of protein-coding and non-coding genes, as well as from intergenic and exonic regions (Rodriguez et al., 2004; Saini et al., 2007). The microprocessor complex is responsible for mediating pri-miRNA cleavage, with the dsRNA-binding protein DGCR8 (DiGeorge syndrome critical region gene 8) binding the pri-miRNA and positioning the catalytic site of Drosha—a ribonuclease III (RNase III) dsRNA-specific endonuclease—11 nucleotides from the base of the duplex stem to mediate nuclear processing to the pre-miRNA transcript (Denli et al., 2004; Han et al., 2006). This produces a pre-miRNA hairpin typically 55–70 nucleotides in length with a two-nucleotide 3′ overhang, characteristic of RNase III-mediated cleavage (Lee et al., 2003). This two-nucleotide overhang facilitates the subsequent exportation of the pre-miRNA from the nucleus to the cytoplasm by a RanGTP/Exportin5-dependent mechanism and is suspected to also facilitate subsequent cleavage by the RNase III endonuclease Dicer (Yi et al., 2003; Bohnsack et al., 2004; Lund et al., 2004). This cleavage requires the interaction of Dicer with the dsRNA-binding protein TRBP [HIV-1 transactivating response (TAR) RNA-binding protein] (Forstemann et al., 2005), and as a result of Dicer processing the terminal base pairs and the loop of the pre-miRNA are excised. This produces a 19–22-nucleotide mature miRNA duplex, which possess two-nucleotide overhangs at each 3′ end (Lee et al., 2002).

 

Figure 1

http://jmcb.oxfordjournals.org/content/early/2013/03/01/jmcb.mjt004/F1.medium.gif

Figure 1

A model for canonical miRNA biogenesis and function in animals. After their transcription by RNA polymerase II, pri-miRNAs are cleaved in the nucleus by Drosha, which forms a microprocessor complex with DGCR8. This generates the pre-miRNA, which is actively exported into the cytoplasm via a RanGTP/Exportin 5-dependent mechanism. In the cytoplasm, Dicer binds the base of the pre-miRNA stem defined in the nucleus by Drosha. Dicer cleavage liberates a mature miRNA duplex that exhibits imperfect complementarity. This miRNA duplex is assembled into the miRISC loading complex, in which the passenger strand is discarded. The miRNA guides the mature miRISC to regions of complementarity within mRNA transcripts, thereby mediating post-transcriptional gene silencing through translational repression and/or mRNA degradation.

miRISC loading

After their maturation into small RNA duplexes, miRNAs are loaded into ribonucleoprotein (RNP) complexes, often referred to as miRNA-induced silencing complexes (miRISCs), RISCs, or miRNPs. The signature components of each miRISC are the miRNA and an Argonaute (AGO) protein. In humans, there are four AGO proteins (AGO1-4), each consisting of the highly conserved P-element-induced wimpy testes (PIWI), middle (MID), and PIWI-AGO-Zwille (PAZ) domains, along with a less-conserved terminal domain. The loading of the miRNA into this protein complex has been proposed to occur in tandem with Dicer-mediated miRNA maturation (Gregory et al., 2005;Maniataki and Mourelatos, 2005) and requires ATP hydrolysis with additional chaperone proteins to create an open conformation to facilitate loading of the miRNA duplex (Liu et al., 2004; Yoda et al., 2010).

A key feature of miRNA is that while both strands of a small RNA duplex are capable of activating the miRISC, typically only one strand will induce silencing (Khvorova et al., 2003). This asymmetry is primarily governed by the relative thermodynamic properties of the RNA duplex, such that the miRISC-associated helicase preferentially unwinds the miRNA duplex from the end with least resistance in terms of inter-strand hydrogen bonding. The strand with its 5′ end at this less thermodynamically stable end is selected as the guide strand, and proteins such as TRBP or protein kinase, interferon-inducible dsRNA-dependent activator (PACT) are proposed to interact with Dicer to sense this thermodynamic asymmetry (Schwarz et al., 2003; Noland et al., 2011). In doing so, the guide strand is retained in the miRISC, while the other strand (the passenger, or the miRNA* strand) is discarded (Hutvagner, 2005; Matranga et al., 2005). miRNA strand selection also appears to be independent of Dicer processing polarity (Preall et al., 2006), where both ends of a duplex have similar thermodynamic properties, both the miRNA and miRNA* act as the guide strand with similar frequencies (Schwarz et al., 2003). However, strand selection does not always occur according to the axiom of thermodynamic strand asymmetry, with tissue-specific factors appearing to play a role in enabling both the miRNA and miRNA* strands to co-accumulate and function as the guide strand (Ro et al., 2007). For this reason, miRNA nomenclature has advanced beyond the miRNA* system, with the adoption of miRNA-5p and -3p names to indicate whether the mature miRNA sequence is derived from the 5′ or 3′ end of the pre-miRNA transcript.

Once the mature miRNA strand has been isolated in the mature miRISC, the AGO protein functions as an interface for the miRNA to interact with its mRNA targets. Recent characterization of human AGO2 has revealed that the 3′ hydroxyl of the miRNA inserts into a hydrophobic pocket of AGO such that the terminal nucleotide stacks against the aromatic ring of a conserved phenylalanine residue in the AGO PAZ domain (Jinek and Doudna, 2009). Meanwhile, the MID domain forms a binding pocket that anchors the miRNA 5′ phosphate such that this terminal nucleotide is distorted and does not interact with the target mRNA (Ma et al., 2005; Parker et al., 2005).

…….

Since being discovered as regulators of developmental timing in C. elegans, it has become widely established that miRNA-mediated regulation of gene expression is a fundamental biological phenomenon required to facilitate key developmental processes such as cellular proliferation, programmed cell death, and cell lineage determination and differentiation (Bartel, 2009; Ambros, 2011). Their significance is such that 60% of the human genome is predicted to be regulated by miRNA function (Friedman et al., 2009), each miRNA estimated to regulate around 200 target genes (Krek et al., 2005).

Figure 2

http://jmcb.oxfordjournals.org/content/early/2013/03/01/jmcb.mjt004/F2.medium.gif

Figure 2

Characteristic miRNA associated with the proliferation and differentiation of specialized cell types. A number of distinct miRNAs are expressed at specific stages through development to play a vital role in mediating cell proliferation, specification, and differentiation. A number of miRNAs involved in the establishment of specialized cell types are illustrated for neurogenesis (Smirnova et al., 2005;Makeyev et al., 2007; Shen and Temple, 2009; Shi et al., 2010; Zhao et al., 2010), myogenesis (Chen et al., 2006; Kim et al., 2006), haematopoiesis (Chen et al., 2004; Georgantas et al., 2007;Vasilatou et al., 2010), oligodendrocyte differentiation (Lau et al., 2008; Dugas et al., 2010), as well as induced pluripotent stem (iPS) cell reprogramming (Miyoshi et al., 2011).

 

miRNAs play a central role in establishing the spatiotemporal gene expression patterns required to establish specialized cell types and promote developmental complexity. The inherent complexity of miRNA function, however, requires a scientific approach in which context-specific miRNA function must be acknowledged if advancements are to be made in understanding how these small regulatory RNA molecules function in various developmental and pathophysiological processes. While this requires an appreciation for mechanistic aspects such as non-redundant miRISC function and the dynamic regulatory outcomes this facilitates, arguably the greatest challenge facing miRNA biology is the identification of the many genes that each miRNA targets and an understanding of the context-specific factors that determine when and how these genes are regulated.

 

 

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Mindful Discoveries

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Schizophrenia and the Synapse

Genetic evidence suggests that overactive synaptic pruning drives development of schizophrenia.

By Ruth Williams | January 27, 2016 … more follows)

http://www.the-scientist.com/?articles.view/articleNo/45189/title/Schizophrenia-and-the-Synapse/

http://www.the-scientist.com/images/News/January2016/Schizophrenia.jpg

C4 (green) at synapses of human neurons

Compared to the brains of healthy individuals, those of people with schizophrenia have higher expression of a gene called C4, according to a paper published inNature today (January 27). The gene encodes an immune protein that moonlights in the brain as an eradicator of unwanted neural connections (synapses). The findings, which suggest increased synaptic pruning is a feature of the disease, are a direct extension of genome-wide association studies (GWASs) that pointed to the major histocompatibility (MHC) locus as a key region associated with schizophrenia risk.

“The MHC [locus] is the first and the strongest genetic association for schizophrenia, but many people have said this finding is not useful,” said psychiatric geneticist Patrick Sullivan of the University of North Carolina School of Medicine who was not involved in the study. “The value of [the present study is] to show that not only is it useful, but it opens up new and extremely interesting ideas about the biology and therapeutics of schizophrenia.”

Schizophrenia has a strong genetic component—it runs in families—yet, because of the complex nature of the condition, no specific genes or mutations have been identified. The pathological processes driving the disease remain a mystery.

Researchers have turned to GWASs in the hope of finding specific genetic variations associated with schizophrenia, but even these have not provided clear candidates.

“There are some instances where genome-wide association will literally hit one base [in the DNA],” explained Sullivan. While a 2014 schizophrenia GWAS highlighted the MHC locus on chromosome 6 as a strong risk area, the association spanned hundreds of possible genes and did not reveal specific nucleotide changes. In short, any hope of pinpointing the MHC association was going to be “really challenging,” said geneticist Steve McCarroll of Harvard who led the new study.

Nevertheless, McCarroll and colleagues zeroed in on the particular region of the MHC with the highest GWAS score—the C4 gene—and set about examining how the area’s structural architecture varied in patients and healthy people.

The C4gene can exist in multiple copies (from one to four) on each copy of chromosome 6, and has four different forms: C4A-short, C4B-short, C4A-long, and C4B-long. The researchers first examined the “structural alleles” of the C4 locus—that is, the combinations and copy numbers of the different C4 forms—in healthy individuals. They then examined how these structural alleles related to expression of both C4Aand C4B messenger RNAs (mRNAs) in postmortem brain tissues.From this the researchers had a clear picture of how the architecture of the C4 locus affected expression ofC4A and C4B. Next, they compared DNA from roughly 30,000 schizophrenia patients with that from 35,000 healthy controls, and a correlation emerged: the alleles most strongly associated with schizophrenia were also those that were associated with the highest C4A expression. Measuring C4A mRNA levels in the brains of 35 schizophrenia patients and 70 controls then revealed that, on average, C4A levels in the patients’ brains were 1.4-fold higher.C4 is an immune system “complement” factor—a small secreted protein that assists immune cells in the targeting and removal of pathogens. The discovery of C4’s association to schizophrenia, said McCarroll, “would have seemed random and puzzling if it wasn’t for work . . . showing that other complement components regulate brain wiring.” Indeed, complement protein C3 locates at synapses that are going to be eliminated in the brain, explained McCarroll, “and C4 was known to interact with C3 . . . so we thought well, actually, this might make sense.”McCarroll’s team went on to perform studies in mice that revealed C4 is necessary for C3 to be deposited at synapses. They also showed that the more copies of the C4 gene present in a mouse, the more the animal’s neurons were pruned.Synaptic pruning is a normal part of development and is thought to reflect the process of learning, where the brain strengthens some connections and eradicates others. Interestingly, the brains of deceased schizophrenia patients exhibit reduced neuron density. The new results, therefore, “make a lot of sense,” said Cardiff University’s Andrew Pocklington who did not participate in the work. They also make sense “in terms of the time period when synaptic pruning is occurring, which sort of overlaps with the period of onset for schizophrenia: around adolescence and early adulthood,” he added.

“[C4] has not been on anybody’s radar for having anything to do with schizophrenia, and now it is and there’s a whole bunch of really neat stuff that could happen,” said Sullivan. For one, he suggested, “this molecule could be something that is amenable to therapeutics.”

A. Sekar et al., “Schizophrenia risk from complexvariation of complement component 4,”Nature,   http://dx.doi.com:/10.1038/nature16549, 2016.     

Tags schizophrenia, neuroscience, gwas, genetics & genomics, disease/medicine and cell & molecular biology

 

Schizophrenia: From genetics to physiology at last

Ryan S. Dhindsa& David B. Goldstein

Nature (2016)  http://dx.doi.org://10.1038/nature16874

The identification of a set of genetic variations that are strongly associated with the risk of developing schizophrenia provides insights into the neurobiology of this destructive disease.

http://www.nytimes.com/2016/01/28/health/schizophrenia-cause-synaptic-pruning-brain-psychiatry.html

 

Genetic study provides first-ever insight into biological origin of schizophrenia

Suspect gene may trigger runaway synaptic pruning during adolescence — NIH-funded study

NIH/NATIONAL INSTITUTE OF MENTAL HEALTH

IMAGE

http://media.eurekalert.org/multimedia_prod/pub/web/107629_web.jpg

The site in Chromosome 6 harboring the gene C4 towers far above other risk-associated areas on schizophrenia’s genomic “skyline,” marking its strongest known genetic influence. The new study is the first to explain how specific gene versions work biologically to confer schizophrenia risk.  CREDIT  Psychiatric Genomics Consortium

Versions of a gene linked to schizophrenia may trigger runaway pruning of the teenage brain’s still-maturing communications infrastructure, NIH-funded researchers have discovered. People with the illness show fewer such connections between neurons, or synapses. The gene switched on more in people with the suspect versions, who faced a higher risk of developing the disorder, characterized by hallucinations, delusions and impaired thinking and emotions.

“Normally, pruning gets rid of excess connections we no longer need, streamlining our brain for optimal performance, but too much pruning can impair mental function,” explained Thomas Lehner, Ph.D., director of the Office of Genomics Research Coordination of the NIH’s National Institute of Mental Health (NIMH), which co-funded the study along with the Stanley Center for Psychiatric Research at the Broad Institute and other NIH components. “It could help explain schizophrenia’s delayed age-of-onset of symptoms in late adolescence/early adulthood and shrinkage of the brain’s working tissue. Interventions that put the brakes on this pruning process-gone-awry could prove transformative.”

The gene, called C4 (complement component 4), sits in by far the tallest tower on schizophrenia’s genomic “skyline” (see graph below) of more than 100 chromosomal sites harboring known genetic risk for the disorder. Affecting about 1 percent of the population, schizophrenia is known to be as much as 90 percent heritable, yet discovering how specific genes work to confer risk has proven elusive, until now.

A team of scientists led by Steve McCarroll, Ph.D., of the Broad Institute and Harvard Medical School, Boston, leveraged the statistical power conferred by analyzing the genomes of 65,000 people, 700 postmortem brains, and the precision of mouse genetic engineering to discover the secrets of schizophrenia’s strongest known genetic risk. C4’s role represents the most compelling evidence, to date, linking specific gene versions to a biological process that could cause at least some cases of the illness.

“Since schizophrenia was first described over a century ago, its underlying biology has been a black box, in part because it has been virtually impossible to model the disorder in cells or animals,” said McCarroll. “The human genome is providing a powerful new way in to this disease. Understanding these genetic effects on risk is a way of prying open that block box, peering inside and starting to see actual biological mechanisms.”

McCarroll’s team, including Harvard colleagues Beth Stevens, Ph.D., Michael Carroll, Ph.D., and Aswin Sekar, report on their findings online Jan. 27, 2016 in the journal Nature.

A swath of chromosome 6 encompassing several genes known to be involved in immune function emerged as the strongest signal associated with schizophrenia risk in genome-wide analyses by the NIMH-funded Psychiatric Genomics Consortium over the past several years. Yet conventional genetics failed to turn up any specific gene versions there linked to schizophrenia.

To discover how the immune-related site confers risk for the mental disorder, McCarroll’s team mounted a search for “cryptic genetic influences” that might generate “unconventional signals.” C4, a gene with known roles in immunity, emerged as a prime suspect because it is unusually variable across individuals. It is not unusual for people to have different numbers of copies of the gene and distinct DNA sequences that result in the gene working differently.

The researchers dug deeply into the complexities of how such structural variation relates to the gene’s level of expression and how that, in turn, might relate to schizophrenia. They discovered structurally distinct versions that affect expression of two main forms of the gene in the brain. The more a version resulted in expression of one of the forms, called C4A, the more it was associated with schizophrenia. The more a person had the suspect versions, the more C4 switched on and the higher their risk of developing schizophrenia. Moreover, in the human brain, the C4 protein turned out to be most prevalent in the cellular machinery that supports connections between neurons.

Adapting mouse molecular genetics techniques for studying synaptic pruning and C4’s role in immune function, the researchers also discovered a previously unknown role for C4 in brain development. During critical periods of postnatal brain maturation, C4 tags a synapse for pruning by depositing a sister protein in it called C3. Again, the more C4 got switched on, the more synapses got eliminated.

In humans, such streamlining/pruning occurs as the brain develops to full maturity in the late teens/early adulthood – conspicuously corresponding to the age-of-onset of schizophrenia symptoms.

Future treatments designed to suppress excessive levels of pruning by counteracting runaway C4 in at risk individuals might nip in the bud a process that could otherwise develop into psychotic illness, suggest the researchers. And thanks to the head start gained in understanding the role of such complement proteins in immune function, such agents are already in development, they note.

“This study marks a crucial turning point in the fight against mental illness. It changes the game,” added acting NIMH director Bruce Cuthbert, Ph.D. “Thanks to this genetic breakthrough, we can finally see the potential for clinical tests, early detection, new treatments and even prevention.”

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VIDEO: Opening Schizophrenia’s Black Box https://youtu.be/s0y4equOTLg

Reference: Sekar A, Biala AR, de Rivera H, Davis A, Hammond TR, Kamitaki N, Tooley K Presumey J Baum M, Van Doren V, Genovese G, Rose SA, Handsaker RE, Schizophrenia Working Group of the Psychiatric Genomics Consortium, Daly MJ, Carroll MC, Stevens B, McCarroll SA. Schizophrenia risk from complex variation of complement component 4.Nature. Jan 27, 2016. DOI: 10.1038/nature16549.

 

Schizophrenia risk from complex variation of complement component 4

Aswin SekarAllison R. BialasHeather de RiveraAvery DavisTimothy R. Hammond, …., Michael C. CarrollBeth Stevens Steven A. McCarroll

Nature(2016)   http://dx.doi.org:/10.1038/nature16549

Schizophrenia is a heritable brain illness with unknown pathogenic mechanisms. Schizophrenia’s strongest genetic association at a population level involves variation in the major histocompatibility complex (MHC) locus, but the genes and molecular mechanisms accounting for this have been challenging to identify. Here we show that this association arises in part from many structurally diverse alleles of the complement component 4 (C4) genes. We found that these alleles generated widely varying levels of C4A and C4B expression in the brain, with each common C4 allele associating with schizophrenia in proportion to its tendency to generate greater expression of C4A. Human C4 protein localized to neuronal synapses, dendrites, axons, and cell bodies. In mice, C4 mediated synapse elimination during postnatal development. These results implicate excessive complement activity in the development of schizophrenia and may help explain the reduced numbers of synapses in the brains of individuals with schizophrenia.

Figure 1: Structural variation of the complement component 4 (C4) gene.

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature16549-f1.jpg

a, Location of the C4 genes within the major histocompatibility complex (MHC) locus on human chromosome 6. b, Human C4 exists as two paralogous genes (isotypes), C4A and C4B; the encoded proteins are distinguished at a key site

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature16549-f3.jpg

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature16549-sf8.jpg

Gene Study Points Toward Therapies for Common Brain Disorders

University of Edinburgh    http://www.dddmag.com/news/2016/01/gene-study-points-toward-therapies-common-brain-disorders

Scientists have pinpointed the cells that are likely to trigger common brain disorders, including Alzheimer’s disease, Multiple Sclerosis and intellectual disabilities.

It is the first time researchers have been able to identify the particular cell types that malfunction in a wide range of brain diseases.

Scientists say the findings offer a roadmap for the development of new therapies to target the conditions.

The researchers from the University of Edinburgh’s Centre for Clinical Brain Sciences used advanced gene analysis techniques to investigate which genes were switched on in specific types of brain cells.

They then compared this information with genes that are known to be linked to each of the most common brain conditions — Alzheimer’s disease, anxiety disorders, autism, intellectual disability, multiple sclerosis, schizophrenia and epilepsy.

Their findings reveal that for some conditions, the support cells rather than the neurons that transmit messages in the brain are most likely to be the first affected.

Alzheimer’s disease, for example, is characterised by damage to the neurons. Previous efforts to treat the condition have focused on trying to repair this damage.

The study found that a different cell type — called microglial cells — are responsible for triggering Alzheimer’s and that damage to the neurons is a secondary symptom of disease progression.

Researchers say that developing medicines that target microglial cells could offer hope for treating the illness.

The approach could also be used to find new treatment targets for other diseases that have a genetic basis, the researchers say.

Dr Nathan Skene, who carried out the study with Professor Seth Grant, said: “The brain is the most complex organ made up from a tangle of many cell types and sorting out which of these cells go wrong in disease is of critical importance to developing new medicines.”

Professor Seth Grant said: “We are in the midst of scientific revolution where advanced molecular methods are disentangling the Gordian Knot of the brain and completely unexpected new pathways to solving diseases are emerging. There is a pressing need to exploit the remarkable insights from the study.”

 

Quantitative multimodal multiparametric imaging in Alzheimer’s disease

Qian Zhao, Xueqi Chen, Yun Zhou      Brain Informatics  http://link.springer.com/article/10.1007/s40708-015-0028-9

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, causing changes in memory, thinking, and other dysfunction of brain functions. More and more people are suffering from the disease. Early neuroimaging techniques of AD are needed to develop. This review provides a preliminary summary of the various neuroimaging techniques that have been explored for in vivo imaging of AD. Recent advances in magnetic resonance (MR) techniques, such as functional MR imaging (fMRI) and diffusion MRI, give opportunities to display not only anatomy and atrophy of the medial temporal lobe, but also at microstructural alterations or perfusion disturbance within the AD lesions. Positron emission tomography (PET) imaging has become the subject of intense research for the diagnosis and facilitation of drug development of AD in both animal models and human trials due to its non-invasive and translational characteristic. Fluorodeoxyglucose (FDG) PET and amyloid PET are applied in clinics and research departments. Amyloid beta (Aβ) imaging using PET has been recognized as one of the most important methods for the early diagnosis of AD, and numerous candidate compounds have been tested for Aβ imaging. Besides in vivo imaging method, a lot of ex vivo modalities are being used in the AD researches. Multiphoton laser scanning microscopy, neuroimaging of metals, and several metal bioimaging methods are also mentioned here. More and more multimodality and multiparametric neuroimaging techniques should improve our understanding of brain function and open new insights into the pathophysiology of AD. We expect exciting results will emerge from new neuroimaging applications that will provide scientific and medical benefits.

Keywords –   Alzheimer’s disease Neuroimaging PET MRI Amyloid beta Multimodal

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that gradually destroys brain cells, causing changes in memory, thinking, and other dysfunction of brain functions [1]. AD is considered to a prolonged preclinical stage where neuropathological changes precede the clinical symptoms [2]. An estimation of 35 million people worldwide is living with this disease. If effective treatments are not discovered in a timely fashion, the number of AD cases is anticipated to rise to 113 million by 2050 [3].

Amyloid beta (Aβ) and tau are two of the major biomarkers of AD, and have important and different roles in association with the progression of AD pathophysiology. Jack et al. established hypothetical models of the major biomarkers of AD. By renewing and modifying the models, they found that the two major proteinopathies underlying AD biomarker changes, Aβ and tau, may be initiated independently in late onset AD where they hypothesize that an incident Aβ pathophysiology can accelerate an antecedent limbic and brainstem tauopathy [4]. MRI technique was used in the article, which revealed that the level of Aβ load was associated with a shorter time-to-progression of AD [5]. This warrants an urgent need to develop early neuroimaging techniques of AD neuropathology that can detect and predict the disease before the onset of dementia, monitor therapeutic efficacy in halting and slowing down progression in the earlier stage of the disease.

There have been various reports on the imaging assessments of AD. Some measurements reflect the pathology of AD directly, including positron emission tomography (PET) amyloid imaging and cerebrospinal fluid (CSF) beta-amyloid 42 (Aβ42), while others reflect neuronal injury associated with AD indirectly, including CSF tau (total and phosphorylated tau), fluorodeoxy-d-glucose (FDG)-PET, and MRI. AD Neuroimaging Initiative (ADNI) has been to establish the optimal panel of clinical assessments, MRI and PET imaging measures, as well as other biomarkers from blood and CSF, to inform clinical trial design for AD therapeutic development. At the same time, it has been highly productive in generating a wealth of data for elucidating disease mechanisms occurring during early stages of preclinical and prodromal AD [6].

Single neuroimaging often reflects limit information of AD. As a result, multimodal neuroimaging is widely used in neuroscience researches, as it overcomes the limitations of individual modalities. Multimodal multiparametric imaging mean the combination of different imaging techniques, such as PET, MRI, simultaneously or separately. The multimodal multiparametric imaging enables the visualization and quantitative analysis of the alterations in brain structure and function, such as PET/CT, and PET/MRI. [7]. In this review article, we summarize and discuss the main applications, findings, perspectives as well as advantages and challenges of different neuroimaging in AD, especially MRI and PET imaging.

2 Magnetic resonance imaging

MRI demonstrates specific volume loss or cortical atrophy patterns with disease progression in AD patients [810]. There are several MRI techniques and analysis methods used in clinical and scientific research of AD. Recent advances in MR techniques, such as functional MRI (fMRI) and diffusion MRI, depict not only anatomy and atrophy of the medial temporal lobe (MTL), but also microstructural alterations or perfusion disturbance within this region.

2.1 Functional MRI

Because of the cognitive reserve (CR), the relationship between severity of AD patients’ brain damage and corresponding clinical symptoms is not always paralleled [11, 12]. Recently, resting-state fMRI (RS-fMRI) is popular for its ability to map brain functional connectivity non-invasively [13]. By using RS-fMRI, Bozzali et al. reported that the CR played a role in modulating the effect of AD pathology on default mode network functional connectivity, which account for the variable clinical symptoms of AD [14]. Moreover, AD patients with higher educated experience were able to recruit compensatory neural mechanisms, which can be measured using RS-fMRI. Arterial spin-labeled (ASL) MRI is another functional brain imaging modality, which measures cerebral blood flow (CBF) by magnetically labeled arterial blood water following through the carotid and vertebral arteries as an endogenous contrast medium. Several studies have concluded the characteristics of CBF changes in AD patients using ASL-MRI [1517].

At some point in time, sufficient brain damage accumulates to result in cognitive symptoms and impairment. Mild cognitive impairment (MCI) is a condition in which subjects are usually only mildly impaired in memory with relative preservation of other cognitive domains and functional activities and do not meet the criteria for dementia [18], or as the prodromal state AD [19]. MCI patients are at a higher risk of developing AD and up to 15 % convert to AD per year [18]. Binnewijzend et al. have reported the pseudocontinuous ASL could distinguish both MCI and AD from healthy controls, and be used in the early diagnosis of AD [20]. In their continuous study, they used quantitative whole brain pseudocontinuous ASL to compare regional CBF (rCBF) distribution patterns in different types of dementia, and concluded that ASL-MRI could be a non-invasive and easily accessible alternative to FDG-PET imaging in the assessment of CBF of AD patients [21].

2.2 Structure MRI

Structural MRI (sMRI) has already been a reliable imaging method in the clinical diagnosis of AD, characterized as gray matter reduction and ventricular enlargement in standard T1-weighted sequences [9]. Locus coeruleus (LC) and substantia nigra (SN) degeneration was seen in AD. By using new quantitative calculating method, Chen et al. presented a new quantitative neuromelanin MRI approach for simultaneous measurement of locus LC and SN of brainstem in living human subjects [22]. The approach they used demonstrated advantages in image acquisition, pre-processing, and quantitative analysis. Numerous transgenic animal models of amyloidosis are available, which can manipulate a lot of neuropathological features of AD progression from the deposition of β-amyloid [23]. Braakman et al. demonstrated the dynamics of amyloid plaque formation and development in a serial MRI study in a transgenic mouse model [24]. Increased iron accumulation in gray matter is frequently observed in AD. Because of the paramagnetic nature of iron, MRI shows nice potential in the investigating iron levels in AD [25]. Quantitative MRI was shown high sensitivity and specificity in mapping cerebral iron deposition, and helped in the research on AD diagnosis [26].

The imaging patterns are always associated with the pathologic changes, such as specific protein markers. Spencer et al. manifested the relationship between quantitative T1 and T2 relaxation time changes and three immunohistochemical markers: β-amyloid, neuron-specific nuclear protein (a marker of neuronal cell load), and myelin basic protein (a marker of myelin load) in AD transgenic mice [27].

High-field MRI has been successfully applied to imaging plaques in transgenic mice for over a decade without contrast agents [24, 2830]. Sillerud et al. devised a method using blood–brain barrier penetrating, amyloid-targeted, superparamagnetic iron oxide nanoparticles (SPIONs) for better imaging of amyloid plaque [31]. Then, they successfully used this SPION-MRI to assess the drug efficacy on the 3D distribution of Aβ plaques in transgenic AD mouse [32].

2.3 Diffusion MRI

Diffusion-weighted imaging (DWI) is a sensitive tool that allows quantifying of physiologic alterations in water diffusion, which result from microscopic structural changes.

Diffusion tensor imaging (DTI) is a well-established and commonly employed diffusion MRI technique in clinical and research on neuroimaging studies, which is based on a Gaussian model of diffusion processes [33]. In general, AD is associated with widespread reduced fractional anisotropy (FA) and increased mean diffusivity (MD) in several regions, most prominently in the frontal and temporal lobes, and along the cingulum, corpus callosum, uncinate fasciculus, superior longitudinal fasciculus, and MTL-associated tracts than healthy controls [3437]. Acosta-Cabronero et al. reported increased axial diffusivity and MD in the splenium, which were the earliest abnormalities in AD [38]. FA and radial diffusivity (DR) differences in the corpus callosum, cingulum, and fornix were found to separate individuals with MCI who converted to AD from non-converters [39]. DTI was also found to be a better predictor of AD-specific MTL atrophy when compared to CSF biomarkers [40]. These findings suggested the potential clinical utility of DTI as early biomarkers of AD and its progression. However, an increase in MD and DR and a decrease in FA with advancing age in selective brain regions have been previously reported [41, 42]. Diffusion MRI can be also used in the classifying of various stages of AD. Multimodal classification method, which combined fMRI and DTI, separated more MCI from healthy controls than single approaches [43].

In recent years, tau has emerged as a potential target for therapeutic intervention. Tau plays a critical role in the neurodegenerative process forming neurofibrillary tangles, which is a major hallmark of AD and correlates with clinical disease progression. Wells et al. applied multiparametric MRI, containing high-resolution structure MRI (sMRI), a novel chemical exchange saturation transfer (CEST) MRI, DTI, and ASL, and glucose CEST to measure changes of tau pathology in AD transgenic mouse [44].

Besides DWI MRI, perfusion-weighted imaging (PWI) is another advanced MR technique, which could measure the cerebral hemodynamics at the capillary level. Zimny et al. evaluated the correlation of MTL with both DWI and PWI in AD and MCI patients [45].

3 Positron emission tomography

PET is a specific imaging technique applying in researches of brain function and neurochemistry of small animals, medium-sized animals, and human subjects [4648]. As a particular brain imaging technique, PET imaging has become the subject of intense research for the diagnosis and facilitation of drug development of AD in both animal models and human trials due to its non-invasive and translational characteristic. PET with various radiotracers is considered as a standard non-invasive quantitative imaging technique to measure CBF, glucose metabolism, and β-amyloid and tau deposition.

3.1 FDG-PET

To date, 18F-FDG is one of the best and widely used neuroimaging tracers of PET, which employed for research and clinical assessment of AD [49]. Typical lower FDG metabolism was shown in the precuneus, posterior cingulate, and temporal and parietal cortex with progression to whole brain reductions with increasing disease progress in AD brains [50, 51]. FDG-PET imaging reflects the cerebral glucose metabolism, neuronal injury, which provides indirect evidence on cognitive function and progression that cannot be provided by amyloid PET imaging.

Schraml et al. [52] identified a significant association between hypometabolic convergence index and phenotypes using ADNI data. Some researchers also used 18F-FDG-PET to analyze genetic information with multiple biomarkers to classify AD status, predicting cognitive decline or MCI to AD conversion [5355]. Trzepacz et al. [56] reported multimodal AD neuroimaging study, using MRI, 11C-PiB PET, and 18F-FDG-PET imaging to predict MCI conversion to AD along with APOE genotype. Zhang et al. [57] compared the genetic modality single-nucleotide polymorphism (SNP) with sMRI, 18F-FDG-PET, and CSF biomarkers, which were used to differentiate healthy control, MCI, and AD. They found FDG-PET is the best modality in terms of accuracy.

3.2 Amyloid beta PET

Aβ, the primary constituent of senile plaques, and tau tangles are hypothesized to play a primary role in the pathogenesis of AD, but it is still hard to identify the fundamental mechanisms [5860]. Aβ plaque in brain is one of the pathological hallmarks of AD [61,62]. Accumulation of Aβ peptide in the cerebral cortex is considered one cause of dementia in AD [63]. Numerous studies have involved in vivo PET imaging assessing cortical β-amyloid burden [6466].

Aβ imaging using PET has been recognized as one of the most important methods for the early diagnosis of AD [67]. Numerous candidate compounds have been tested for Aβ imaging, such as 11C-PiB [68], 18F-FDDNP [69], 11C-SB-13 [70], 18F-BAY94-9172 [71], 18F-AV-45 [72], 18F-flutemetamol [73, 74], 11C-AZD2184 [75], and 18F-ADZ4694 [76], 11C-BF227 and 18F-FACT [77].

Several amyloid PET studies examined genotypes, phenotypes, or gene–gene interactions. Ramanan et al. [78] reported the GWAS results with 18F-AV-45 reflecting the cerebral amyloid metabolism in AD for the first time. Swaminathan et al. [79] revealed the association between plasma Aβ from peripheral blood and cortical amyloid deposition on 11C-PiB. Hohman et al. [80] reported the relationship between SNPs involved in amyloid and tau pathophysiology with 18F-AV-45 PET.

Among the PET tracers, 11C-PiB, which has a high affinity for fibrillar Aβ, is a reliable biomarker of underlying AD pathology [68, 81]. It shows cortical uptake well paralleled with AD pathology [82, 83], has recently been approved for use by the Food and Drug Administration (FDA, April 2012) and the European Medicines Agency (January 2013). 18F-GE-067 (flutemetamol) and 18F-BAY94-9172 (florbetaben) have also been approved by the US FDA in the last 2 years [84, 85].

18F-Florbetapir (also known as 18F-AV-45) exhibits high affinity specific binding to amyloid plaques. 18F-AV-45 labels Aβ plaques in sections from patients with pathologically confirmed AD [72].

It was reported in several research groups that 18F-AV-45 PET imaging showed a reliability of both qualitative and quantitative assessments in AD patients, and Aβ+ increased with diagnostic category (healthy control < MCI < AD) [82, 86, 87]. Johnson et al. used 18F-AV-45 PET imaging to evaluate the amyloid deposition in both MCI and AD patients qualitatively and quantitatively, and found that amyloid burden increased with diagnostic category (MCI < AD), age, and APOEε4 carrier status [88]. Payoux et al. reported the equivocal amyloid PET scans using 18F-AV-45 associated with a specific pattern of clinical signs in a large population of non-demented older adults more than 70 years old [89].

More and more researchers consider combination and comparison of multiple PET tracers targeting amyloid plaque imaging together. Bruck et al. compared the prognostic ability of 11C-PiB PET, 18F-FDG-PET, and quantitative hippocampal volumes measured with MR imaging in predicting MCI to AD conversion. They found that the FDG-PET and 11C-PiB PET imaging are better in predicting MCI to AD conversion [90]. Hatashita et al. used 11C-PiB and FDG-PET imaging to identify MCI due to AD, 11C-PiB showed a higher sensitivity of 96.6 %, and FDG-PET added diagnostic value in predicting AD over a short period [91].

Besides, new Aβ imaging agents were radiosynthesized. Yousefi et al. radiosynthesized a new Aβ imaging agent 18F-FIBT, and compared the three different Aβ-targeted radiopharmaceuticals for PET imaging, including 18F-FIBT, 18F-florbetaben, and 11C-PiB [92]. 11C-AZD2184 is another new PET tracer developed for amyloid senile plaque imaging, and the kinetic behavior of 11C-AZD2184 is suitable for quantitative analysis and can be used in clinical examination without input function [75,93, 94].

4 Multimodality imaging: PET/MRI

Several diagnostic techniques, including MRI and PET, are employed for the diagnosis and monitoring of AD [95]. Multimodal imaging could provide more information in the formation and key molecular event of AD than single method. It drives the progression of neuroimaging research due to the recognition of the clinical benefits of multimodal data [96], and the better access to hybrid devices, such as PET/MRI [97].

Maier et al. evaluated the dynamics of 11C-PiB PET, 15O-H2O-PET, and ASL-MRI in transgenic AD mice and concluded that the AD-related decline of rCBF was caused by the cerebral Aβ angiopathy [98]. Edison et al. systematically compared 11C-PiB PET and MRI in AD, MCI patients, and controls. They thought that 11C-PiB PET was adequate for clinical diagnostic purpose, while MRI remained more appropriate for clinical research [99]. Zhou et al. investigated the interactions between multimodal PET/MRI in elder patients with MCI, AD, and healthy controls, and confirmed the invaluable application of amyloid PET and MRI in early diagnosis of AD [100]. Kim et al. reported that Aβ-weighted cortical thickness, which incorporates data from both MRI and amyloid PET imaging, is a consistent and objective imaging biomarker in AD [101].

5 Other imaging modalities

Multiphoton non-linear optical microscope imaging systems using ultrafast lasers have powerful advantages such as label-free detection, deep penetration of thick samples, high sensitivity, subcellular spatial resolution, 3D optical sectioning, chemical specificity, and minimum sample destruction [102, 103]. Coherent anti-Stokes–Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second-harmonic generation (SHG) microscopy are the most widely used biomedical imaging techniques [104106].

 

Quantitative electroencephalographic and neuropsychological investigation of an alternative measure of frontal lobe executive functions: the Figure Trail Making Test

 Paul S. Foster, Valeria Drago, Brad J. Ferguson, Patti Kelly Harrison,David W. Harrison 

Brain Informatis    http://dx.doi.org:/10.1007/s40708-015-0025-z    http://link.springer.com/article/10.1007/s40708-015-0025-z/fulltext.html

The most frequently used measures of executive functioning are either sensitive to left frontal lobe functioning or bilateral frontal functioning. Relatively little is known about right frontal lobe contributions to executive functioning given the paucity of measures sensitive to right frontal functioning. The present investigation reports the development and initial validation of a new measure designed to be sensitive to right frontal lobe functioning, the Figure Trail Making Test (FTMT). The FTMT, the classic Trial Making Test, and the Ruff Figural Fluency Test (RFFT) were administered to 42 right-handed men. The results indicated a significant relationship between the FTMT and both the TMT and the RFFT. Performance on the FTMT was also related to high beta EEG over the right frontal lobe. Thus, the FTMT appears to be an equivalent measure of executive functioning that may be sensitive to right frontal lobe functioning. Applications for use in frontotemporal dementia, Alzheimer’s disease, and other patient populations are discussed.

Keywords – Frontal lobes, Executive functioning, Trail making test, Sequencing, Behavioral speed, Designs, Nonverbal, Neuropsychological assessment, Regulatory control, Effortful control

A recent survey indicated that the vast majority of neuropsychologists frequently assess executive functioning as part of their neuropsychological evaluations [1]. Surveys of neuropsychologists have indicated that the Trail Making Test (TMT), Controlled Oral Word Association Test (COWAT), Wisconsin Card Sorting Test (WCST), and the Stroop Color-Word Test (SCWT) are among the most commonly used instruments [1,2]. Further, the Rabin et al. [1] survey indicated that these same tests are among the most frequently used by neuropsychologists when specifically assessing executive or frontal lobe functioning. The frequent use of the TMT, WCST, and the SCWT, as well as the assumption that they are measures of executive functioning, led Demakis (2003–2004) to conduct a series of meta-analyses to determine the sensitivity of these test to detect frontal lobe dysfunction, particularly lateralized frontal lobe dysfunction. The findings indicated that the SCWT and Part A of the TMT [3], as well as the WCST [4], were all sensitive to frontal lobe dysfunction. However, only the SCWT differentiated between left and right frontal lobe dysfunction, with the worst performance among those with left frontal lobe dysfunction [3].

The finding of the Demakis [4] meta-analysis, that the WCST was not sensitive to lateralized frontal lobe dysfunction, is not surprising given the equivocal findings that have been reported. Whereas performance on the WCST is sensitive to frontal lobe dysfunction [5, 6], demonstration of lateralized frontal dysfunction has been quite problematic. Unilateral left or right dorsolateral frontal dysfunction has been associated with impaired performance on the WCST [6]. Fallgatter and Strik [7] found bilateral frontal lobe activation during performance of the WCST. However, other imaging studies have found right lateralized frontal lobe activation [8] and left lateralized frontal activation [9] in response to performance on the WCST. Further, left frontal lobe alpha power is negatively correlated with performance on the WCST [10]. Finally, patients with left frontal lobe tumors exhibit more impaired performance on the WCST than those with right frontal tumors [11].

Unlike the data for the WCST, more consistent findings have been reported regarding lateralized frontal lobe functioning for the other commonly used measures of executive functioning. For instance, as with the Demakis [3] study, many investigations have found the SCWT to be sensitive to left frontal lobe functioning, although the precise localization within the left frontal lobe has varied. Impaired performance on the SCWT results from left frontal lesions [12] and specifically from lesions localized to the left dorsolateral frontal lobe [13, 14], though bilateral frontal lesions have also yielded impaired performance [13, 14]. Further, studies using neuroimaging to investigate the neural basis of performance on the SCWT have indicated involvement of the left anterior cingulated cortex [15], left lateral prefrontal cortex [16], left inferior precentral sulcus [17], and the left dorsolateral frontal lobe [18].

Wide agreement exists among investigations of the frontal lateralization of verbal or lexical fluency to confrontation. Specifically, patients with left frontal lobe lesions are known to exhibit impaired performance on lexical fluency to confrontation tasks, relative to either patients with right frontal lesions [12, 19, 20] or controls [21]. A recent meta-analysis also indicated that the largest deficits in performance on measures of lexical fluency are associated with left frontal lobe lesions [22]. Troster et al. [23] found that, relative to patients with right pallidotomy, patients with left pallidotomy exhibited more impaired lexical fluency. Several neuroimaging investigations have further supported the role of the left frontal lobe in lexical fluency tasks [15, 2427]. Performance on lexical fluency tasks also varies as a function of lateral frontal lobe asymmetry, as assessed by electroencephalography [28].

The Trail Making Test is certainly among the most widely used tests [1] and perhaps the most widely researched. Various norms exist for the TMT (see [29]), with Tombaugh [30] providing the most recent comprehensive set of normative data. Different methods of analyzing and interpreting the data have also been proposed and used, including error analysis [13, 14, 3133], subtraction scores [13, 14, 34], and ratio scores [13, 14, 35].

Several different language versions of the test have been developed and reported, including Arabic [36], Chinese [37, 38], Greek [39], and Hebrew [40]. Numerous alternative versions of the TMT have been developed to address perceived shortcomings of the original TMT. For instance, the Symbol Trail Making Test [41] was developed to reduce the cultural confounds associated with the use of the Arabic numeral system and English alphabet in the original TMT. The Color Trails Test (CTT; [42]) was also developed to control for cultural confounds, although mixed results have been reported regarding whether the CTT is indeed analogous to the TMT [4345]. A version of the TMT for preschool children, the TRAILS-P, has also been reported [46].

Additionally, the Comprehensive Trail Making Test [47] was developed to control for perceived psychometric shortcomings of the original TMT (for a review see [48] and the Oral Trail Making Test (OTMT; [49]) was developed to reduce confounds associated with motor speed and visual search abilities, with research supporting the OTMT as an equivalent measure [50, 51]. Alternate forms of the TMT have also been developed to permit successive administrations [32, 52] and to assess the relative contributions of the requisite cognitive skills [53].

Delis et al. [54] stated that the continued development of new instrumentation for improving diagnosis and treatment is a critical undertaking in all health-related fields. Further, in their view, the field of neuropsychology has recognized the importance of continually striving to develop new clinical measures. Delis and colleagues developed the extensive Delis-Kaplan Executive Functioning System (D-KEFS; [55]) in the spirit of advancing the instrumentation of neuropsychology. The D-KEFS includes a Trail Making Test consisting of five separate conditions. The Number-Letter Switching condition involves a sequencing procedure similar to that of the classic TMT. The other four conditions are designed to assess the component processes involved in completing the Number-Letter Switching condition so that a precise analysis of the nature of any underlying dysfunction may be accomplished. Specifically, these additional components include Visual Scanning, Number Sequencing, Letter Sequencing, and Motor Speed.

Given that the TMT comprises numbers and letters and is a measure of executive functioning, it may preferentially involve the left frontal lobe. Although the literature is somewhat controversial, neuropsychological and neuroimaging studies seem to provide support for the sensitivity of the TMT to detect left frontal dysfunction [56]. Recent clinically oriented studies investigating frontal lobe involvement of the TMT using transcranial magnetic stimulation (TMS) and near-infrared spectroscopy (NIRS) also support this localization [57]. Performance on Part B of the TMT improved following repetitive TMS applied to the left dorsolateral frontal lobe [57].

With 9–13-year-old boys performing TMT Part B, Weber et al. [58] found a left lateralized increase in the prefrontal cortex in deoxygenated hemoglobin, an indicator of increased oxygen consumption. Moll et al. [59] demonstrated increased activation specific to the prefrontal cortex, especially the left prefrontal region, in healthy controls performing Part B of the TMT. Foster et al. [60] found a significant positive correlation between performance on Part A of the TMT and low beta (13–21 Hz) magnitude (μV) at the left lateral frontal lobe, but not at the right lateral frontal lobe. Finally, Stuss et al. [13, 14] found that patients with left dorsolateral frontal dysfunction evidenced more errors than patients with lesions in other areas of the frontal lobes and those patients with left frontal lesions were the slowest to complete the test.

Taken together, the possibility exists that the aforementioned tests are largely associated with left frontal lobe activity and the TMT, in particular, provides information concerning mental processing speed as well as cognitive flexibility and set-shifting. While some studies have found that deficits in visuomotor set-shifting are specific to the frontal lobe damage [61], others investigators have reported such impairment in patients with posterior brain lesions and widespread cerebral dysfunctions, including cerebellar damage [62] and Alzheimer disease [63]. Thus, it remains unclear whether impairments in visuomotor set-shifting are specific to frontal lobe dysfunction or whether they are non-specific and can result from more posterior or widespread brain dysfunction.

Compared to the collective knowledge we have regarding the cognitive roles of the left frontal lobe, relatively little is known about right frontal lobe contributions to executive functioning. This is likely a result of the dearth of tests that are associated with right frontal activity. The Ruff Figural Fluency Test (RFFT; [64]) is among the few standardized tests of right frontal lobe functioning and was listed as the 14th most commonly used instrument to assess executive functioning in the Rabin et al. [1] survey. The RFFT is known to be sensitive to right frontal lobe functioning [65, 66]; see also [67] pp. 297–298), as is a measure based on the RFFT [19].

The present investigation, with the same intent and spirit as that reported by Delis et al. [54], sought to develop and initially validate a measure of right frontal lobe functioning in an effort to attain a greater understanding of right frontal contributions to executive functioning and to advance the instrumentation of neuropsychology. To meet this objective, a version of the Trail Making Test comprising figures, as opposed to numbers and letters, was developed. The TMT was used as a model for the new test, referred to as the Figure Trail Making Test (FTMT), due to the high frequency of use, the volume of research conducted, and the ease of administration of the TMT. Given that the TMT and the FTMT are both measuring executive functioning, we felt that a moderate correlation would exist between these two measures. Specifically, we hypothesized that performance on the FTMT would be positively correlated with performance on the TMT, in terms of the total time required to complete each part of the tests, an additive and subtractive score, and a ratio score. The total time required to complete each part of the FTMT was also hypothesized to be negatively correlated with the total number of unique designs produced on the RFFT and positively correlated with the number of perseverative errors committed on the RFFT and the perseverative error ratio. We also sought to determine whether the TMT and the FTMT were measuring different constructs by conducting a factor analysis, anticipating that the two tests would load on separate factors.

Additionally, we sought to obtain neurophysiological evidence that the FTMT is sensitive to right frontal lobe functioning. Specifically, we used quantitative electroencephalography (QEEG) to measure electrical activity over the left and right frontal lobes. A previous investigation we conducted found that performance on Part A of the TMT was related to left frontal lobe (F7) low beta magnitude [60]. For the present investigation, we predicted that significant negative correlations would exist between performance on Parts A and B of the TMT and both low and high beta magnitude at the F7 electrode site. We further predicted that significant negative correlations would exist between performance on Parts C and D of the FTMT and both low and high beta magnitude at the F8 electrode site.

3 Discussion

The need for additional measures of executive functions and especially instruments which may provide implications relevant to cerebral laterality is clear. There remains especially a void for neuropsychological instruments using a TMT format, which may provide information pertaining to the functional integrity of the right frontal region. Consistent with the hypotheses forwarded, significant correlations were found between performance on the TMT and the FTMT, in terms of the raw time required to complete each respective part of the tests as well as the additive and subtraction scores. The fact that the ratio scores were not significantly correlated is not surprising given that research has generally indicated a lack of clinical utility for this score [13, 14, 35]. Given the present findings, the TMT and the FTMT appear to be equivalent measures of executive functioning. Further, the present findings not only suggest that the FTMT may be a measure of executive functioning but also extend the realm of executive functioning to the sequencing and set-shifting of nonverbal stimuli.

However, the finding of significant correlations between the TMT and the FTMT represents somewhat of a caveat in that the TMT has been found to be sensitive to left frontal lobe functioning [13, 14, 57, 59]. This would seem to suggest the possibility that the FTMT is also sensitive to left frontal lobe functioning. The possibility that FTMT is related to left frontal lobe functioning is tempered, though, by the fact that the many of the hypothesized correlations between performance on the RFFT and the FTMT were also significant. Performance on the RFFT is related to right frontal lobe functioning [65,66]. Thus, the significant correlations between the RFFT and the FTMT suggest that the FTMT may also be sensitive to right frontal lobe functioning. Additionally, it should also be noted that the TMT was not significantly correlated with performance on the RFFT, with the exception of the significant correlation between performance on the TMT Part A and the total number of unique designs produced on the RFFT. Taken together, the results suggest that the FTMT may be a measure of right frontal executive functioning.

Additional support for the sensitivity of the FTMT to right frontal lobe functioning is provided by the finding of a significant negative correlation between performance on Part D of the FTMT and high beta magnitude. We have previously used QEEG to provide neurophysiological validation of the RFFT [65] and the Rey Auditory Verbal Learning Test [70] and the present findings provide further support for the use of QEEG in validating neuropsychological tests. The lack of significant correlations between the TMT and either low or high beta magnitude may be related to a restricted range of scores on the TMT. As a whole, performance on the FTMT was more variable than performance on the TMT and this relatively restricted range for the TMT may have impacted the obtained correlations. Given the present findings, together with those of the Foster et al. [65, 70] investigations, further support is also provided for the use of EEG in establishing neurophysiological validation for neuropsychological tests.

The results from the factor analysis provide support for the contention that the FMT may be a measure of right frontal lobe activity and also provide initial discriminant validity for the FTMT. Specifically, Parts C and D of the FTMT were found to load on the same factor as the number of designs generated on the RFFT, although the time required to complete Part A of the TMT is also included. Additionally, the number of errors committed on Parts C and D of the FTMT comprises a single factor, separate from either the TMT or the RFFT. Although these results support the FTMT as a measure of nonverbal executive functioning, it would be helpful to conduct an additional factor analysis including additional measures of right frontal functioning, and perhaps other measures of right hemisphere functioning as marker variables.

We sought to develop a measure sensitive to right frontal lobe functioning due to the paucity of such tests and the potentially important uses that right frontal lobe tests may have clinically. Tests of right frontal lobe functioning may, for instance, be useful in identifying and distinguishing left versus right frontotemporal dementia (FTD). Research has indicated that FTD is associated with cerebral atrophy at the right dorsolateral frontal and left premotor cortices [71]. Fukui and Kertesz [72] found right frontal lobe volume reduction in FTD relative to Alzheimer’s disease and progressive nonfluent aphasia. Some have suggested that FTD should not be considered as a unitary disorder and that neuropsychological testing may aid in differentially diagnosing left versus right FTD [73].

Whereas right FTD has been associated with more errors and perseverative responses on the Wisconsin Card Sorting Test (WCST), left FTD has been associated with significantly worse performance on the Boston Naming Test (BNT) and the Stroop Color-Word test [73]. Razani et al. [74] also distinguished between left and right FTD in finding that left FTD performed worse on the BNT and the right FTD patients performed worse on the WCST. However, as noted earlier, the WCST has been associated with left frontal activity [9], right frontal activation [8], and bilateral frontal activation [7]. Further, patients with left frontal tumors perform worse than those with right frontal tumors [11].

Patients with FTD that predominantly involves the right frontotemporal region have behavioral and emotional abnormalities and those with predominantly left frontotemporal region damage have a loss of lexical semantic knowledge. Patients, in whom neural degeneration begins on the left side, often present to the clinicians at an early stage of the disease due to the presence of language abnormalities, but maintain their emotion processing abilities, being preserved the right anterior temporal lobe. However, as this disease advances, the disease may progress to the right frontotemporal regions. Tests sensitive to right frontal lobe functioning may be useful tools to identify in advance the course of the disease, providing immediate and specific treatments and informing the caregivers on the possible prospective frame of the disease.

A potentially more important use of tests sensitive to right frontal lobe functioning, though, may be in predicting dementia patients that will develop significant and disruptive behavioral deficits. Research has found that approximately 92 % of right-sided FTD patients exhibit socially undesirable behaviors as their initial symptom, as compared to only 11 % of left-sided FTD patients [75]. Behavioral deficits in FTD are associated with gray matter loss at the dorsomedial frontal region, particularly on the right [76].

Alzheimer’s disease (AD) is also often associated with significant behavioral disturbances. Even AD patients with mild dementia are noted to exhibit behavioral deficits such as delusions, hallucinations, agitation, dysphoria, anxiety, apathy, and irritability [77]. Indeed, Shimabukuro et al. [77] found that regardless of dementia severity, over half of all AD patients exhibited apathy, delusions, irritability, dysphoria, and anxiety. Delusions in AD patients are associated with relative right frontal hypoperfusion as indicated by SPECT imaging [78, 79]. Further, positron emission tomography (PET) has indicated that AD patients exhibiting delusions exhibit hypometabolism at the right superior dorsolateral frontal and right inferior frontal pole [80].

Although research clearly implicates right frontal lobe dysfunction in the expression of behavioral deficits, data from neuropsychological testing are not as clear. Negative symptoms in patients with AD and FTD have been related to measures of nonverbal and verbal executive functioning as well as verbal memory [81]. Positive symptoms, in contrast, were related to constructional skills and attention. However, Staff et al. [78] failed to dissociate patients with delusions from those without delusions based on neuropsychological test performance, despite significant differences existing in right frontal and limbic functioning as revealed by functional imaging. The inclusion of other measures of right frontal lobe functioning may result in improved neuropsychological differentiation of dementia patients with and without significant behavioral disturbances. Further, it may be possible to predict early in the disease process those patients that will ultimately develop behavioral disturbances with improved measures of right frontal functioning. Predicting those that may develop behavioral problems will permit earlier treatment and will provide the family with more time to prepare for the potential emergence of such difficulties. Certainly, future research needs to be conducted that incorporates measures of right and left frontal lobe functioning in regression analyses to determine the plausibility of such prediction.

Tests sensitive to right frontal lobe functioning may also be useful in identifying more subtle right frontal lobe dysfunction and the cognitive and behavioral changes that follow. The right frontal lobe mediates language melody or prosody and forms a cohesive discourse, interprets abstract communication in spoken and written languages, and interprets the inferred relationships involved in communications. Subtle difficulties in interpreting abstract meaning in communication, comprehending metaphors, and even understanding jokes that are often seen in right frontal lobe stroke patients may not be detected by the family and may also be under diagnosed by clinicians [82]. Further, patients with right frontal lobe lesions are generally more euphoric and unconcerned, often minimizing their symptoms [82] or denying the illness, which may delay referral to a clinician and diagnosis.

Attention deficit hyperactivity disorder (ADHD) is a neurological disease characterized by motor inhibition deficit, problems with cognitive flexibility, social disruption, and emotional disinhibition [83, 84]. Functional MRI studies reveal reduced right prefrontal activation during “frontal tasks,” such as go/no go [85], Stroop [86], and attention task performance [87]. The right frontal lobe deficit hypothesis is further supported by structural studies [88, 89]. Tests of right frontal lobe functioning may be useful in further characterizing the nature of this deficit and in specifying the likely hemispheric locus of dysfunction.

To summarize, we feel that right frontal lobe functioning has been relatively neglected in neuropsychological assessment and that many uses for such tests exist. Our intent was to develop a test purportedly sensitive to right frontal functioning that would be easy and quick to administer in a clinical setting. However, we are certainly not meaning to assert that our FTMT would be applicable in all the aforementioned conditions. Additional research should be conducted to determine the precise clinical utility of the FTMT.

Further validation of the FTMT should also be undertaken. Establishing convergent validation may involve correlating tests measuring the same domain, such as executive functioning. This was initially accomplished in the present investigation through the significant correlations between the TMT and the FTMT. Additionally, convergent validation may also involve correlating tests that purportedly measure the same region of the brain. This was also initially accomplished in the present investigation through the significant correlations between the FTMT and the RFFT. However, additional convergent validation certainly needs to be obtained, as well as validation using patient populations and neurophysiological validation.

We are currently collecting data that hopefully will provide neurophysiological validation of the FTMT. Certainly, though, it is hoped that the present investigation will not only stimulate further research seeking to validate the FTMT and provide more comprehensive normative data, but also stimulate research investigating whether the FTMT or other measures of right frontal lobe functioning may be used to predict patients that will develop behavioral disturbances.

 

World’s Greatest Literature Reveals Multifractals, Cascades of Consciousness

http://www.scientificcomputing.com/news/2016/01/worlds-greatest-literature-reveals-multifractals-cascades-consciousness

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Multifractal analysis of Finnegan’s Wake by James Joyce. The ideal shape of the graph is virtually indistinguishable from the results for purely mathematical multifractals. The horizontal axis represents the degree of singularity, and the vertical axis shows the spectrum of singularity. Courtesy of IFJ PAN

Arthur Conan Doyle, Charles Dickens, James Joyce, William Shakespeare and JRR Tolkien. Regardless of the language they were working in, some of the world’s greatest writers appear to be, in some respects, constructing fractals. Statistical analysis, however, revealed something even more intriguing. The composition of works from within a particular genre was characterized by the exceptional dynamics of a cascading (avalanche) narrative structure. This type of narrative turns out to be multifractal. That is, fractals of fractals are created.

As far as many bookworms are concerned, advanced equations and graphs are the last things which would hold their interest, but there’s no escape from the math. Physicists from the Institute of Nuclear Physics of the Polish Academy of Sciences (IFJ) in Cracow, Poland, performed a detailed statistical analysis of more than one hundred famous works of world literature, written in several languages and representing various literary genres. The books, tested for revealing correlations in variations of sentence length, proved to be governed by the dynamics of a cascade. This means that the construction of these books is, in fact, a fractal. In the case of several works, their mathematical complexity proved to be exceptional, comparable to the structure of complex mathematical objects considered to be multifractal. Interestingly, in the analyzed pool of all the works, one genre turned out to be exceptionally multifractal in nature.

Fractals are self-similar mathematical objects: when we begin to expand one fragment or another, what eventually emerges is a structure that resembles the original object. Typical fractals, especially those widely known as the Sierpinski triangle and the Mandelbrot set, are monofractals, meaning that the pace of enlargement in any place of a fractal is the same, linear: if they at some point were rescaled x number of times to reveal a structure similar to the original, the same increase in another place would also reveal a similar structure.

Multifractals are more highly advanced mathematical structures: fractals of fractals. They arise from fractals ‘interwoven’ with each other in an appropriate manner and in appropriate proportions. Multifractals are not simply the sum of fractals and cannot be divided to return back to their original components, because the way they weave is fractal in nature. The result is that, in order to see a structure similar to the original, different portions of a multifractal need to expand at different rates. A multifractal is, therefore, non-linear in nature.

“Analyses on multiple scales, carried out using fractals, allow us to neatly grasp information on correlations among data at various levels of complexity of tested systems. As a result, they point to the hierarchical organization of phenomena and structures found in nature. So, we can expect natural language, which represents a major evolutionary leap of the natural world, to show such correlations as well. Their existence in literary works, however, had not yet been convincingly documented. Meanwhile, it turned out that, when you look at these works from the proper perspective, these correlations appear to be not only common, but in some works they take on a particularly sophisticated mathematical complexity,” says Professor Stanislaw Drozdz, IFJ PAN, Cracow University of Technology.

The study involved 113 literary works written in English, French, German, Italian, Polish, Russian and Spanish by such famous figures as Honore de Balzac, Arthur Conan Doyle, Julio Cortazar, Charles Dickens, Fyodor Dostoevsky, Alexandre Dumas, Umberto Eco, George Elliot, Victor Hugo, James Joyce, Thomas Mann, Marcel Proust, Wladyslaw Reymont, William Shakespeare, Henryk Sienkiewicz, JRR Tolkien, Leo Tolstoy and Virginia Woolf, among others. The selected works were no less than 5,000 sentences long, in order to ensure statistical reliability.

To convert the texts to numerical sequences, sentence length was measured by the number of words (an alternative method of counting characters in the sentence turned out to have no major impact on the conclusions). The dependences were then searched for in the data — beginning with the simplest, i.e. linear. This is the posited question: if a sentence of a given length is x times longer than the sentences of different lengths, is the same aspect ratio preserved when looking at sentences respectively longer or shorter?

“All of the examined works showed self-similarity in terms of organization of the lengths of sentences. Some were more expressive — here The Ambassadors by Henry James stood out — while others to far less of an extreme, as in the case of the French seventeenth-century romance Artamene ou le Grand Cyrus. However, correlations were evident and, therefore, these texts were the construction of a fractal,” comments Dr. Pawel Oswiecimka (IFJ PAN), who also noted that fractality of a literary text will, in practice, never be as perfect as in the world of mathematics. It is possible to magnify mathematical fractals up to infinity, while the number of sentences in each book is finite and, at a certain stage of scaling, there will always be a cut-off in the form of the end of the dataset.

Things took a particularly interesting turn when physicists from IFJ PAN began tracking non-linear dependence, which in most of the studied works was present to a slight or moderate degree. However, more than a dozen works revealed a very clear multifractal structure, and almost all of these proved to be representative of one genre, that of stream of consciousness. The only exception was the Bible, specifically the Old Testament, which has, so far, never been associated with this literary genre.

“The absolute record in terms of multifractality turned out to be Finnegan’s Wakeby James Joyce. The results of our analysis of this text are virtually indistinguishable from ideal, purely mathematical multifractals,” says Drozdz.

The most multifractal works also included A Heartbreaking Work of Staggering Genius by Dave Eggers, Rayuela by Julio Cortazar, The US Trilogy by John Dos Passos, The Waves by Virginia Woolf, 2666 by Roberto Bolano, and Joyce’sUlysses. At the same time, a lot of works usually regarded as stream of consciousness turned out to show little correlation to multifractality, as it was hardly noticeable in books such as Atlas Shrugged by Ayn Rand and A la recherche du temps perdu by Marcel Proust.

“It is not entirely clear whether stream of consciousness writing actually reveals the deeper qualities of our consciousness, or rather the imagination of the writers. It is hardly surprising that ascribing a work to a particular genre is, for whatever reason, sometimes subjective. We see, moreover, the possibility of an interesting application of our methodology: it may someday help in a more objective assignment of books to one genre or another,” notes Drozdz.

Multifractal analyses of literary texts carried out by the IFJ PAN have been published in Information Sciences, the journal of computer science. The publication has undergone rigorous verification: given the interdisciplinary nature of the subject, editors immediately appointed up to six reviewers.

Citation: “Quantifying origin and character of long-range correlations in narrative texts” S. Drożdż, P. Oświęcimka, A. Kulig, J. Kwapień, K. Bazarnik, I. Grabska-Gradzińska, J. Rybicki, M. Stanuszek; Information Sciences, vol. 331, 32–44, 20 February 2016; DOI: 10.1016/j.ins.2015.10.023

 

New Quantum Approach to Big Data could make Impossibly Complex Problems Solvable

David L. Chandler, MIT

http://www.scientificcomputing.com/news/2016/01/new-quantum-approach-big-data-could-make-impossibly-complex-problems-solvable

 

http://www.scientificcomputing.com/sites/scientificcomputing.com/files/New_Quantum_Approach_to_Big_Data_could_make_Impossibly_Complex_Problems_Solvable_440.jpg

This diagram demonstrates the simplified results that can be obtained by using quantum analysis on enormous, complex sets of data. Shown here are the connections between different regions of the brain in a control subject (left) and a subject under the influence of the psychedelic compound psilocybin (right). This demonstrates a dramatic increase in connectivity, which explains some of the drug’s effects (such as “hearing” colors or “seeing” smells). Such an analysis, involving billions of brain cells, would be too complex for conventional techniques, but could be handled easily by the new quantum approach, the researchers say. Courtesy of the researchers

From gene mapping to space exploration, humanity continues to generate ever-larger sets of data — far more information than people can actually process, manage or understand.

Machine learning systems can help researchers deal with this ever-growing flood of information. Some of the most powerful of these analytical tools are based on a strange branch of geometry called topology, which deals with properties that stay the same even when something is bent and stretched every which way.

Such topological systems are especially useful for analyzing the connections in complex networks, such as the internal wiring of the brain, the U.S. power grid, or the global interconnections of the Internet. But even with the most powerful modern supercomputers, such problems remain daunting and impractical to solve. Now, a new approach that would use quantum computers to streamline these problems has been developed by researchers at MIT, the University of Waterloo, and the University of Southern California.

The team describes their theoretical proposal this week in the journal Nature Communications. Seth Lloyd, the paper’s lead author and the Nam P. Suh Professor of Mechanical Engineering, explains that algebraic topology is key to the new method. This approach, he says, helps to reduce the impact of the inevitable distortions that arise every time someone collects data about the real world.

In a topological description, basic features of the data (How many holes does it have? How are the different parts connected?) are considered the same no matter how much they are stretched, compressed, or distorted. Lloyd explains that it is often these fundamental topological attributes “that are important in trying to reconstruct the underlying patterns in the real world that the data are supposed to represent.”

It doesn’t matter what kind of dataset is being analyzed, he says. The topological approach to looking for connections and holes “works whether it’s an actual physical hole, or the data represents a logical argument and there’s a hole in the argument. This will find both kinds of holes.”

Using conventional computers, that approach is too demanding for all but the simplest situations. Topological analysis “represents a crucial way of getting at the significant features of the data, but it’s computationally very expensive,” Lloyd says. “This is where quantum mechanics kicks in.” The new quantum-based approach, he says, could exponentially speed up such calculations.

Lloyd offers an example to illustrate that potential speedup: If you have a dataset with 300 points, a conventional approach to analyzing all the topological features in that system would require “a computer the size of the universe,” he says. That is, it would take 2300 (two to the 300th power) processing units — approximately the number of all the particles in the universe. In other words, the problem is simply not solvable in that way.

“That’s where our algorithm kicks in,” he says. Solving the same problem with the new system, using a quantum computer, would require just 300 quantum bits — and a device this size may be achieved in the next few years, according to Lloyd.

“Our algorithm shows that you don’t need a big quantum computer to kick some serious topological butt,” he says.

There are many important kinds of huge datasets where the quantum-topological approach could be useful, Lloyd says, for example understanding interconnections in the brain. “By applying topological analysis to datasets gleaned by electroencephalography or functional MRI, you can reveal the complex connectivity and topology of the sequences of firing neurons that underlie our thought processes,” he says.

The same approach could be used for analyzing many other kinds of information. “You could apply it to the world’s economy, or to social networks, or almost any system that involves long-range transport of goods or information,” Lloyd says. But the limits of classical computation have prevented such approaches from being applied before.

While this work is theoretical, “experimentalists have already contacted us about trying prototypes,” he says. “You could find the topology of simple structures on a very simple quantum computer. People are trying proof-of-concept experiments.”

Ignacio Cirac, a professor at the Max Planck Institute of Quantum Optics in Munich, Germany, who was not involved in this research, calls it “a very original idea, and I think that it has a great potential.” He adds “I guess that it has to be further developed and adapted to particular problems. In any case, I think that this is top-quality research.”

The team also included Silvano Garnerone of the University of Waterloo in Ontario, Canada, and Paolo Zanardi of the Center for Quantum Information Science and Technology at the University of Southern California. The work was supported by the Army Research Office, Air Force Office of Scientific Research, Defense Advanced Research Projects Agency, Multidisciplinary University Research Initiative of the Office of Naval Research, and the National Science Foundation.

 

Beyond Chess: Computer Beats Human in Ancient Chinese Game

http://www.rdmag.com/news/2016/01/beyond-chess-computer-beats-human-ancient-chinese-game

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A player places a black stone while his opponent waits to place a white one as they play Go, a game of strategy, in the Seattle Go Center, Tuesday, April 30, 2002. The game, which originated in China more than 2,500 years ago, involves two players who take turns putting markers on a grid. The object is to surround more area on the board with the markers than one’s opponent, as well as capturing the opponent’s pieces by surrounding them. A paper released Wednesday, Jan. 27, 2016 describes how a computer program has beaten a human master at the complex board game, marking significant advance for development of artificial intelligence. (AP Photo/Cheryl Hatch)

 

A computer program has beaten a human champion at the ancient Chinese board game Go, marking a significant advance for development of artificial intelligence.

The program had taught itself how to win, and its developers say its learning strategy may someday let computers help solve real-world problems like making medical diagnoses and pursuing scientific research.

The program and its victory are described in a paper released Wednesday by the journal Nature.

Computers previously have surpassed humans for other games, including chess, checkers and backgammon. But among classic games, Go has long been viewed as the most challenging for artificial intelligence to master.

Go, which originated in China more than 2,500 years ago, involves two players who take turns putting markers on a checkerboard-like grid. The object is to surround more area on the board with the markers than one’s opponent, as well as capturing the opponent’s pieces by surrounding them.

While the rules are simple, playing it well is not. It’s “probably the most complex game ever devised by humans,” Dennis Hassabis of Google DeepMind in London, one of the study authors, told reporters Tuesday.

The new program, AlphaGo, defeated the European champion in all five games of a match in October, the Nature paper reports.

In March, AlphaGo will face legendary player Lee Sedol in Seoul, South Korea, for a $1 million prize, Hassabis said.

Martin Mueller, a computing science professor at the University of Alberta in Canada who has worked on Go programs for 30 years but didn’t participate in AlphaGo, said the new program “is really a big step up from everything else we’ve seen…. It’s a very, very impressive piece of work.”

 

 

Biological Origin of Schizophrenia

Excessive ‘pruning’ of connections between neurons in brain predisposes to disease

http://hms.harvard.edu/sites/default/files/uploads/news/McCarroll_C4_600x400.jpg

Imaging studies showed C4 (in green) located at the synapses of primary human neurons. Image: Heather de Rivera, McCarroll lab

 PAUL GOLDSMITH    http://hms.harvard.edu/news/biological-origin-schizophrenia

The risk of schizophrenia increases if a person inherits specific variants in a gene related to “synaptic pruning”—the elimination of connections between neurons—according to a study from Harvard Medical School, the Broad Institute and Boston Children’s Hospital. The findings were based on genetic analysis of nearly 65,000 people.

The study represents the first time that the origin of this psychiatric disease has been causally linked to specific gene variants and a biological process.

Get more HMS news here

It also helps explain two decades-old observations: synaptic pruning is particularly active during adolescence, which is the typical period of onset for symptoms of schizophrenia, and the brains of schizophrenic patients tend to show fewer connections between neurons.

The gene, complement component 4 (C4), plays a well-known role in the immune system. It has now been shown to also play a key role in brain development and schizophrenia risk. The insight may allow future therapeutic strategies to be directed at the disorder’s roots, rather than just its symptoms.

The study, which appears online Jan. 27 in Nature, was led by HMS researchers at the Broad Institute’s Stanley Center for Psychiatric Research and Boston Children’s. They include senior author Steven McCarroll, HMS associate professor of genetics and director of genetics for the Stanley Center; Beth Stevens, HMS assistant professor of neurology at Boston Children’s and institute member at the Broad; Michael Carroll, HMS professor of pediatrics at Boston Children’s; and first author Aswin Sekar, an MD-PhD student at HMS.

The study has the potential to reinvigorate translational research on a debilitating disease. Schizophrenia afflicts approximately 1 percent people worldwide and is characterized by hallucinations, emotional withdrawal and a decline in cognitive function. These symptoms most frequently begin in patients when they are teenagers or young adults.

“These results show that it is possible to go from genetic data to a new way of thinking about how a disease develops—something that has been greatly needed.”

First described more than 130 years ago, schizophrenia lacks highly effective treatments and has seen few biological or medical breakthroughs over the past half-century.

In the summer of 2014, an international consortium led by researchers at the Stanley Center identified more than 100 regions in the human genome that carry risk factors for schizophrenia.

The newly published study now reports the discovery of the specific gene underlying the strongest of these risk factors and links it to a specific biological process in the brain.

“Since schizophrenia was first described over a century ago, its underlying biology has been a black box, in part because it has been virtually impossible to model the disorder in cells or animals,” said McCarroll. “The human genome is providing a powerful new way in to this disease. Understanding these genetic effects on risk is a way of prying open that black box, peering inside and starting to see actual biological mechanisms.”

“This study marks a crucial turning point in the fight against mental illness,” said Bruce Cuthbert, acting director of the National Institute of Mental Health. “Because the molecular origins of psychiatric diseases are little-understood, efforts by pharmaceutical companies to pursue new therapeutics are few and far between. This study changes the game. Thanks to this genetic breakthrough we can finally see the potential for clinical tests, early detection, new treatments and even prevention.”

The path to discovery

The discovery involved the collection of DNA from more than 100,000 people, detailed analysis of complex genetic variation in more than 65,000 human genomes, development of an innovative analytical strategy, examination of postmortem brain samples from hundreds of people and the use of animal models to show that a protein from the immune system also plays a previously unsuspected role in the brain.

Over the past five years, Stanley Center geneticists and collaborators around the world collected more than 100,000 human DNA samples from 30 different countries to locate regions of the human genome harboring genetic variants that increase the risk of schizophrenia. The strongest signal by far was on chromosome 6, in a region of DNA long associated with infectious disease. This caused some observers to suggest that schizophrenia might be triggered by an infectious agent. But researchers had no idea which of the hundreds of genes in the region was actually responsible or how it acted.

Based on analyses of the genetic data, McCarroll and Sekar focused on a region containing the C4 gene. Unlike most genes, C4 has a high degree of structural variability. Different people have different numbers of copies and different types of the gene.

McCarroll and Sekar developed a new molecular technique to characterize the C4 gene structure in human DNA samples. They also measured C4 gene activity in nearly 700 post-mortem brain samples.

They found that the C4 gene structure (DNA) could predict the C4 gene activity (RNA) in each person’s brain. They then used this information to infer C4 gene activity from genome data from 65,000 people with and without schizophrenia.

These data revealed a striking correlation. People who had particular structural forms of the C4 gene showed higher expression of that gene and, in turn, had a higher risk of developing schizophrenia.

Connecting cause and effect through neuroscience

But how exactly does C4—a protein known to mark infectious microbes for destruction by immune cells—affect the risk of schizophrenia?

Answering this question required synthesizing genetics and neurobiology.

Stevens, a recent recipient of a MacArthur Foundation “genius grant,” had found that other complement proteins in the immune system also played a role in brain development. These results came from studying an experimental model of synaptic pruning in the mouse visual system.

“This discovery enriches our understanding of the complement system in brain development and in disease, and we could not have made that leap without the genetics.”

Carroll had long studied C4 for its role in immune disease, and developed mice with different numbers of copies of C4.

The three labs set out to study the role of C4 in the brain.

They found that C4 played a key role in pruning synapses during maturation of the brain. In particular, they found that C4 was necessary for another protein—a complement component called C3—to be deposited onto synapses as a signal that the synapses should be pruned. The data also suggested that the more C4 activity an animal had, the more synapses were eliminated in its brain at a key time in development.

The findings may help explain the longstanding mystery of why the brains of people with schizophrenia tend to have a thinner cerebral cortex (the brain’s outer layer, responsible for many aspects of cognition) with fewer synapses than do brains of unaffected individuals. The work may also help explain why the onset of schizophrenia symptoms tends to occur in late adolescence.

The human brain normally undergoes widespread synapse pruning during adolescence, especially in the cerebral cortex. Excessive synaptic pruning during adolescence and early adulthood, due to increased complement (C4) activity, could lead to the cognitive symptoms seen in schizophrenia.

“Once we had the genetic findings in front of us we started thinking about the possibility that complement molecules are excessively tagging synapses in the developing brain,” Stevens said.

“This discovery enriches our understanding of the complement system in brain development and in disease, and we could not have made that leap without the genetics,” she said. “We’re far from having a treatment based on this, but it’s exciting to think that one day we might be able to turn down the pruning process in some individuals and decrease their risk.”

Opening a path toward early detection and potential therapies

Beyond providing the first insights into the biological origins of schizophrenia, the work raises the possibility that therapies might someday be developed that could turn down the level of synaptic pruning in people who show early symptoms of schizophrenia.

This would be a dramatically different approach from current medical therapies, which address only a specific symptom of schizophrenia—psychosis—rather than the disorder’s root causes, and which do not stop cognitive decline or other symptoms of the illness.

The researchers emphasize that therapies based on these findings are still years down the road. Still, the fact that much is already known about the role of complement proteins in the immune system means that researchers can tap into a wealth of existing knowledge to identify possible therapeutic approaches. For example, anticomplement drugs are already under development for treating other diseases.

“In this area of science, our dream has been to find disease mechanisms that lead to new kinds of treatments,” said McCarroll. “These results show that it is possible to go from genetic data to a new way of thinking about how a disease develops—something that has been greatly needed.”

This work was supported by the Broad Institute’s Stanley Center for Psychiatric Research and by the National Institutes of Health (grants U01MH105641, R01MH077139 and T32GM007753).

Adapted from a Broad Institute news release.

 

Scientists open the ‘black box’ of schizophrenia with dramatic genetic discovery

Amy Ellis Nutt    https://www.washingtonpost.com/news/speaking-of-science/wp/2016/01/27/scientists-open-the-black-box-of-schizophrenia-with-dramatic-genetic-finding/

 

Scientists Prune Away Schizophrenia’s Hidden Genetic Mechanisms

http://www.genengnews.com/gen-news-highlights/scientists-prune-away-schizophrenia-s-hidden-genetic-mechanisms/81252297/

https://youtu.be/s0y4equOTLg

A landmark study has revealed that a person’s risk of schizophrenia is increased if they inherit specific variants in a gene related to “synaptic pruning”—the elimination of connections between neurons. The findings represent the first time that the origin of this devastating psychiatric disease has been causally linked to specific gene variants and a biological process.

http://www.genengnews.com/Media/images/GENHighlight/thumb_107629_web2209513618.jpg

The site in Chromosome 6 harboring the gene C4 towers far above other risk-associated areas on schizophrenia’s genomic “skyline,” marking its strongest known genetic influence. The new study is the first to explain how specific gene versions work biologically to confer schizophrenia risk. [Psychiatric Genomics Consortium]

  • A new study by researchers at the Broad Institute’s Stanley Center for Psychiatric Research, Harvard Medical School, and Boston Children’s Hospital genetically analyzed nearly 65,000 people and revealed that an individual’s risk of schizophrenia is increased if they inherited distinct variants in a gene related to “synaptic pruning”—the elimination of connections between neurons. This new data represents the first time that the origin of this psychiatric disease has been causally linked to particular gene variants and a biological process.

The investigators discovered that versions of a gene commonly thought to be involved in immune function might trigger a runaway pruning of an adolescent brain’s still-maturing communications infrastructure. The researchers described a scenario where patients with schizophrenia show fewer such connections between neurons or synapses.

“Normally, pruning gets rid of excess connections we no longer need, streamlining our brain for optimal performance, but too much pruning can impair mental function,” explained Thomas Lehner, Ph.D., director of the Office of Genomics Research Coordination at the NIH’s National Institute of Mental Health (NIMH), which co-funded the study along with the Stanley Center for Psychiatric Research at the Broad Institute and other NIH components. “It could help explain schizophrenia’s delayed age-of-onset of symptoms in late adolescence and early adulthood and shrinkage of the brain’s working tissue. Interventions that put the brakes on this pruning process-gone-awry could prove transformative.”

The gene the research team called into question, dubbed C4 (complement component 4), was associated with the largest risk for the disorder. C4’s role represents some of the most compelling evidence, to date, linking specific gene versions to a biological process that could cause at least some cases of the illness.

The findings from this study were published recently in Nature through an article entitled “Schizophrenia risk from complex variation of complement component 4.”

“Since schizophrenia was first described over a century ago, its underlying biology has been a black box, in part because it has been virtually impossible to model the disorder in cells or animals,” noted senior study author Steven McCarroll, Ph.D., director of genetics for the Stanley Center and an associate professor of genetics at Harvard Medical School. “The human genome is providing a powerful new way into this disease. Understanding these genetic effects on risk is a way of prying open that block box, peering inside and starting to see actual biological mechanisms.”

Dr. McCarroll and his colleagues found that a stretch of chromosome 6 encompassing several genes known to be involved in immune function emerged as the strongest signal associated with schizophrenia risk in genome-wide analyses. Yet conventional genetics failed to turn up any specific gene versions there that were linked to schizophrenia.

In order to uncover how the immune-related site confers risk for the mental disorder, the scientists mounted a search for cryptic genetic influences that might generate unconventional signals. C4, a gene with known roles in immunity, emerged as a prime suspect because it is unusually variable across individuals.

Upon further investigation into the complexities of how such structural variation relates to the gene’s level of expression and how that, in turn, might link to schizophrenia, the team discovered structurally distinct versions that affect expression of two main forms of the gene within the brain. The more a version resulted in expression of one of the forms, called C4A, the more it was associated with schizophrenia. The greater number of copies an individual had of the suspect versions, the more C4 switched on and the higher their risk of developing schizophrenia. Furthermore, the C4 protein turned out to be most prevalent within the cellular machinery that supports connections between neurons.

“Once we had the genetic findings in front of us we started thinking about the possibility that complement molecules are excessively tagging synapses in the developing brain,” remarked co-author Beth Stevens, Ph.D. a neuroscientist and assistant professor of neurology at Boston Children’s Hospital and institute member at the Broad. “This discovery enriches our understanding of the complement system in brain development and disease, and we could not have made that leap without the genetics. We’re far from having a treatment based on this, but it’s exciting to think that one day we might be able to turn down the pruning process in some individuals and decrease their risk.”

“This study marks a crucial turning point in the fight against mental illness. It changes the game,” added acting NIMH director Bruce Cuthbert, Ph.D. “Because the molecular origins of psychiatric diseases are little-understood, efforts by pharmaceutical companies to pursue new therapeutics are few and far between. This study changes the game. Thanks to this genetic breakthrough, we can finally see the potential for clinical tests, early detection, new treatments, and even prevention.”

 

Connecting cause and effect through neuroscience

But how exactly does C4—a protein known to mark infectious microbes for destruction by immune cells—affect the risk of schizophrenia?

Answering this question required synthesizing genetics and neurobiology.

Stevens, a recent recipient of a MacArthur Foundation “genius grant,” had found that other complement proteins in the immune system also played a role in brain development. These results came from studying an experimental model of synaptic pruning in the mouse visual system.

“This discovery enriches our understanding of the complement system in brain development and in disease, and we could not have made that leap without the genetics.”

Carroll had long studied C4 for its role in immune disease, and developed mice with different numbers of copies of C4.

The three labs set out to study the role of C4 in the brain.

They found that C4 played a key role in pruning synapses during maturation of the brain. In particular, they found that C4 was necessary for another protein—a complement component called C3—to be deposited onto synapses as a signal that the synapses should be pruned. The data also suggested that the more C4 activity an animal had, the more synapses were eliminated in its brain at a key time in development.

The findings may help explain the longstanding mystery of why the brains of people with schizophrenia tend to have a thinner cerebral cortex (the brain’s outer layer, responsible for many aspects of cognition) with fewer synapses than do brains of unaffected individuals. The work may also help explain why the onset of schizophrenia symptoms tends to occur in late adolescence.

The human brain normally undergoes widespread synapse pruning during adolescence, especially in the cerebral cortex. Excessive synaptic pruning during adolescence and early adulthood, due to increased complement (C4) activity, could lead to the cognitive symptoms seen in schizophrenia.

“Once we had the genetic findings in front of us we started thinking about the possibility that complement molecules are excessively tagging synapses in the developing brain,” Stevens said.

“This discovery enriches our understanding of the complement system in brain development and in disease, and we could not have made that leap without the genetics,” she said. “We’re far from having a treatment based on this, but it’s exciting to think that one day we might be able to turn down the pruning process in some individuals and decrease their risk.”

Opening a path toward early detection and potential therapies

Beyond providing the first insights into the biological origins of schizophrenia, the work raises the possibility that therapies might someday be developed that could turn down the level of synaptic pruning in people who show early symptoms of schizophrenia.

This would be a dramatically different approach from current medical therapies, which address only a specific symptom of schizophrenia—psychosis—rather than the disorder’s root causes, and which do not stop cognitive decline or other symptoms of the illness.

The researchers emphasize that therapies based on these findings are still years down the road. Still, the fact that much is already known about the role of complement proteins in the immune system means that researchers can tap into a wealth of existing knowledge to identify possible therapeutic approaches. For example, anticomplement drugs are already under development for treating other diseases.

“In this area of science, our dream has been to find disease mechanisms that lead to new kinds of treatments,” said McCarroll. “These results show that it is possible to go from genetic data to a new way of thinking about how a disease develops—something that has been greatly needed.”

This work was supported by the Broad Institute’s Stanley Center for Psychiatric Research and by the National Institutes of Health (grants U01MH105641, R01MH077139 and T32GM007753).

Adapted from a Broad Institute news release.

 

 

https://img.washingtonpost.com/wp-apps/imrs.php?src=https://img.washingtonpost.com/rf/image_908w/2010-2019/WashingtonPost/2011/09/27/Production/Sunday/SunBiz/Images/mental2b.jpg&w=1484

This post has been updated.

For the first time, scientists have pinned down a molecular process in the brain that helps to trigger schizophrenia. The researchers involved in the landmark study, which was published Wednesday in the journal Nature, say the discovery of this new genetic pathway probably reveals what goes wrong neurologically in a young person diagnosed with the devastating disorder.

The study marks a watershed moment, with the potential for early detection and new treatments that were unthinkable just a year ago, according to Steven Hyman, director of the Stanley Center for Psychiatric Research at the Broad Institute at MIT. Hyman, a former director of the National Institute of Mental Health, calls it “the most significant mechanistic study about schizophrenia ever.”

“I’m a crusty, old, curmudgeonly skeptic,” he said. “But I’m almost giddy about these findings.”

The researchers, chiefly from the Broad Institute, Harvard Medical School and Boston Children’s Hospital, found that a person’s risk of schizophrenia is dramatically increased if they inherit variants of a gene important to “synaptic pruning” — the healthy reduction during adolescence of brain cell connections that are no longer needed.

[Schizophrenic patients have different oral bacteria than non-mentally ill individuals]

In patients with schizophrenia, a variation in a single position in the DNA sequence marks too many synapses for removal and that pruning goes out of control. The result is an abnormal loss of gray matter.

The genes involved coat the neurons with “eat-me signals,” said study co-author Beth Stevens, a neuroscientist at Children’s Hospital and Broad. “They are tagging too many synapses. And they’re gobbled up.

The Institute’s founding director, Eric Lander, believes the research represents an astonishing breakthrough. “It’s taking what has been a black box…and letting us peek inside for the first time. And that is amazingly consequential,” he said.

The timeline for this discovery has been relatively fast. In July 2014, Broad researchers published the results of the largest genomic study on the disorder and found more than 100 genetic locations linked to schizophrenia. Based on that research, Harvard and Broad geneticist Steven McCarroll analyzed data from about 29,000 schizophrenia cases, 36,000 controls and 700 post mortem brains. The information was drawn from dozens of studies performed in 22 countries, all of which contribute to the worldwide database called the Psychiatric Genomics Consortium.

[Influential government-appointed panel recommends depression screening for everyone]

One area in particular, when graphed, showed the strongest association. It was dubbed the “Manhattan plot” for its resemblance to New York City’s towering buildings. The highest peak was on chromosome 6, where McCarroll’s team discovered the gene variant. C4 was “a dark corner of the human genome,” he said, an area difficult to decipher because of its “astonishing level” of diversity.

C4 and numerous other genes reside in a region of chromosome 6 involved in the immune system, which clears out pathogens and similar cellular debris from the brain. The study’s researchers found that one of C4’s variants, C4A, was most associated with a risk for schizophrenia.

More than 25 million people around the globe are affected by schizophrenia, according to the World Health Organization, including 2 million to 3 million Americans. Highly hereditable, it is one of the most severe mental illnesses, with an annual economic burden in this country of tens of billions of dollars.

“This paper is really exciting,” said Jacqueline Feldman, associate medical director of the National Alliance on Mental Illness. “We as scientists and physicians have to temper our enthusiasm because we’ve gone down this path before. But this is profoundly interesting.”

There have been hundreds of theories about schizophrenia over the years, but one of the enduring mysteries has been how three prominent findings related to each other: the apparent involvement of immune molecules, the disorder’s typical onset in late adolescence and early adulthood, and the thinning of gray matter seen in autopsies of patients.

[A low-tech way to help treat young schizophrenic patients]

“The thing about this result,” said McCarroll, the lead author, ” it makes a lot of other things understandable. To have a result to connect to these observations and to have a molecule and strong level of genetic evidence from tens of thousands of research participants, I think that combination sets [this study] apart.”

The authors stressed that their findings, which combine basic science with large-scale analysis of genetic studies, depended on an unusual level of cooperation among experts in genetics, molecular biology, developmental neurobiology and immunology.

“This could not have been done five years ago,” said Hyman. “This required the ability to reference a very large dataset . …When I was [NIMH] director, people really resisted collaborating. They were still in the Pharaoh era. They wanted to be buried with their data.”

The study offers a new approach to schizophrenia research, which has been largely stagnant for decades.  Most psychiatric drugs seek to interrupt psychotic thinking, but experts agree that psychosis is just a single symptom — and a late-occurring one at that. One of the chief difficulties for psychiatric researchers, setting them apart from most other medical investigators, is that they can’t cut schizophrenia out of the brain and look at it under a microscope. Nor are there any good animal models.

All that now has changed, according to Stevens. “We now have a strong molecular handle, a pathway and a gene, to develop better models,” he said.

Which isn’t to say a cure is right around the corner.

“This is the first exciting  clue, maybe even the most important we’ll ever have, but it will be decades” before a true cure is found,” Hyman said. “Hope is a wonderful thing. False promise is not.”

Insight Pharma Report

Three neurodegenerative disorders that are heavily focused on in this report include: Alzheimer’s Disease/Mild Cognitive Impairment, Parkinson’s Disease, and Amyotrophic Lateral Sclerosis. Part II of the report will include all three of these disorders, highlighting specifics including background, history, and development of the disease. Deeper into the chapters, the report will unfold biomarkers under investigation, genetic targets, and an analysis of multiple studies investigating these elements.

Experts interviewed in these chapters include:

  • Dr. Jens Wendland, Head of Neuroscience Genetics, Precision Medicine, Clinical Research, Pfizer Worldwide R&D
  • Dr. Howard J. Federoff, Executive Vice President for Health Sciences, Georgetown University
  • Dr. Andrew West, Associate Professor of Neurology and Neurobiology and Co-Director, Center for Neurodegeneration and Experimental Therapeutics
  • Dr. Merit Ester Cudkowicz, Chief of Neurology at Massachusetts General Hospital

Part III of the report makes a shift from neurobiomarkers to neurodiagnostics. This section highlights several diagnostics in play and in the making from a number of companies, identifying company strategies, research underway, hypotheses, and institution goals. Elite researchers and companies highlighted in this part include:

  • Dr. Xuemei Huang, Professor and Vice Chair, Department of Neurology; Professor of Neurosurgery, Radiology,  Pharmacology, and Kinesiology Director; Hershey Brain Analysis Research Laboratory for Neurodegenerative Disorders, Penn State University-Milton, S. Hershey Medical Center Department of Neurology
  • Dr. Andreas Jeromin, CSO and President of Atlantic Biomarkers
  • Julien Bradley, Senior Director, Sales & Marketing, Quanterix
  • Dr. Scott Marshall, Head of Bioanalytics, and Dr. Jared Kohler, Head of Biomarker Statistics, BioStat Solutions, Inc.

Further analysis appears in Part IV. This section includes a survey exclusively conducted for this report. With over 30 figures and graphics and an in depth analysis, this part features insight into targets under investigation, challenges, advantages, and desired features of future diagnostic applications. Furthermore, the survey covers more than just the featured neurodegenerative disorders in this report, expanding to Multiple Sclerosis and Huntington’s Disease.

Finally, Insight Pharma Reports concludes this report with clinical trial and pipeline data featuring targets and products from over 300 companies working in Alzheimer’s Disease, Parkinson’s Disease and Amyotrophic Lateral Sclerosis.

 

Epigenome Tapped to Understand Rise of Subtype of Brain Medulloblastoma

http://www.genengnews.com/gen-news-highlights/epigenome-tapped-to-understand-rise-of-subtype-of-brain-medulloblastoma/81252294/

Scientists have identified the cells that likely give rise to the brain tumor subtype Group 4 medulloblastoma. [V. Yakobchuk/ Fotolia]

http://www.genengnews.com/Media/images/GENHighlight/thumb_Jan_28_2016_Fotolia_6761569_ColorfulBrain_4412824411.jpg

An international team of scientists say they have identified the cells that likely give rise to the brain tumor subtype Group 4 medulloblastoma. The believe their study (“Active medulloblastoma enhancers reveal subgroup-specific cellular origins”), published in Nature, removes a barrier to developing more effective targeted therapies against the brain tumor’s most common subtype.

Medulloblastoma occurs in infants, children, and adults, but it is the most common malignant pediatric brain tumor. The disease includes four biologically and clinically distinct subtypes, of which Group 4 is the most common. In children, about half of medulloblastoma patients are of the Group 4 subtype. Efforts to improve patient outcomes, particularly for those with high-risk Group 4 medulloblastoma, have been hampered by the lack of accurate animal models.

Evidence from this study suggests Group 4 tumors begin in neural stem cells that are born in a region of the developing cerebellum called the upper rhomic lip (uRL), according to the researchers.

“Pinpointing the cell(s) of origin for Group 4 medulloblastoma will help us to better understand normal cerebellar development and dramatically improve our chances of developing genetically faithful preclinical mouse models. These models are desperately needed for learning more about Group 4 medulloblastoma biology and evaluating rational, molecularly targeted therapies to improve patient outcomes,” said Paul Northcott, Ph.D., an assistant member of the St. Jude department of developmental neurobiology. Dr. Northcott, Stefan Pfister, M.D., of the German Cancer Research Center (DKFZ), and James Bradner, M.D., of Dana-Farber Cancer Institute, are the corresponding authors.

The discovery and other findings about the missteps fueling tumor growth came from studying the epigenome. Researchers used the analytic tool ChiP-seq to identify and track medulloblastoma subtype differences based on the activity of epigenetic regulators, which included proteins known as master regulator transcription factors. They bind to DNA enhancers and super-enhancers. The master regulator transcription factors and super-enhancers work together to regulate the expression of critical genes, such as those responsible for cell identity.

Those and other tools helped investigators identify more than 3,000 super-enhancers in 28 medulloblastoma tumors as well as evidence that the activity of super-enhancers varied by subtype. The super-enhancers switched on known cancer genes, including genes like ALK, MYC, SMO, and OTX2 that are associated with medulloblastoma, the researchers reported.

Knowledge of the subtype super-enhancers led to identification of the transcription factors that regulate their activity. Using computational methods, researchers applied that information to reconstruct the transcription factor networks responsible for medulloblastoma subtype diversity and identity, providing previously unknown insights into the regulatory landscape and transcriptional output of the different medulloblastoma subtypes.

The approach helped to discover and nominate Lmx1A as a master regulator transcription factor of Group 4 tumors, which led to the identification of the likely Group 4 tumor cells of origin. Lmx1A was known to play an important role in normal development of cells in the uRL and cerebellum. Additional studies performed in mice with and without Lmx1A in this study supported uRL cells as the likely source of Group 4 tumors.

“By studying the epigenome, we also identified new pathways and molecular dependencies not apparent in previous gene expression and mutational studies,” explained Dr. Northcott. “The findings open new therapeutic avenues, particularly for the Group 3 and 4 subtypes where patient outcomes are inferior for the majority of affected children.”

For example, researchers identified increased enhancer activity targeting the TGFbeta pathway. The finding adds to evidence that the pathway may drive Group 3 medulloblastoma, currently the subtype with the worst prognosis. The pathway regulates cell growth, cell death, and other functions that are often disrupted in cancer, but it’s role in medulloblastoma is poorly understood.

The analysis included samples from 28 medulloblastoma tumors representing the four subtypes. Researchers believe it is the largest epigenetic study yet for any single cancer type and, importantly, the first to use a large cohort of primary patient tumor tissues instead of cell lines grown in the laboratory. Previous studies have suggested that cell lines may be of limited use for studying the tumor epigenome. The three Group 3 medulloblastoma cell lines used in this study reinforced the observation, highlighting significant differences in epigenetic regulators at work in medulloblastoma cell lines versus tumor samples.

 

 

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Identical Twin Brother Develops Schizophrenia

Author: Larry H. Bernstein, MD, FCAP

 

 

 

My identical twin brother develops schizophrenia

I graduated college as a chemistry undergraduate prior to entrance into medical school in 1973, and my brother had not exhibited signs of serious mental distress until that point.  He was dating a young woman who rejected him, and he was also under pressure from our father, who thought he did not have direction. The oldest daughter was married and was well prepared in piano and in mathematics, and she has prepared students in piano to the present day. The triplet sister was married to a medical student who went on to become a psychiatrist. The two sons were still living at home.  There was considerable pressure on my brother to complete his studies.   The trigger seemed to be the breakup of his relationship. It was in the months prior to my graduation that my mother was deeply concerned and our parents pursued a psychiatric evaluation.   He was put on chlorpromazine, but then developed jaundice. Schizophrenia was not understood in those years, and for many years was an illness that brought shame to the parents.  I shared a bedroom with my brother for all the years prior to this event and I only saw it develop at the surface.

We both had worked as waiters at a resort on Lake Michigan for some years prior to my entrance to medical school, and Leslie had an interest in biology.  He was closer to our younger sister, and I was trying to keep up with Sharon, who was 2 years older and had an infant that I visited often.  I had a close friend who was my buddy.  I could talk to him often, and we compared notes after a double date.  Leslie had a friend who we had played chess with in high school.  My brother showed no progress and his psychiatric visits were costly. My father was a dental technician who was skilled at making dentures.

It was the summer prior to my entry into medical school that I worked in a biochemistry research laboratory under the supervision of my brother in law.  The first year medical studies were pressured with anatomy, biochemistry, inborn errors of metabolism, neuroanatomy and embryology, and dissection of cadavers.  Leslie was admitted to the Lafayette Clinic at Wayne State University. He was now receiving the best care available. I visited him at that time, and he played chess with the attendant.
It was also during the first year of medical school that the progressive Rabbi Adler, at Rodeph Shalom who had a national reputation was shot in front of the Bima by Richard Wishnetsky, a troubled man our age who was mentally ill, probably with a mood disorder. My good friend was home from Berkeley and tried to avoid the problem, but he was released by a law school student. Richard’s parents were leaders in the congregation.  My friend and I knew there was a problem early because Richard had received a Woodrow Wilson scholarship, and he considered graduate studies under a faculty member at the Catholic University in Detroit, but he did go to the University of Michigan.

At the end of the first year, the triplets went to Washington, DC to participate in an ongoing Schizophrenia twin study.  I was engaged in studies of radiation on virus in an NIH lab during the study. Three years later, when I was rotating through psychiatry at Herman Kiefer Hospital in my third year having taking time out for a Master of Science degree in Anatomy (the evolution of the proteins of the eye lens), I found myself in the Detroit riots.

 

My brother grew a beard and became somewhat disheveled. He had hallucinations, and he could tell about his dreams.  For instance, so and so visited him.  He began living in an apartment on Woodward Avenue, the largest street in Detroit. He became very spiritual, and he wrote poetry.  One day he stood in the middle of Woodward Avenue wearing a tallis (prayer shawl) and directed traffic.  He did manage to finish his undergraduate studies, but when he took a job teaching biology, he just couldn’t. He also knew that a Croatian girl who graduated high school with us, who was a talented dancer, developed schizophrenia.

My mother was very stressed. She was helping to care for my older sister’s daughter, and she was grieving over her son.  She developed abdominal pain in 1978, when I graduated and went to my residency in Pathology at University of Kansas Medical Center in Kansas City, in what was to be a residency and PhD program. I had joined the Berry Plan during my medical school years and when I graduated I was randomly selected to go into the Navy, but got a deferment to complete my studies.

It was during that time that I learned that my mother had had an exploratory laparotomy for what turned out to be an infiltrating carcinoma of the stomach, through the wall and on the peritoneal surface (linitis plastic).  No biopsy was taken.  I flew home frequently until the end.  She was on morphine to ease the pain.  I began seeing a woman I had known in high school, who was now teaching. We were married in December of 1979, after my mother died.  My mother’s father had always been well and was a mechanic in Cleveland. I was told that he died of a broken heart with the loss of my mother.

I went to University of California, San Diego in January, 1980, to work in Enzymology, the inhibition of the pyridine nucleotide linked malate dehydrogenase reaction, under Nathan O. Kaplan, and there I also completed my residency.  It happens at that time, my brother had moved to San Diego, and he was looked after our triplet sister.  It was a fortunate circumstance for the triplets.

 

 

 

 

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Biochemistry and Dysmetabolism of Aging and Serious Illness

Curator: Larry H. Bernstein, MD, FCAP

 

White Matter Lipids as a Ketogenic Fuel Supply in Aging Female Brain: Implications for Alzheimer’s Disease

Lauren P. Klosinski, Jia Yao, Fei Yin, Alfred N. Fonteh, Michael G. Harrington, Trace A. Christensen, Eugenia Trushina, Roberta Diaz Brinton
http://www.ebiomedicine.com/article/S2352-3964(15)30192-4/abstract      DOI: http://dx.doi.org/10.1016/j.ebiom.2015.11.002
Highlights
  • Mitochondrial dysfunction activates mechanisms for catabolism of myelin lipids to generate ketone bodies for ATP production.
  • Mechanisms leading to ketone body driven energy production in brain coincide with stages of reproductive aging in females.
  • Sequential activation of myelin catabolism pathway during aging provides multiple therapeutic targets and windows of efficacy.

The mechanisms underlying white matter degeneration, a hallmark of multiple neurodegenerative diseases including Alzheimer’s, remain unclear. Herein we provide a mechanistic pathway, spanning multiple transitions of aging, that links mitochondrial dysfunction early in aging with later age white matter degeneration. Catabolism of myelin lipids to generate ketone bodies can be viewed as an adaptive survival response to address brain fuel and energy demand. Women are at greatest risk of late-onset-AD, thus, our analyses in female brain address mechanisms of AD pathology and therapeutic targets to prevent, delay and treat AD in the sex most affected with potential relevance to men.

 

White matter degeneration is a pathological hallmark of neurodegenerative diseases including Alzheimer’s. Age remains the greatest risk factor for Alzheimer’s and the prevalence of age-related late onset Alzheimer’s is greatest in females. We investigated mechanisms underlying white matter degeneration in an animal model consistent with the sex at greatest Alzheimer’s risk. Results of these analyses demonstrated decline in mitochondrial respiration, increased mitochondrial hydrogen peroxide production and cytosolic-phospholipase-A2 sphingomyelinase pathway activation during female brain aging. Electron microscopic and lipidomic analyses confirmed myelin degeneration. An increase in fatty acids and mitochondrial fatty acid metabolism machinery was coincident with a rise in brain ketone bodies and decline in plasma ketone bodies. This mechanistic pathway and its chronologically phased activation, links mitochondrial dysfunction early in aging with later age development of white matter degeneration. The catabolism of myelin lipids to generate ketone bodies can be viewed as a systems level adaptive response to address brain fuel and energy demand. Elucidation of the initiating factors and the mechanistic pathway leading to white matter catabolism in the aging female brain provides potential therapeutic targets to prevent and treat demyelinating diseases such as Alzheimer’s and multiple sclerosis. Targeting stages of disease and associated mechanisms will be critical.

3. Results

  1. 3.1. Pathway of Mitochondrial Deficits, H2O2 Production and cPLA2 Activation in the Aging Female Brain
  2. 3.2. cPLA2-sphingomyelinase Pathway Activation in White Matter Astrocytes During Reproductive Senescence
  3. 3.3. Investigation of White Matter Gene Expression Profile During Reproductive Senescence
  4. 3.4. Ultra Structural Analysis of Myelin Sheath During Reproductive Senescence
  5. 3.5. Analysis of the Lipid Profile of Brain During the Transition to Reproductive Senescence
  6. 3.6. Fatty Acid Metabolism and Ketone Generation Following the Transition to Reproductive Senescence

 

4. Discussion

Age remains the greatest risk factor for developing AD (Hansson et al., 2006, Alzheimer’s, 2015). Thus, investigation of transitions in the aging brain is a reasoned strategy for elucidating mechanisms and pathways of vulnerability for developing AD. Aging, while typically perceived as a linear process, is likely composed of dynamic transition states, which can protect against or exacerbate vulnerability to AD (Brinton et al., 2015). An aging transition unique to the female is the perimenopausal to menopausal conversion (Brinton et al., 2015). The bioenergetic similarities between the menopausal transition in women and the early appearance of hypometabolism in persons at risk for AD make the aging female a rational model to investigate mechanisms underlying risk of late onset AD.

Findings from this study replicate our earlier findings that age of reproductive senescence is associated with decline in mitochondrial respiration, increased H2O2 production and shift to ketogenic metabolism in brain (Yao et al., 2010, Ding et al., 2013, Yin et al., 2015). These well established early age-related changes in mitochondrial function and shift to ketone body utilization in brain, are now linked to a mechanistic pathway that connects early decline in mitochondrial respiration and H2O2 production to activation of the cPLA2-sphingomyelinase pathway to catabolize myelin lipids resulting in WM degeneration (Fig. 12). These lipids are sequestered in lipid droplets for subsequent use as a local source of ketone body generation via astrocyte mediated beta-oxidation of fatty acids. Astrocyte derived ketone bodies can then be transported to neurons where they undergo ketolysis to generate acetyl-CoA for TCA derived ATP generation required for synaptic and cell function (Fig. 12).

Thumbnail image of Fig. 12. Opens large image

http://www.ebiomedicine.com/cms/attachment/2040395791/2053874721/gr12.sml

Fig. 12

Schematic model of mitochondrial H2O2 activation of cPLA2-sphingomyelinase pathway as an adaptive response to provide myelin derived fatty acids as a substrate for ketone body generation: The cPLA2-sphingomyelinase pathway is proposed as a mechanistic pathway that links an early event, mitochondrial dysfunction and H2O2, in the prodromal/preclinical phase of Alzheimer’s with later stage development of pathology, white matter degeneration. Our findings demonstrate that an age dependent deficit in mitochondrial respiration and a concomitant rise in oxidative stress activate an adaptive cPLA2-sphingomyelinase pathway to provide myelin derived fatty acids as a substrate for ketone body generation to fuel an energetically compromised brain.

Biochemical evidence obtained from isolated whole brain mitochondria confirms that during reproductive senescence and in response to estrogen deprivation brain mitochondria decline in respiratory capacity (Yao et al., 2009, Yao et al., 2010, Brinton, 2008a, Brinton, 2008b, Swerdlow and Khan, 2009). A well-documented consequence of mitochondrial dysfunction is increased production of reactive oxygen species (ROS), specifically H2O2 (Boveris and Chance, 1973, Beal, 2005, Yin et al., 2014, Yap et al., 2009). While most research focuses on the damage generated by free radicals, in this case H2O2 functions as a signaling molecule to activate cPLA2, the initiating enzyme in the cPLA2-sphingomyelinase pathway (Farooqui and Horrocks, 2006, Han et al., 2003, Sun et al., 2004). In AD brain, increased cPLA2 immunoreactivity is detected almost exclusively in astrocytes suggesting that activation of the cPLA2-sphingomyelinase pathway is localized to astrocytes in AD, as opposed to the neuronal or oligodendroglial localization that is observed during apoptosis (Sun et al., 2004, Malaplate-Armand et al., 2006, Di Paolo and Kim, 2011, Stephenson et al., 1996,Stephenson et al., 1999). In our analysis, cPLA2 (Sanchez-Mejia and Mucke, 2010) activation followed the age-dependent rise in H2O2 production and was sustained at an elevated level.

Direct and robust activation of astrocytic cPLA2 by physiologically relevant concentrations of H2O2 was confirmed in vitro. Astrocytic involvement in the cPLA2-sphingomyelinase pathway was also indicated by an increase in cPLA2 positive astrocyte reactivity in WM tracts of reproductively incompetent mice. These data are consistent with findings from brains of persons with AD that demonstrate the same striking localization of cPLA2immunoreactivity within astrocytes, specifically in the hippocampal formation (Farooqui and Horrocks, 2004). While neurons and astrocytes contain endogenous levels of cPLA2, neuronal cPLA2 is activated by an influx of intracellular calcium, whereas astrocytic cPLA2 is directly activated by excessive generation of H2O2 (Sun et al., 2004, Xu et al., 2003, Tournier et al., 1997). Evidence of this cell type specific activation was confirmed by the activation of cPLA2 in astrocytes by H2O2 and the lack of activation in neurons. These data support that astrocytic, not neuronal, cPLA2 is the cellular mediator of the H2O2 dependent cPLA2-sphingomyelinase pathway activation and provide associative evidence supporting a role of astrocytic mitochondrial H2O2 in age-related WM catabolism.

The pattern of gene expression during the shift to reproductive senescence in the female mouse hippocampus recapitulates key observations in human AD brain tissue, specifically elevation in cPLA2, sphingomyelinase and ceramidase (Schaeffer et al., 2010, He et al., 2010, Li et al., 2014). Further, up-regulation of myelin synthesis, lipid metabolism and inflammatory genes in reproductively incompetent female mice is consistent with the gene expression pattern previously reported from aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). In these analyses of gene expression in aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus down regulation of genes related to mitochondrial function, and up-regulation in multiple genes encoding for enzymes involved in ketone body metabolism occurred (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). The comparability across data derived from aging female mouse hippocampus reported herein and those derived from male rodent brain, female nonhuman brain and human AD brain strongly suggest that cPLA2-sphingomyelinase pathway activation, myelin sheath degeneration and fatty acid metabolism leading to ketone body generation is a metabolic adaptation that is generalizable across these naturally aging models and are evident in aged human AD brain. Collectively, these data support the translational relevance of findings reported herein.

Data obtained via immunohistochemistry, electron microscopy and MBP protein analyses demonstrated an age-related loss in myelin sheath integrity. Evidence for a loss of myelin structural integrity emerged in reproductively incompetent mice following activation of the cPLA2-sphingomyelinase pathway. The unraveling myelin phenotype observed following reproductive senescence and aging reported herein is consistent with the degenerative phenotype that emerges following exposure to the chemotherapy drug bortezomib which induces mitochondrial dysfunction and increased ROS generation (Carozzi et al., 2010, Cavaletti et al., 2007,Ling et al., 2003). In parallel to the decline in myelin integrity, lipid droplet density increased. In aged mice, accumulation of lipid droplets declined in parallel to the rise in ketone bodies consistent with the utilization of myelin-derived fatty acids to generate ketone bodies. Due to the sequential relationship between WM degeneration and lipid droplet formation, we posit that lipid droplets serve as a temporary storage site for myelin-derived fatty acids prior to undergoing β-oxidation in astrocytes to generate ketone bodies.

Microstructural alterations in myelin integrity were associated with alterations in the lipid profile of brain, indicative of WM degeneration resulting in release of myelin lipids. Sphingomyelin and galactocerebroside are two main lipids that compose the myelin sheath (Baumann and Pham-Dinh, 2001). Ceramide is common to both galactocerebroside and sphingomyelin and is composed of sphingosine coupled to a fatty acid. Ceramide levels increase in aging, in states of ketosis and in neurodegeneration (Filippov et al., 2012, Blazquez et al., 1999, Costantini et al., 2005). Specifically, ceramide levels are elevated at the earliest clinically recognizable stage of AD, indicating a degree of WM degeneration early in disease progression (Di Paolo and Kim, 2011,Han et al., 2002, Costantini et al., 2005). Sphingosine is statistically significantly elevated in the brains of AD patients compared to healthy controls; a rise that was significantly correlated with acid sphingomyelinase activity, Aβ levels and tau hyperphosphorylation (He et al., 2010). In our analyses, a rise in ceramides was first observed early in the aging process in reproductively incompetent mice. The rise in ceramides was coincident with the emergence of loss of myelin integrity consistent with the release of myelin ceramides from sphingomyelin via sphingomyelinase activation. Following the rise in ceramides, sphingosine and fatty acid levels increased. The temporal sequence of the lipid profile was consistent with gene expression indicating activation of ceramidase for catabolism of ceramide into sphingosine and fatty acid during reproductive senescence. Once released from ceramide, fatty acids can be transported into the mitochondrial matrix of astrocytes via CPT-1, where β-oxidation of fatty acids leads to the generation of acetyl-CoA (Glatz et al., 2010). It is well documented that acetyl-CoA cannot cross the inner mitochondrial membrane, thus posing a barrier to direct transport of acetyl-CoA generated by β-oxidation into neurons. In response, the newly generated acetyl-CoA undergoes ketogenesis to generate ketone bodies to fuel energy demands of neurons (Morris, 2005,Guzman and Blazquez, 2004, Stacpoole, 2012). Because astrocytes serve as the primary location of β-oxidation in brain they are critical to maintaining neuronal metabolic viability during periods of reduced glucose utilization (Panov et al., 2014, Ebert et al., 2003, Guzman and Blazquez, 2004).

Once fatty acids are released from myelin ceramides, they are transported into astrocytic mitochondria by CPT1 to undergo β-oxidation. The mitochondrial trifunctional protein HADHA catalyzes the last three steps of mitochondrial β-oxidation of long chain fatty acids, while mitochondrial ABAD (aka SCHAD—short chain fatty acid dehydrogenase) metabolizes short chain fatty acids. Concurrent with the release of myelin fatty acids in aged female mice, CPT1, HADHA and ABAD protein expression as well as ketone body generation increased significantly. These findings indicate that astrocytes play a pivotal role in the response to bioenergetic crisis in brain to activate an adaptive compensatory system that activates catabolism of myelin lipids and the metabolism of those lipids into fatty acids to generate ketone bodies necessary to fuel neuronal demand for acetyl-CoA and ATP.

Collectively, these findings provide a mechanistic pathway that links mitochondrial dysfunction and H2O2generation in brain early in the aging process to later stage white matter degeneration. Astrocytes play a pivotal role in providing a mechanistic strategy to address the bioenergetic demand of neurons in the aging female brain. While this pathway is coincident with reproductive aging in the female brain, it is likely to have mechanistic translatability to the aging male brain. Further, the mechanistic link between bioenergetic decline and WM degeneration has potential relevance to other neurological diseases involving white matter in which postmenopausal women are at greater risk, such as multiple sclerosis. The mechanistic pathway reported herein spans time and is characterized by a progression of early adaptive changes in the bioenergetic system of the brain leading to WM degeneration and ketone body production. Translationally, effective therapeutics to prevent, delay and treat WM degeneration during aging and Alzheimer’s disease will need to specifically target stages within the mechanistic pathway described herein. The fundamental initiating event is a bioenergetic switch from being a glucose dependent brain to a glucose and ketone body dependent brain. It remains to be determined whether it is possible to prevent conversion to or reversal of a ketone dependent brain. Effective therapeutic strategies to intervene in this process require biomarkers of bioenergetic phenotype of the brain and stage of mechanistic progression. The mechanistic pathway reported herein may have relevance to other age-related neurodegenerative diseases characterized by white matter degeneration such as multiple sclerosis.

Blood. 2015 Oct 15;126(16):1925-9.    http://dx.doi.org:/10.1182/blood-2014-12-617498. Epub 2015 Aug 14.
Targeting the leukemia cell metabolism by the CPT1a inhibition: functional preclinical effects in leukemias.
Cancer cells are characterized by perturbations of their metabolic processes. Recent observations demonstrated that the fatty acid oxidation (FAO) pathway may represent an alternative carbon source for anabolic processes in different tumors, therefore appearing particularly promising for therapeutic purposes. Because the carnitine palmitoyl transferase 1a (CPT1a) is a protein that catalyzes the rate-limiting step of FAO, here we investigated the in vitro antileukemic activity of the novel CPT1a inhibitor ST1326 on leukemia cell lines and primary cells obtained from patients with hematologic malignancies. By real-time metabolic analysis, we documented that ST1326 inhibited FAO in leukemia cell lines associated with a dose- and time-dependent cell growth arrest, mitochondrial damage, and apoptosis induction. Data obtained on primary hematopoietic malignant cells confirmed the FAO inhibition and cytotoxic activity of ST1326, particularly on acute myeloid leukemia cells. These data suggest that leukemia treatment may be carried out by targeting metabolic processes.
Oncogene. 2015 Oct 12.   http://dx.doi.org:/10.1038/onc.2015.394. [Epub ahead of print]
Tumour-suppression function of KLF12 through regulation of anoikis.
Suppression of detachment-induced cell death, known as anoikis, is an essential step for cancer metastasis to occur. We report here that expression of KLF12, a member of the Kruppel-like family of transcription factors, is downregulated in lung cancer cell lines that have been selected to grow in the absence of cell adhesion. Knockdown of KLF12 in parental cells results in decreased apoptosis following cell detachment from matrix. KLF12 regulates anoikis by promoting the cell cycle transition through S phase and therefore cell proliferation. Reduced expression levels of KLF12 results in increased ability of lung cancer cells to form tumours in vivo and is associated with poorer survival in lung cancer patients. We therefore identify KLF12 as a novel metastasis-suppressor gene whose loss of function is associated with anoikis resistance through control of the cell cycle.
Mol Cell. 2015 Oct 14. pii: S1097-2765(15)00764-9. doi: 10.1016/j.molcel.2015.09.025. [Epub ahead of print]
PEPCK Coordinates the Regulation of Central Carbon Metabolism to Promote Cancer Cell Growth.
Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid, and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.
Mol Cancer Res. 2015 Oct;13(10):1408-20.   http://dx.doi.org:/10.1158/1541-7786.MCR-15-0048. Epub 2015 Jun 16.
Disruption of Proline Synthesis in Melanoma Inhibits Protein Production Mediated by the GCN2 Pathway.
Many processes are deregulated in melanoma cells and one of those is protein production. Although much is known about protein synthesis in cancer cells, effective ways of therapeutically targeting this process remain an understudied area of research. A process that is upregulated in melanoma compared with normal melanocytes is proline biosynthesis, which has been linked to both oncogene and tumor suppressor pathways, suggesting an important convergent point for therapeutic intervention. Therefore, an RNAi screen of a kinase library was undertaken, identifying aldehyde dehydrogenase 18 family, member A1 (ALDH18A1) as a critically important gene in regulating melanoma cell growth through proline biosynthesis. Inhibition of ALDH18A1, the gene encoding pyrroline-5-carboxylate synthase (P5CS), significantly decreased cultured melanoma cell viability and tumor growth. Knockdown of P5CS using siRNA had no effect on apoptosis, autophagy, or the cell cycle but cell-doubling time increased dramatically suggesting that there was a general slowdown in cellular metabolism. Mechanistically, targeting ALDH18A1 activated the serine/threonine protein kinase GCN2 (general control nonderepressible 2) to inhibit protein synthesis, which could be reversed with proline supplementation. Thus, targeting ALDH18A1 in melanoma can be used to disrupt proline biosynthesis to limit cell metabolism thereby increasing the cellular doubling time mediated through the GCN2 pathway.  This study demonstrates that melanoma cells are sensitive to disruption of proline synthesis and provides a proof-of-concept that the proline synthesis pathway can be therapeutically targeted in melanoma tumors for tumor inhibitory efficacy. Mol Cancer Res; 13(10); 1408-20. ©2015 AACR.
SDHB-Deficient Cancers: The Role of Mutations That Impair Iron Sulfur Cluster Delivery.
BACKGROUND:  Mutations in the Fe-S cluster-containing SDHB subunit of succinate dehydrogenase cause familial cancer syndromes. Recently the tripeptide motif L(I)YR was identified in the Fe-S recipient protein SDHB, to which the cochaperone HSC20 binds.
METHODS:   In order to characterize the metabolic basis of SDH-deficient cancers we performed stable isotope-resolved metabolomics in a novel SDHB-deficient renal cell carcinoma cell line and conducted bioinformatics and biochemical screening to analyze Fe-S cluster acquisition and assembly of SDH in the presence of other cancer-causing SDHB mutations.

RESULTS:

We found that the SDHB(R46Q) mutation in UOK269 cells disrupted binding of HSC20, causing rapid degradation of SDHB. In the absence of SDHB, respiration was undetectable in UOK269 cells, succinate was elevated to 351.4±63.2 nmol/mg cellular protein, and glutamine became the main source of TCA cycle metabolites through reductive carboxylation. Furthermore, HIF1α, but not HIF2α, increased markedly and the cells showed a strong DNA CpG island methylator phenotype (CIMP). Biochemical and bioinformatic screening revealed that 37% of disease-causing missense mutations in SDHB were located in either the L(I)YR Fe-S transfer motifs or in the 11 Fe-S cluster-ligating cysteines.

CONCLUSIONS:

These findings provide a conceptual framework for understanding how particular mutations disproportionately cause the loss of SDH activity, resulting in accumulation of succinate and metabolic remodeling in SDHB cancer syndromes.

 

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMPK-mTOR Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

  1. L. Figarola, J. Singhal, J. D. Tompkins, G. W. Rogers, C. Warden, D. Horne, A. D. Riggs, S. Awasthi and S. S. Singhal.

J Biol Chem. 2015 Nov 3, [epub ahead of print]

 

CD47 Receptor Globally Regulates Metabolic Pathways That Control Resistance to Ionizing Radiation

  1. W. Miller, D. R. Soto-Pantoja, A. L. Schwartz, J. M. Sipes, W. G. DeGraff, L. A. Ridnour, D. A. Wink and D. D. Roberts.

J Biol Chem. 2015 Oct 9, 290 (41): 24858-74.

 

Knockdown of PKM2 Suppresses Tumor Growth and Invasion in Lung Adenocarcinoma

  1. Sun, A. Zhu, L. Zhang, J. Zhang, Z. Zhong and F. Wang.

Int J Mol Sci. 2015 Oct 15, 16 (10): 24574-87.

 

EglN2 associates with the NRF1-PGC1alpha complex and controls mitochondrial function in breast cancer

  1. Zhang, C. Wang, X. Chen, M. Takada, C. Fan, X. Zheng, H. Wen, Y. Liu, C. Wang, R. G. Pestell, K. M. Aird, W. G. Kaelin, Jr., X. S. Liu and Q. Zhang.

EMBO J. 2015 Oct 22, [epub ahead of print]

 

Mitochondrial Genetics Regulate Breast Cancer Tumorigenicity and Metastatic Potential.

Current paradigms of carcinogenic risk suggest that genetic, hormonal, and environmental factors influence an individual’s predilection for developing metastatic breast cancer. Investigations of tumor latency and metastasis in mice have illustrated differences between inbred strains, but the possibility that mitochondrial genetic inheritance may contribute to such differences in vivo has not been directly tested. In this study, we tested this hypothesis in mitochondrial-nuclear exchange mice we generated, where cohorts shared identical nuclear backgrounds but different mtDNA genomes on the background of the PyMT transgenic mouse model of spontaneous mammary carcinoma. In this setting, we found that primary tumor latency and metastasis segregated with mtDNA, suggesting that mtDNA influences disease progression to a far greater extent than previously appreciated. Our findings prompt further investigation into metabolic differences controlled by mitochondrial process as a basis for understanding tumor development and metastasis in individual subjects. Importantly, differences in mitochondrial DNA are sufficient to fundamentally alter disease course in the PyMT mouse mammary tumor model, suggesting that functional metabolic differences direct early tumor growth and metastatic efficiency. Cancer Res; 75(20); 4429-36. ©2015 AACR.

 

Cancer Lett. 2015 Oct 29. pii: S0304-3835(15)00656-4.    http://dx.doi.org:/10.1016/j.canlet.2015.10.025. [Epub ahead of print]
Carboxyamidotriazole inhibits oxidative phosphorylation in cancer cells and exerts synergistic anti-cancer effect with glycolysis inhibition.

Targeting cancer cell metabolism is a promising strategy against cancer. Here, we confirmed that the anti-cancer drug carboxyamidotriazole (CAI) inhibited mitochondrial respiration in cancer cells for the first time and found a way to enhance its anti-cancer activity by further disturbing the energy metabolism. CAI promoted glucose uptake and lactate production when incubated with cancer cells. The oxidative phosphorylation (OXPHOS) in cancer cells was inhibited by CAI, and the decrease in the activity of the respiratory chain complex I could be one explanation. The anti-cancer effect of CAI was greatly potentiated when being combined with 2-deoxyglucose (2-DG). The cancer cells treated with the combination of CAI and 2-DG were arrested in G2/M phase. The apoptosis and necrosis rates were also increased. In a mouse xenograft model, this combination was well tolerated and retarded the tumor growth. The impairment of cancer cell survival was associated with significant cellular ATP decrease, suggesting that the combination of CAI and 2-DG could be one of the strategies to cause dual inhibition of energy pathways, which might be an effective therapeutic approach for a broad spectrum of tumors.

 

Cancer Immunol Res. 2015 Nov;3(11):1236-47.    http://dx.doi.org:/10.1158/2326-6066.CIR-15-0036. Epub 2015 May 29.
Inhibition of Fatty Acid Oxidation Modulates Immunosuppressive Functions of Myeloid-Derived Suppressor Cells and Enhances Cancer Therapies.

Myeloid-derived suppressor cells (MDSC) promote tumor growth by inhibiting T-cell immunity and promoting malignant cell proliferation and migration. The therapeutic potential of blocking MDSC in tumors has been limited by their heterogeneity, plasticity, and resistance to various chemotherapy agents. Recent studies have highlighted the role of energy metabolic pathways in the differentiation and function of immune cells; however, the metabolic characteristics regulating MDSC remain unclear. We aimed to determine the energy metabolic pathway(s) used by MDSC, establish its impact on their immunosuppressive function, and test whether its inhibition blocks MDSC and enhances antitumor therapies. Using several murine tumor models, we found that tumor-infiltrating MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation (FAO). This was accompanied by an increased mitochondrial mass, upregulation of key FAO enzymes, and increased oxygen consumption rate. Pharmacologic inhibition of FAO blocked immune inhibitory pathways and functions in T-MDSC and decreased their production of inhibitory cytokines. FAO inhibition alone significantly delayed tumor growth in a T-cell-dependent manner and enhanced the antitumor effect of adoptive T-cell therapy. Furthermore, FAO inhibition combined with low-dose chemotherapy completely inhibited T-MDSC immunosuppressive effects and induced a significant antitumor effect. Interestingly, a similar increase in fatty acid uptake and expression of FAO-related enzymes was found in human MDSC in peripheral blood and tumors. These results support the possibility of testing FAO inhibition as a novel approach to block MDSC and enhance various cancer therapies. Cancer Immunol Res; 3(11); 1236-47. ©2015 AACR.

 

Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase

  1. L. Judge, K. M. Owens, S. J. Pollock, C. F. Woeller, T. H. Thatcher, J. P. Williams, R. P. Phipps, P. J. Sime and R. M. Kottmann.

Am J Physiol Lung Cell Mol Physiol. 2015 Oct 15, 309 (8): L879-87.

 

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH

  1. Yun, E. Mullarky, C. Lu, K. N. Bosch, A. Kavalier, K. Rivera, J. Roper, Chio, II, E. G. Giannopoulou, C. Rago, A. Muley, J. M. Asara, J. Paik, O. Elemento, Z. Chen, D. J. Pappin, L. E. Dow, N. Papadopoulos, S. S. Gross and L. C. Cantley.

Science. 2015 Nov 5, [epub ahead of print]

 

Down-regulation of FBP1 by ZEB1-mediated repression confers to growth and invasion in lung cancer cells

  1. Zhang, J. Wang, H. Xing, Q. Li, Q. Zhao and J. Li.

Mol Cell Biochem. 2015 Nov 6, [epub ahead of print]

 

J Mol Cell Cardiol. 2015 Oct 23. pii: S0022-2828(15)30073-0.     http://dx.doi.org:/10.1016/j.yjmcc.2015.10.002. [Epub ahead of print]
GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels.

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.

 

Br J Pharmacol. 2015 Oct 27. doi: 10.1111/bph.13377. [Epub ahead of print]
All-trans retinoic acid protects against doxorubicin-induced cardiotoxicity by activating the Erk2 signalling pathway.
BACKGROUND AND PURPOSE:

Doxorubicin (Dox) is a powerful antineoplastic agent for treating a wide range of cancers. However, doxorubicin cardiotoxicity of the heart has largely limited its clinical use. It is known that all-trans retinoic acid (ATRA) plays important roles in many cardiac biological processes, however, the protective effects of ATRA on doxorubicin cardiotoxicity remain unknown. Here, we studied the effect of ATRA on doxorubicin cardiotoxicity and underlying mechanisms.

EXPERIMENTAL APPROACHES:

Cellular viability assays, western blotting and mitochondrial respiration analyses were employed to evaluate the cellular response to ATRA in H9c2 cells and primary cardiomyocytes. Quantitative PCR (Polymerase Chain Reaction) and gene knockdown were performed to investigate the underlying molecular mechanisms of ATRA’s effects on doxorubicin cardiotoxicity.

KEY RESULTS:

ATRA significantly inhibited doxorubicin-induced apoptosis in H9c2 cells and primary cardiomyocytes. ATRA was more effective against doxorubicin cardiotoxicity than resveratrol and dexrazoxane. ATRA also suppressed reactive oxygen species (ROS) generation, and restored the expression level of mRNA and proteins in phase II detoxifying enzyme system: Nrf2 (nuclear factor-E2-related factor 2), MnSOD (manganese superoxide dismutase), HO-1 (heme oxygenase1) as well as mitochondrial function (mitochondrial membrane integrity, mitochondrial DNA copy numbers, mitochondrial respiration capacity, biogenesis and dynamics). Both Erk1/2 (extracellular signal-regulated kinase1/2) inhibitor (U0126) and Erk2 siRNA, but not Erk1 siRNA, abolished the protective effect of ATRA against doxorubicin-induced toxicity in H9c2 cells. Remarkably, ATRA did not compromise the anticancer efficacy of doxorubicin in gastric carcinoma cells.

CONCLUSION AND IMPLICATION:

ATRA protected cardiomyocytes against doxorubicin-induced toxicity by activating the Erk2 pathway without compromising the anticancer efficacy of doxorubicin. Therefore, ATRA may be a promising candidate as a cardioprotective agent against doxorubicin cardiotoxicity.

 

Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation

  1. Colak, O. Pougovkina, L. Dai, M. Tan, H. Te Brinke, H. Huang, Z. Cheng, J. Park, X. Wan, X. Liu, W. W. Yue, R. J. Wanders, J. W. Locasale, D. B. Lombard, V. C. de Boer and Y. Zhao.

Mol Cell Proteomics. 2015 Nov 1, 14 (11): 3056-71.

 

Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

  1. Pancrazi, G. Di Benedetto, L. Colombaioni, G. Della Sala, G. Testa, F. Olimpico, A. Reyes, M. Zeviani, T. Pozzan and M. Costa.

Proc Natl Acad Sci U S A. 2015 Oct 27, 112(45): 13910-5.

 

Evidence of Mitochondrial Dysfunction within the Complex Genetic Etiology of Schizophrenia

  1. E. Hjelm, B. Rollins, F. Mamdani, J. C. Lauterborn, G. Kirov, G. Lynch, C. M. Gall, A. Sequeira and M. P. Vawter.

Mol Neuropsychiatry. 2015 Nov 1, 1 (4): 201-219.

 

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation

  1. Bernard, N. J. Logsdon, S. Ravi, N. Xie, B. P. Persons, S. Rangarajan, J. W. Zmijewski, K. Mitra, G. Liu, V. M. Darley-Usmar and V. J. Thannickal.

J Biol Chem. 2015 Oct 16, 290 (42): 25427-38.

 

J Biol Chem. 2015 Oct 23;290(43):25834-46.    http://dx.doi.org:/10.1074/jbc.M115.658815. Epub 2015 Sep 4.
Kinome Screen Identifies PFKFB3 and Glucose Metabolism as Important Regulators of the Insulin/Insulin-like Growth Factor (IGF)-1 Signaling Pathway.

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling.

 

FASEB J. 2015 Oct 19.    http://dx.doi.org:/pii: fj.15-276360. [Epub ahead of print]
Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

Skeletal muscle mitochondrial content and oxidative capacity are important determinants of muscle function and whole-body health. Mitochondrial content and function are enhanced by endurance exercise and impaired in states or diseases where muscle function is compromised, such as myopathies, muscular dystrophies, neuromuscular diseases, and age-related muscle atrophy. Hence, elucidating the mechanisms that control muscle mitochondrial content and oxidative function can provide new insights into states and diseases that affect muscle health. In past studies, we identified Perm1 (PPARGC1- and ESRR-induced regulator, muscle 1) as a gene induced by endurance exercise in skeletal muscle, and regulating mitochondrial oxidative function in cultured myotubes. The capacity of Perm1 to regulate muscle mitochondrial content and function in vivo is not yet known. In this study, we use adeno-associated viral (AAV) vectors to increase Perm1 expression in skeletal muscles of 4-wk-old mice. Compared to control vector, AAV1-Perm1 leads to significant increases in mitochondrial content and oxidative capacity (by 40-80%). Moreover, AAV1-Perm1-transduced muscles show increased capillary density and resistance to fatigue (by 33 and 31%, respectively), without prominent changes in fiber-type composition. These findings suggest that Perm1 selectively regulates mitochondrial biogenesis and oxidative function, and implicate Perm1 in muscle adaptations that also occur in response to endurance exercise.-Cho, Y., Hazen, B. C., Gandra, P. G., Ward, S. R., Schenk, S., Russell, A. P., Kralli, A. Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

 

A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.
Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.

 

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Schizophrenic hallucinations

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Update 11/23/2015

Brain Fold Tied to Hallucinations

A shorter crease in the medial prefrontal cortex is linked with a higher risk of schizophrenics experiencing hallucinations.

By Kerry Grens | November 19, 2015

http://www.the-scientist.com//?articles.view/articleNo/44547/title/Brain-Fold-Tied-to-Hallucinations/

WIKIMEDIA, DATABASE CENTER FOR LIFE SCIENCE

People with schizophrenia who experience hallucinations are more likely to have a certain contour to their brain—specifically, a shorter groove in the medial prefrontal cortex called the paracingulate sulcus (PCS). That’s according to a study published this week (November 17) in Nature Communications of 153 people, some of whom had schizophrenia with and without hallucinations and some who did not.

“We think that the PCS is involved in brain networks that help us recognize information that has been generated ourselves,” Jane Garrison, the lead author of the study and a researcher at the University of Cambridge, said in a press release. “People with a shorter PCS seem less able to distinguish the origin of such information, and appear more likely to experience it as having been generated externally.”

Garrison and her colleagues used MRI scans to gather PCS length. They found that schizophrenics who experienced hallucinations tended to have a shorter PCS, and a 1-cm reduction in the fold related to a 20 percent higher chance of having hallucinations. People with schizophrenia who did not have hallucinations and the healthy controls did not differ in their PCS length.

“We’ve known for some time that disorders like schizophrenia are not down to a single region of the brain. Changes are seen throughout various different areas. To be able to pin such a key symptom to a relatively specific part of the brain is quite unusual,” study coauthor Jon Simons of Cambridge told BBC News.

The study could not determine whether PCS length is a causal factor in hallucinations in schizophrenia.

 

Paracingulate sulcus morphology is associated with hallucinations in the human brain

Jane R. GarrisonCharles FernyhoughSimon McCarthy-JonesMark HaggardThe Australian Schizophrenia Research Bank & Jon S. Simons

Nature Communications  2015; 6(8956).      http://dx.doi.org:/10.1038/ncomms9956

 

Hallucinations are common in psychiatric disorders, and are also experienced by many individuals who are not mentally ill. Here, in 153 participants, we investigate brain structural markers that predict the occurrence of hallucinations by comparing patients with schizophrenia who have experienced hallucinations against patients who have not, matched on a number of demographic and clinical variables. Using both newly validated visual classification techniques and automated, data-driven methods, hallucinations were associated with specific brain morphology differences in the paracingulate sulcus, a fold in the medial prefrontal cortex, with a 1cm reduction in sulcal length increasing the likelihood of hallucinations by 19.9%, regardless of the sensory modality in which they were experienced. The findings suggest a specific morphological basis for a pervasive feature of typical and atypical human experience.

 

PCS measurement for two example images.

Figure 1: PCS measurement for two example images.

The paracingulate sulcus (PCS), marked in red, lies dorsal and parallel to the cingulate sulcus (CS), itself dorsal to the corpus callosum. (a) In this image, the PCS is continuous and is measured from its origin in the first quadrant (indicated by the cross-hairs at y=0 and z=0) to its end. (b) In this example, the PCS appears less distinct; it is measured from the point at which it runs in a posterior direction, dorsal to the cingulate sulcus.

 

PCS length by group.

Figure 2: PCS length by group

(a) Total PCS length across both hemispheres. (b) PCS length in the left hemisphere. (c) PCS length in the right hemisphere. ***P<0.001, **P<0.01, *P<0.05. Error bars represent standard error of the mean. Controls: 40 healthy control subjects; no hallucinations: 34 patients with schizophrenia who had not experienced hallucinations; hallucinations: 79 patients with schizophrenia who experienced hallucinations in any modality.

 

Hallucinations are a primary symptom of numerous mental health disorders, as well as featuring in the experience of many individuals within the healthy population. Previous attempts to characterize the brain mechanisms of hallucinations have often been confounded by designs relying on comparisons between patients and non-patients1. However, around 30% of patients who meet diagnostic criteria for schizophrenia never report such anomalous experiences, providing the potential for the discovery of brain structural markers that are specifically associated with the occurrence of hallucinations.

Undoubtedly, many neurobiological factors underlie hallucinations. In the present study, we focused on the paracingulate sulcus (PCS) in the medial prefrontal cortex (mPFC) given its previously established role in reality monitoring2, among other cognitive functions, defined as the ability to discriminate between real and imagined information3. Reality monitoring is impaired in patients with schizophrenia with hallucinations4, 5, 6 and in non-patients prone to hallucinations7. In the study by Buda et al.2, we previously identified that bilateral absence of the PCS was associated with reductions in reality monitoring performance in healthy individuals with no neurological damage. The PCS is one of the last sulci to develop in utero, providing the potential for individual differences in its morphology, such as sulcal length, to be particularly informative about functional variation in an area of the brain extensively implicated in reality monitoring judgments8, 9.

Here, we investigate PCS length in both hemispheres in three matched groups: schizophrenia patients with a history of hallucinations, schizophrenia patients with no history of hallucinations and healthy controls (see Table 1 for participant details). PCS length was measured from structural MRI scans using a newly validated visual classification technique and data-driven whole-brain analysis methods, carried out blind to diagnosis (see Methods section for details). Examples of long and short PCS images are displayed in Fig. 1. We report converging results from across methods indicating that hallucination status can be determined by specific brain morphology differences in the PCS.

Validation of PCS measurement protocol

To validate the new PCS measurement protocol, it was first applied to 53 healthy volunteer structural scans previously analysed by Buda et al.2, with the analysis undertaken blind to the ratings in the earlier study, to give 106 measurements of sulcal length across hemispheres. The left and right hemisphere PCS for each individual was classified as ‘prominent’ if the length was >40mm, ‘absent’ if PCS length was <20mm and ‘present’ if PCS length fell between these two limits, based on the earlier protocols2, 10. The PCS classifications obtained were then compared with the original ratings from the study by Buda et al.2 In all, 94 out of the 106 measurements matched precisely, resulting in a Cohen’s Kappa of 0.79 (P<0.001), 95% CI (0.68, 0.84), indicating ‘substantial agreement’11 between the two protocols.

To validate the measurement protocol further, and verify its sensitivity to morphological variations in schizophrenia, we measured PCS length in a small, locally acquired independent sample of 19 patients with schizophrenia, all of whom experienced hallucinations, as well as in 19 matched control participants. Informed consent was obtained from these participants in a manner approved by the UK National Research Ethics Service. Total PCS length was significantly reduced in the patients with schizophrenia (mean=84.1mm, s.d.=30.5mm) compared with controls (mean=110.2mm, s.d.=38.5mm), t(36)=2.31, P=0.027, d=0.77. These independent validations provide grounds for confidence about the reliability of our measurement protocol, and the likelihood that it will be sufficiently sensitive to identify morphological differences in our larger sample of 153 patients with schizophrenia and controls that may distinguish those who experienced hallucinations from those without hallucinations.

PCS measurement differences associated with hallucinations

Turning to the principal analysis of PCS morphology differences as a function of hallucination status, we compared PCS length between three large matched groups (patients with schizophrenia who had experienced hallucinations, patients with schizophrenia who had not experienced hallucinations and matched healthy controls; see Methods section for participant details and matching procedure). There was a main effect of group on total PCS length, summed across both hemispheres, F(2, 150)=8.90, P<0.001, ηp2=0.106, which survived the addition of cortical surface area as a covariate, F(2, 149)=7.03, P=0.001, ηp2=0.086. Other potential covariates such as age, IQ, intracranial volume and global brain gyrification index had no significant effect on PCS length and were removed from the model.

Planned comparisons revealed that patients with schizophrenia who experienced hallucinations exhibited significantly reduced PCS length compared with the patients without hallucinations (mean reduction=19.2mm), t(111)=2.531, P=0.013, d=0.519 and healthy controls (mean reduction=29.2mm), t(117)=4.149, P<0.001, d=0.805, whereas sulcal length between patients who did not experience hallucinations and healthy controls did not differ significantly, t(72)=1.07,P=0.288, d=0.246 (Fig. 2a).

With earlier research providing conflicting evidence of differential cortical-folding patterns between the two cerebral hemispheres in schizophrenia, we next investigated possible laterality effects on PCS length. There were main effects of hemisphere, F(1,150)=9.978, P=0.002, ηp2=0.062, and group, F(2,150)=8.900, P<0.001, ηp2=0.106, on PCS length, but no interaction between hemisphere and group. PCS length was greater in the left than the right hemisphere across all subject groups, t(152)=2.959, P=0.004, d=0.317 (Fig. 2b,c). Patients with schizophrenia who had experienced hallucinations exhibited reduced PCS length compared with the healthy controls in both hemispheres, t>2.636, P<0.01, d>0.546. The difference in PCS length between patients with schizophrenia who had experienced hallucinations and patients who had not experienced hallucinations was significant only in the left hemisphere, t(111)=2.464, P=0.015, d=0.505.

We tested the modality specificity of the observed relations by comparing PCS length between patients with auditory hallucinations and patients with hallucinations limited to other modalities (for example, visual, tactile, olfactory). The PCS reductions could not be differentiated according to hallucination modality, either summed across both hemispheres, t(77)=0.067, P=0.947, d=0.015, or within the left, t(77)=0.600, P=0.551, d=0.135, or right, t(77)=0.822, P=0.413, d=0.185, hemispheres alone, consistent with a generalized role for reality monitoring impairment in the formation of hallucinations, regardless of the sensory modality in which they occur.

……..

Data-driven whole-brain analyses

To further validate the PCS measurement protocol and to determine whether between-group differences in PCS length were accompanied by structural variations elsewhere in the brain, we conducted separate automated whole-brain analyses of surface-based cortical gyrification and of voxel-based grey matter volume (see Methods section for details). Confirming the results of the PCS measurement method, significant differences in local gyrification index were observed in the mPFC regions of interest surrounding the PCS, namely bilateral frontopolar, medial orbitofrontal, superior frontal and paracentral cortices, with patients with schizophrenia who experienced hallucinations exhibiting significantly reduced gyrification in these regions compared with patients without hallucinations, t(111)=2.165, P=0.033, d=0.448 (Fig. 3). No significant regional group differences elsewhere in the brain survived correction for multiple comparisons

 

Figure 3: Whole-brain cortical gyrification differences as a function of hallucination status.

Whole-brain cortical gyrification differences as a function of hallucination status.

http://www.nature.com/ncomms/2015/151117/ncomms9956/images_article/ncomms9956-f3.jpg

a) mPFC regions surrounding the PCS exhibiting significantly reduced gyrification in 79 patients who experienced hallucinations compared with 34 patients without hallucinations, rendered on a canonical pial cortical surface, viewed from the midline. (b) Local gyrification index in regions surrounding the PCS significantly differentiates patients with schizophrenia as a function of hallucination status, t(111)=2.165,P=0.033, d=0.448. Error bars represent standard error of the mean.

Consistent with reductions in mPFC cortical folding in hallucinations, grey matter volume was significantly greater in the functionally defined 8-mm sphere mPFC region of interest surrounding the anterior PCS in patients with schizophrenia who experienced hallucinations than in those who did not (x=6, y=54, z=−5; BA 10; Z=2.82; P=0.036 (small volume corrected), Fig. 4). The region identified as significant using this voxel-based method was smaller than the region that emerged in the surface-based gyrification analysis, which may be attributable to the different properties of cortical morphology measured, as well as any of numerous statistical and methodological differences between the two techniques (see Methods section for details). In any event, no significant grey matter volume differences elsewhere in the brain, associated with the occurrence of hallucinations, survived correction for multiple comparisons.

 

Figure 4: Grey matter volume differences measured with voxel-based morphometry.

Grey matter volume differences measured with voxel-based morphometry.

http://www.nature.com/ncomms/2015/151117/ncomms9956/images_article/ncomms9956-f4.jpg

(a) Significantly greater grey matter volume in 79 patients who experienced hallucinations than in 34 patients without hallucinations in the mPFC region of interest in the vicinity of the anterior PCS (circled), rendered on an inflated canonical cortical surface, viewed from the front. (b) Grey matter volume in PCS region significantly differentiates patients with schizophrenia as a function of hallucination status, Z=2.82;P=0.036 (small volume corrected). Error bars represent standard error of the mean.

 

Using newly validated visual classification techniques and automated, data-driven analysis methods, the present study identified that hallucinations were associated with specific brain morphology differences in the PCS region of the mPFC. Because the connection between PCS reduction and hallucinations was evident in participants who all had diagnoses of schizophrenia, our findings avoid confounding with patient status, as can occur in case–control comparisons. The hallucinating and non-hallucinating groups with schizophrenia in our study were matched for age, sex, handedness, IQ, duration of illness, antipsychotic medication and incidence of delusions and negative symptoms. In identifying that hallucinations can be distinguished by structural brain imaging data, we demonstrate that a multifactorial phenomenon which is defined experientially can be related to a single morphological change in the mPFC. As a tertiary sulcus forming around 36 weeks of gestation12, the 19.2mm mean reduction in PCS length that distinguished patients who hallucinated from those who did not hallucinate might arise from genetic factors that influence primary folding of the cortex through a disruption to neurodevelopmental pathways. Alternatively, the variability in PCS length might be a non-genetic consequence of some disturbance in primary sulcal development, or might represent extremes of normal statistical variation in the development of primary and secondary sulci.

Our results go beyond previous findings of changes in cortical-folding patterns associated with schizophrenia. Several previous studies have reported differences in PCS morphology in patients with schizophrenia compared with healthy controls13, 14, or investigated differences in global measures of cortical gyrification or sulcation associated with hallucination status15. The present study is the first to identify that PCS morphology changes can discriminate between hallucinating and non-hallucinating groups that are matched for overall brain volume, cortical surface area and global gyrification index, among other variables. The present findings are consistent with earlier research suggesting that leftward PCS hemispheric asymmetries in schizophrenia might be similar to those typically observed in healthy controls14, 16, though some previous studies have reported reduced PCS asymmetry in schizophrenia13, 17. In the present data, comparable laterality effects were observed in all subject groups, with significantly greater PCS length in the left than right hemisphere, and group differences evident across both hemispheres. Methodological differences might explain the discrepancies between previous studies, motivating the development of common measurement protocols, preferably incorporating both visual classification and automated, data-driven components, to optimize the identification and measurement of sometimes relatively indistinct or discontinuous anatomical landmarks such as the PCS.

Evidence from research in healthy individuals indicates that PCS reductions are associated with increased grey matter volume in the surrounding anterior cingulate cortex18, with Buda et al.2reporting that increased grey matter volume in the mPFC correlated negatively with an individual’s reality monitoring ability. Such findings fit with the present results, in which reduced mPFC surface-based gyrification and concomitant increased voxel-based grey matter volume were the only significant differences in the brain to be associated with the occurrence of hallucinations. Together with the results by Buda et al., these findings are consistent with a role for reality monitoring impairment in the generation of hallucinations, with a structural basis for that ability in the region of the PCS. An influence of reduced paracingulate folding and greater surrounding cortical volume may arise from weakened connectivity between the mPFC and both proximal and distal brain regions. Prominent theories of morphogenesis suggest that cortical folding in the human brain, which begins at around the 26th week of gestation19, 20, results either from differential mechanical tension along white matter axons linking disparate brain areas21, 22 or from variable tangential expansion of the cortical surface23.

Altered PCS morphology could thus lead to hallucinations through changes in connectivity between cortical regions involved in processing sensory representations and mPFC areas that support decision-making processes such as distinguishing real experiences from those that might have been imagined, among other cognitive functions8, 9, 24. This hypothesis has yet to be tested directly, although there is evidence of impaired anterior cingulate modulation of fronto–temporal connectivity in schizophrenia25. Investigating functional and structural connectivity between the broader mPFC and, for example, posterior auditory and language regions around the superior temporal gyrus, would further inform models of hallucination formation. Hallucinations are likely to be a multifactorial phenomenon5, and theoretical models implicate a range of cognitive and affective variables in their occurrence26, 27. It is possible that modality-general risk factors, such as reduced PCS length, may interact in some individuals with modality-specific risk factors, such as reduced arcuate fasciculus integrity in the case of auditory hallucinations28, to produce hallucinations in specific sensory modalities. Information on neurodevelopmental models of schizophrenia could also be gained by comparing PCS morphology in family studies and during disease development.

Our findings support modality-general views of hallucinations as stemming from atypicalities in reality monitoring. They raise important questions for cognitive models of hallucinations including how the internal ‘raw material’ of reality monitoring errors might be defined. In the case of auditory hallucinations, there is compelling evidence that hallucinations arise through the misattribution of internal events (for example, inner speech) as external auditory stimuli. A modality-general account would need to specify analogous internal events that could be misattributed as external ones in, for example, the visual or tactile modalities. A modality-general account would also have to explain considerable phenomenological variability in the experience as it is described in all modalities. Moreover, as with all theories proposing brain structural or functional changes associated with hallucinations, a reality monitoring account must explain why hallucinations are often transient phenomena rather than being experienced constantly. Susceptibility to hallucinations, and their triggering and maintenance by psychological and environmental factors, are likely to be multifactorial, complex processes. We show that a simple morphological variation is an important factor in determining why some individuals can have quasi-perceptual experience of entities that are not physically present.

 

How hallucinations emerge from trying to make sense of an ambiguous world

http://www.neuroscience.cam.ac.uk/news/article.php?permalink=edbb8aa287

Take a look at the black and white image. It probably looks like a meaningless pattern of black and white blotches. But now take a look at the image below and then return to the picture: it’s likely that you can now make sense of the black and white image. It is this ability that scientists at Cardiff University and the University of Cambridge believe could help explain why some people are prone to hallucinations.

http://www.cam.ac.uk/sites/www.cam.ac.uk/files/inner-images/press_twotone_sml.jpg

 

http://www.cam.ac.uk/sites/www.cam.ac.uk/files/inner-images/press_colour_smal.jpg

 

Adapted from University of Cambridge News
Press coverage: BBC News Cambridge News Daily Mail Motherboard ITV News Irish Examiner Belfast Telegraph

Posted on 10/13/2015

A bewildering and often very frightening experience in some mental illnesses is psychosis – a loss of contact with external reality. This often results in a difficulty in making sense of the world, which can appear threatening, intrusive and confusing. Psychosis is sometimes accompanied by drastic changes in perception, to the extent that people may see, feel, smell and taste things that are not actually there – so-called hallucinations. These hallucinations may be accompanied by beliefs that others find irrational and impossible to comprehend.

In research published today in the journal Proceedings of National Academy of Sciences (PNAS), a team of researchers based at Cardiff University and the University of Cambridge explore the idea that hallucinations arise due to an enhancement of our normal tendency to interpret the world around us by making use of prior knowledge and predictions.

In order to make sense of and interact with our physical and social environment, we need appropriate information about the world around us, for example the size or location of a nearby object. However, we have no direct access to this information and are forced to interpret potentially ambiguous and incomplete information from our senses. This challenge is overcome in the brain – for example in our visual system – by combining ambiguous sensory information with our prior knowledge of the environment to generate a robust and unambiguous representation of the world around us. For example, when we enter our living room, we may have little difficulty discerning a fast-moving black shape as the cat, even though the visual input was little more than a blur that rapidly disappeared behind the sofa: the actual sensory input was minimal and our prior knowledge did all the creative work.

“Vision is a constructive process – in other words, our brain makes up the world that we ‘see’,” explains first author Dr Christoph Teufel from the School of Psychology at Cardiff University. “It fills in the blanks, ignoring the things that don’t quite fit, and presents to us an image of the world that has been edited and made to fit with what we expect.”

“Having a predictive brain is very useful – it makes us efficient and adept at creating a coherent picture of an ambiguous and complex world,”adds senior author Professor Paul Fletcher from the Department of Psychiatry at the University of Cambridge. “But it also means that we are not very far away from perceiving things that aren’t actually there, which is the definition of a hallucination.

“In fact, in recent years we’ve come to realise that such altered perceptual experiences are by no means restricted to people with mental illness. They are relatively common, in a milder form, across the entire population. Many of us will have heard or seen things that aren’t there.”

In order to address the question of whether such predictive processes contribute to the emergence of psychosis, the researchers worked with 18 individuals who had been referred to a mental health service run by the NHS Cambridgeshire and Peterborough Foundation Trust, and led by Dr Jesus Perez, one of the co-authors on the study, and who suffered from very early signs of psychosis. They examined how these individuals, as well as a group of 16 healthy volunteers, were able to use predictions in order to make sense of ambiguous, incomplete black and white images, similar to the one shown above.

The volunteers were asked to look at a series of these black and white images, some of which contained a person, and then to say for a given image whether or not it contained a person. Because of the ambiguous nature of the images, the task was very difficult at first. Participants were then shown a series of full colour original images, including those from which the black and white images had been derived: this information could be used to improve the brain’s ability to make sense of the ambiguous image. The researchers reasoned that, since hallucinations may come from a greater tendency to superimpose one’s predictions on the world, people who were prone to hallucinations would be better at using this information because, in this task, such a strategy would be an advantage.

The researchers found a larger performance improvement in people with very early signs of psychosis in comparison to the healthy control group. This suggested that people from the clinical group were indeed relying more strongly on the information that they had been given to make sense of the ambiguous pictures.

When the researchers presented the same task to a larger group of 40 healthy people, they found a continuum in task performance that correlated with the participants’ scores on tests of psychosis-proneness. In other words, the shift in information processing that favours prior knowledge over sensory input during perception can be detected even before the onset of early psychotic symptoms.

“These findings are important because they tell us that the emergence of key symptoms of mental illness can be understood in terms of an altered balance in normal brain functions,” says Naresh Subramaniam from the Department of Psychiatry at the University of Cambridge. “Importantly, they also suggest that these symptoms and experiences do not reflect a ‘broken’ brain but rather one that is striving – in a very natural way – to make sense of incoming data that are ambiguous.”

The study was carried out in collaboration with Dr Veronika Dobler and Professor Ian Goodyer from the Department of Child and Adolescent Psychiatry at the University of Cambridge. The research was funded by the Wellcome Trust and the Bernard Wolfe Health Neuroscience Fund. It was carried out within the Cambridge and Peterborough NHS Foundation Trust. Additional support for the Behavioural and Clinical Neuroscience Institute at the University of Cambridge came from the Wellcome Trust and the Medical Research Council.

Shift toward prior knowledge confers a perceptual advantage in early psychosis and psychosis-prone healthy individuals

Christoph Teufela,b,1Naresh SubramaniambVeronika Doblerc,dJesus Perezc,dJohanna Finnemannb,ePuja R. Mehtab, et al.

PNAS 2013; 112(43): 13401–13406    http//dx.doi.org:/10.1073/pnas.1503916112

Significance

Perceiving things that are not there and holding unfounded, bizarre beliefs (hallucinations and delusions, respectively) are psychotic symptoms that occur in particular syndromes including affective psychoses, paranoid states, and schizophrenia. We studied the emergence of this loss of contact with reality based on current models of normal brain function. Working with clinical individuals experiencing early psychosis and nonclinical individuals with high levels of psychosis proneness, we show that their visual perception is characterized by a shift that favors prior knowledge over incoming sensory evidence. Given that these alterations in information processing are evident early on in psychosis and even in association with subtle perceptual changes indicating psychosis proneness, they may be important factors contributing to the emergence of severe mental illnesses.

 

Many neuropsychiatric illnesses are associated with psychosis, i.e., hallucinations (perceptions in the absence of causative stimuli) and delusions (irrational, often bizarre beliefs). Current models of brain function view perception as a combination of two distinct sources of information: bottom-up sensory input and top-down influences from prior knowledge. This framework may explain hallucinations and delusions. Here, we characterized the balance between visual bottom-up and top-down processing in people with early psychosis (study 1) and in psychosis-prone, healthy individuals (study 2) to elucidate the mechanisms that might contribute to the emergence of psychotic experiences. Through a specialized mental-health service, we identified unmedicated individuals who experience early psychotic symptoms but fall below the threshold for a categorical diagnosis. We observed that, in early psychosis, there was a shift in information processing favoring prior knowledge over incoming sensory evidence. In the complementary study, we capitalized on subtle variations in perception and belief in the general population that exhibit graded similarity with psychotic experiences (schizotypy). We observed that the degree of psychosis proneness in healthy individuals, and, specifically, the presence of subtle perceptual alterations, is also associated with stronger reliance on prior knowledge. Although, in the current experimental studies, this shift conferred a performance benefit, under most natural viewing situations, it may provoke anomalous perceptual experiences. Overall, we show that early psychosis and psychosis proneness both entail a basic shift in visual information processing, favoring prior knowledge over incoming sensory evidence. The studies provide complementary insights to a mechanism by which psychotic symptoms may emerge.

 

To interact successfully with our physical and social environment, we need appropriate information about relevant states of the world, such as the size, location, or distance of an object. However, there is no direct access to this information, only to sensory stimulation caused by the environment. This sensory information is inherently ambiguous and, on its own, rarely suffices to uniquely specify our surroundings (1). The human visual system overcomes this challenge by combining ambiguous sensory information with prior knowledge of the environment to generate a robust and unambiguous representation of the world around us (17). This insight has been formalized under the tenets of Bayesian decision theory and is typically modeled within a predictive coding framework. Here, the notion is that expectations based on prior knowledge are fed back from higher to lower levels of information processing, thereby shaping the way incoming signals are treated by lower-level mechanisms. This influence is labeled top-down processing. The present study tests the hypothesis that psychotic experiences arise from an increased use of prior knowledge in constructing meaningful percepts from ambiguous sensory inputs.

Psychosis—a loss of contact with external reality—is characterized by delusions (irrational, often bizarre beliefs) and hallucinations (perceptions in the absence of causative stimuli). Conceptual and computational models of psychosis have hypothesized that an imbalance in the combination of bottom-up sensory evidence and top-down prior knowledge is at the core of this altered state of mind (812). According to such models, at the perceptual level, an undue reliance on prior knowledge in perception may lead to the emergence of aberrant perceptions such as hallucinations. The current study tests this hypothesis in the visual domain by characterizing the impact of prior knowledge on the perception of ambiguous stimuli in two groups of people: a clinical group with early psychotic experiences (study 1) and healthy volunteers showing differing levels of proneness to such experiences (study 2). Although the conventional view focuses preferentially on auditory hallucinations in psychosis, epidemiological evidence indicates that hallucinations in the visual domain are very common in, for example, schizophrenia (13). In fact, vision seems to play a prominent role in the development of psychosis given that basic visual symptoms identified before illness onset are one of the most powerful predictors of the emergence of later psychotic disorders (14).

To determine mechanisms for the emergence of perceptual psychotic symptoms as purely as possible, we conducted two complementary studies. First, using a case-control study design, we characterized the balance between visual bottom-up and top-down processing in a group of patients with early psychotic experiences and matched healthy controls (SI Materials and Methods and Table S1). Individuals in our clinical group were recruited from a dedicated mental health service identifying help-seeking people who have low-level but measurable psychotic experiences. Although, at the time of testing, these individuals fell below the threshold for a categorical diagnosis, they already showed symptoms and have an increased risk for transitioning to a severe mental illness such as schizophrenia or an affective disorder (15). Importantly, working with such a group of patients and comparing them to controls enabled us to focus on the features of early psychosis before any formal categorical diagnosis. Moreover, and also critically, this comparison is not confounded by the effects of antipsychotic medication or the impact of chronic illness, allowing us, as purely as possible, to explore the mechanisms of early psychosis.

In a second study, we explored psychosis proneness in healthy participants characterized according to the presence of perceptual (16) and belief-related schizotypal features (17). Schizotypy refers to a personality measure that has established predictive value for psychotic and other mental illnesses (18). Although it has been traditionally considered a specific risk measure for schizophrenia, more recently it has been proposed to reflect a general psychosis proneness. A number of schizotypy scales have been devised to characterize various dimensions of psychosis. In the current study, we focused on individual variation in measures relating to perception and belief (16, 17) because they most clearly relate to the key features of psychosis. These measures provided us with a fine-grained index for relevant perceptual experience and beliefs, allowing us to characterize the bottom-up/top-down balance in relation to subtle, nonclinical but specific and measurable markers associated with psychosis proneness.

Characterizing these two situations enabled us to pursue our central aim of exploring information-processing mechanisms that are altered in association with the occurrence of early symptoms (study 1) and also identifiable even before such symptoms arise (study 2). As well as offering a purer assessment of the emergence of psychotic experiences, this approach is inspired by growing evidence suggesting that psychosis lies on a continuum with normality (19, 20) and is associated with a range of different psychiatric disorders (15, 21). According to this perspective, existing diagnostic categories group biologically heterogeneous syndromes with potentially different pathophysiological mechanisms into one disorder (22); this may obfuscate our attempts to understand the neurobiological underpinnings of mental illness. In keeping with a broader move within the field, the aim of this approach is therefore to characterize deeper dimensions in their own right, such as psychosis as in the current study, irrespective of diagnostic categorization to advance our mechanistic understanding of specific symptom clusters.

In summary, we explored how the use of prior knowledge in visual information processing is related to early psychosis and to psychosis proneness. Importantly, given our hypothesis, we predicted that the putative mechanism associated with the emergence of psychosis would confer a relative advantage in this task, given that successful performance required the use of prior knowledge to discriminate ambiguous stimuli. Together, the two studies provide evidence to suggest that early psychosis and psychosis proneness is associated with a shift in visual processing that favors prior knowledge over incoming sensory evidence. We also demonstrate that this relation is specific to atypical perceptual experiences rather than being linked to psychotic experiences more generally.

 

Our studies were designed to characterize, in complementary ways, the balance between visual bottom-up and top-down processing in clinical individuals with early psychosis and healthy people prone to developing psychotic symptoms. A relative advantage in using prior knowledge to discriminate between ambiguous images was observed in both situations. This finding is especially striking in the clinical group in study 1 given that performance in this group (as in psychiatrically ill individuals more generally) is typically impaired. Such a result is rare and revealing in that it highlights a specific information-processing atypicality rather than a general performance deficit. Study 2 allowed us to characterize these alterations in visual function more completely by adopting an individual differences approach with healthy participants and by capitalizing on subtle variations in perception and belief that exhibit graded similarity with psychotic experiences. In line with our clinical findings, we uncovered a relation between an individual’s visual performance benefit due to prior knowledge and their scores on two scales of psychosis proneness. Importantly, also, our data suggest that this relation is primarily driven by perceptual alterations rather than unusual beliefs. Taken together, these results indicate that visual function in early psychosis and in healthy people who are prone to such experiences is characterized by a basic information-processing shift that favors existing knowledge over incoming sensory evidence. Although, in the current experimental task, this shift conferred a performance benefit, under most natural viewing situations, it may provoke anomalous perceptual experiences. Specifically, it might impose prior expectations on inputs to the extent that, ultimately, formed percepts are generated that have no direct sensory cause: hallucinations.

These findings fit neatly with and support current conceptual and computational models of psychotic symptoms (812). For instance, it has been hypothesized that a single core disturbance relating to the balance between bottom-up and top-down processing can explain both the hallucinatory experiences and the bizarre delusional beliefs of psychotic patients (8, 11). Importantly, we show that, on the perceptual level, a shift in this balance toward prior knowledge is present both in a clinical group of individuals with early psychosis and even associated with psychosis proneness in the general population. Although schizotypy is a marker for psychosis proneness as ascertained by previous longitudinal studies (18), it is important to acknowledge that individuals in study 2 were not suffering from psychosis or even a diagnosed mental illness. Rather, those individuals scoring high on the scales identified a number of unusual perceptual experiences. It is therefore striking that the same information-processing shift was observed as was found in early psychosis. Indeed, even in the early psychosis group, no formal, categorical diagnosis was applicable (although it is known that such groups have a high risk of transition to full psychiatric illness) (15). The findings may therefore suggest that the altered balance is a fundamental trait that contributes to the emergence of psychosis rather than a reflection or consequence of the psychotic state.

The specificity of the relation between performance on our task and perceptual aspects of schizotypy is of particular interest. It has long been known that altered perceptual experiences form a key part of the emergence of psychosis (29). Given that the CAPS is selective for measuring schizotypal perceptual phenomena rather than targeting schizotypy in general (16), our findings indicate that a shift in visual information processing that favors prior knowledge over sensory evidence might be a marker for the mechanisms underlying this observation. The finding that healthy individuals that score high on this scale share this marker with our clinical group is in line with the growing belief that psychotic mental illnesses are part of a continuum with normality (19, 20). It supports the idea that the putative atypicality underlying the emergence of perceptual psychotic experiences relates directly to normal function of the system. In other words, the potential for psychotic experiences such as hallucinations might be a logical consequence of the way in which our brain deals with the inherent ambiguity of sensory information by incorporating prior knowledge into our perceptual processing. The current study uncovered an imbalance of this processing type that shows its effects at the perceptual level. However, within a hierarchical and recurrent information-processing system such as the human brain, an imbalance at any level will, in time, propagate up and down the hierarchy and affects the whole system (8, 30), a notion that might ultimately account for atypicalities in both lower-level perceptual processing and higher-level belief formation in severe mental illnesses and psychosis proneness (30).

 

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Schizophrenia Brain

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

http://health-innovations.org/2015/10/27/neuroimaging-matches-specific-schizophrenia-behaviour-to-the-brains-anatomy/

Neuroimaging studies using fMRI and PET to examine functional differences in brain activity in patients with schizophrenia have shown that differences seem to most commonly occur in the frontal lobes, hippocampus, and temporal lobes. These differences are heavily linked to the neurocognitive deficits which often occur with schizophrenia, particularly in areas of memory, attention, problem solving, executive function and social cognition.

Earlier studies from the researchers reported evidence suggesting that schizophrenia is not a single disease but a group of eight genetically distinct disorders, each with its own set of symptoms. Results found that distinct sets of genes were strongly associated with particular clinical symptoms.

The current study investigates the brain’s anatomy and shows that there are distinct subgroups of patients with a schizophrenia diagnosis that correlates with symptoms.  This also explains the difficulty in past studies to identify a single set of biomarkers for a single type of schizophrenia.

The current study evaluated scans taken with magnetic resonance imaging (MRI) and a technique called diffusion tensor imaging in 36 healthy volunteers and 47 people with schizophrenia. Results show that the scans of patients with schizophrenia had various abnormalities in portions of the corpus callosum, a bundle of fibers that connects the left and right hemispheres of the brain and is considered critical to neural communication. Characteristics across the corpus callosum revealed in the brain scans matched specific symptoms of schizophrenia. Patients with specific features in one part of the corpus callosum typically displayed bizarre and disorganized behaviour. In other patients, irregularities in a different part of that structure were associated with disorganized thinking and speech and symptoms such as a lack of emotion; other brain abnormalities in the corpus callosum were associated with delusions or hallucinations.  The lab conclude that their findings provide further evidence that schizophrenia is a heterogeneous group of disorders rather than a single disorder.

The team surmise that they didn’t start with people who had certain symptoms and then look to see whether they had corresponding abnormalities in the brain. They note that they just looked at the data, and the patterns began to emerge. They go ony to add that this kind of granular information, combined with data about the genetics of schizophrenia, one day will help physicians treat the disorder in a more precise way.

Many genes responsible for the creation of synaptic proteins have previously shown to be strongly linked to schizophrenia and other brain disorders, however, until now the reasons have not been understood.  Now, researchers from Cardiff University have identified a critical function of what they believe to be schizophrenia’s ‘Rosetta Stone’ gene that could hold the key to decoding the function of all genes involved in the disease.  The team state that the breakthrough has revealed a vulnerable period in the early stages of the brain’s development that they hope can be targeted for future efforts in reversing schizophrenia.  The study is published in the journal Science.

The gene identified in the current study is known as ‘disrupted in schizophrenia-1’ (DISC-1). Earlier studies have shown that when mutated, the gene is a high risk factor for mental illness including schizophrenia, major clinical depression and bipolar disorder.  The aim of the current study was to determine whether DISC-1’s interactions with other proteins early on in the brain’s development had a bearing on the brain’s ability to adapt its structure and function, also known as ‘plasticity’, later on in adulthood.

In order for healthy development of the brain’s synapses to take place, the DISC-1 gene first needs to bind with two other molecules known as ‘Lis’ and ‘Nudel’.  The experiments in mice revealed that by preventing DISC-1 from binding with these molecules prevents cortical neurons in the brain’s largest region from being able to form synapses.  The ability to form coherent thoughts and to properly perceive the world is damaged as a consequence of this.

Preventing DISC-1 from binding with ‘Lis’ and ‘Nudel’ molecules when the brain was fully formed had no effect on its plasticity. However, the researchers were able to pinpoint a seven-day window early on in the brain’s development, one week after birth, where failure to bind had an irreversible effect on the brain’s plasticity later on in life.

The researchers hypothesize that DISC-1 is schizophrenia’s Rosetta Stone gene and could hold the master key to help unlock the understanding of the role played by all risk genes involved in the disease.  They go on to add that they have identified a critical period during brain development that will assist in testing whether other schizophrenia risk genes affecting different regions of the brain create their malfunction during their own critical period.

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