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Archive for the ‘Human aging’ Category


Live 11:00 AM- 12:00 Mediterranean Diet and Lifestyle: A Symposium on Diet and Human Health : Opening Remarks October 19, 2018

Reporter: Stephen J. Williams, Ph.D.

11:00 Welcome

 

 

Prof. Antonio Giordano, MD, PhD.

Director and President of the Sbarro Health Research Organization, College of Science and Technology, Temple University

Welcome to this symposium on Italian lifestyle and health.  This is similar to a symposium we had organized in New York.  A year ago Bloomberg came out with a study on higher longevity of the italian population and this study was concluded that this increased longevity was due to the italian lifestyle and diet especially in the southern part of Italy, a region which is older than Rome (actually founded by Greeks and Estonians).  However this symposium will delve into the components of this healthy Italian lifestyle which contributes to this longevity effect.  Some of this work was done in collaboration with Temple University and sponsored by the Italian Consulate General in Philadelphia ( which sponsors programs in this area called Ciao Philadelphia).

Greetings: Fucsia Nissoli Fitzgerald, Deputy elected in the Foreign Circumscription – North and Central America Division

Speaking for the Consulate General is Francesca  Cardurani-Meloni.   I would like to talk briefly about the Italian cuisine and its evolution, from the influence of the North and South Italy, economic factors, and influence by other cultures.  Italian cooking is about simplicity, cooking with what is in season and freshest.  The meal is not about the food but about comfort around the table, and comparible to a cullinary heaven, about sharing with family and friends, and bringing the freshest ingredients to the table.

Consul General, Honorable Pier Attinio Forlano, General Consul of Italy in Philadelphia

 

11:30 The Impact of Environment and Life Style in Human Disease

Prof. Antonio Giordano MD, PhD.

 

 

 

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Anti-Müllerian Hormone (AMH), is secreted by growing follicles that contains the egg or ovum. According to regular practice low AMH and high Follicle Stimulating Hormone (FSH) are generally considered as indicators of diminished egg quantity in a female. But, there are several cases the female conceived absolutely normally without any support even after low AMH was reported.

 

Therefore, a new research published in the Journal of the American Medical Association declares that AMH doesn’t dictate a woman’s reproductive potential. Although AMH testing is one of the most common ways that doctors assess a woman’s fertility. Present research says that all it takes is one egg each cycle and AMH is not a marker of whether a female can or cannot become pregnant. So, for women who haven’t yet tried to get pregnant and who are wondering whether they are fertile, an AMH value isn’t going to be helpful in that context. In addition, AMH is not necessarily a good marker to predict that whether one has to cryopreserve her eggs. So, practically doctors don’t yet have a way to definitively predict egg quality or a woman’s long-term ability to conceive, but age is obviously one of the most important factors.

 

The above mentioned study followed 750 women between the ages of 30 and 44 who had been trying to conceive for three months or less. During the 12-month observation period, those with low AMH values of less than 0.7 were not less likely to conceive than those who had normal AMH values. The study had various limitations, however, that are worth noting. The researchers only included women who did not have a history of infertility. Women who sought fertility treatments (about 6 percent) were withdrawn. And only 12 percent of the women were in the 38-to-44 age range. In addition, the number of live births was unavailable.

 

Among women aged 30 to 44 years without a history of infertility who had been trying to conceive for 3 months or less, biomarkers indicating diminished ovarian reserve compared with normal ovarian reserve were not associated with reduced fertility. These findings do not support the use of urinary or blood FSH tests or AMH levels to assess natural fertility for women with these characteristics. The researchers’ next want to see whether low AMH is associated with a higher risk of miscarriage among the women who conceived.

 

Although AMH testing isn’t designed to be an overall gauge of a woman’s fertility, it can still provide valuable information, especially for women who are infertile and seeking treatment. It can assist in diagnosing polycystic ovarian syndrome, and identify when a woman is getting closer to menopause. Previous research also showed that AMH is good predictor of a woman’s response to ovarian stimulation for in vitro fertilization and therefore it can predict the probability of conceiving via in vitro fertilization (I.V.F.).

 

References:

 

https://jamanetwork.com/journals/jama/article-abstract/2656811?JamaNetworkReader=True

 

https://www.nytimes.com/2017/10/16/health/fertility-test-ovarian-reserve.html

 

https://academic.oup.com/humrep/article/26/11/2925/656065

 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3339896/

 

https://www.ncbi.nlm.nih.gov/pubmed/27179263

 

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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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Effect of mitochondrial stress on epigenetic modifiers

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Early Mitochondrial Stress Alters Epigenetics, Secures Lifelong Health Benefits

GEN 5/3/2016  http://www.genengnews.com/gen-news-highlights/early-mitochondrial-stress-alters-epigenetics-secures-lifelong-health-benefits/81252685/

A little adversity builds character, or so the saying goes. True or not, the saying does seem an apt description of a developmental phenomenon that shapes gene expression. While it knows nothing of character, the gene expression apparatus appears to respond well to short-term mitochondrial stress that occurs early in development. In fact, transient stress seems to result in lasting benefits. These benefits, which include improved metabolic function and increased longevity, have been observed in both worms and mice, and may even occur—or be made to occur—in humans.

Gene expression is known to be subject to reprogramming by epigenetic modifiers, but such modifiers generally affect metabolism or lifespan, not both. A new set of epigenetic modifiers, however, has been found to trigger changes that do just that—both improve metabolism and extend lifespan.

Scientists based at the University of California, Berkeley, and the École Polytechnique Fédérale de Lausanne (EPFL) have discovered enzymes that are ramped up after mild stress during early development and continue to affect the expression of genes throughout the animal’s life. When the scientists looked at strains of inbred mice that have radically different lifespans, those with the longest lifespans had significantly higher expression of these enzymes than did the short-lived mice.

“Two of the enzymes we discovered are highly, highly correlated with lifespan; it is the biggest genetic correlation that has ever been found for lifespan in mice, and they’re both naturally occurring variants,” said Andrew Dillin, a UC Berkeley professor of molecular and cell biology. “Based on what we see in worms, boosting these enzymes could reprogram your metabolism to create better health, with a possible side effect of altering lifespan.”

Details of the work, which appeared online April 29 in the journal Cell, are presented in a pair of papers. One paper (“Two Conserved Histone Demethylases Regulate Mitochondrial Stress-Induced Longevity”) resulted from an effort led by Dillin and the EPFL’s Johan Auwerx. The other paper (“Mitochondrial Stress Induces Chromatin Reorganization to Promote Longevity and UPRmt”) resulted from an effort led by Dillin and his UC Berkeley colleague Barbara Meyer.

According to these papers, mitochondrial stress activates enzymes in the brain that affect DNA folding, exposing a segment of DNA that contains the 1500 genes involved in the work of the mitochondria. A second set of enzymes then tags these genes, affecting their activation for much or all of the lifetime of the animal and causing permanent changes in how the mitochondria generates energy.

The first set of enzymes—methylases, in particular LIN-65—add methyl groups to the DNA, which can silence promoters and thus suppress gene expression. By also opening up the mitochondrial genes, these methylases set the stage for the second set of enzymes—demethylases, in this case jmjd-1.2 and jmjd-3.1—to ramp up transcription of the mitochondrial genes. When the researchers artificially increased production of the demethylases in worms, all the worms lived longer, a result identical to what is observed after mitochondrial stress.

“By changing the epigenetic state, these enzymes are able to switch genes on and off,” Dillin noted. This happens only in the brain of the worm, however, in areas that sense hunger or satiety. “These genes are expressed in neurons that are sensing the nutritional status of the animal, and these signals emanate out to the periphery to change peripheral metabolism,” he continued.

When the scientists profiled enzymes in short- and long-lived mice, they found upregulation of these genes in the brains of long-lived mice, but not in other tissues or in the brains of short-lived mice. “These genes are expressed in the hypothalamus, exactly where, when you eat, the signals are generated that tell you that you are full. And when you are hungry, signals in that region tell you to go and eat,” Dillin explained said. “These genes are all involved in peripheral feedback.”

Among the mitochondrial genes activated by these enzymes are those involved in the body’s response to proteins that unfold, which is a sign of stress. Increased activity of the proteins that refold other proteins is another hallmark of longer life.

These observations suggest that the reversal of aging by epigenetic enzymes could also take place in humans.

“It seems that, while extreme metabolic stress can lead to problems later in life, mild stress early in development says to the body, ‘Whoa, things are a little bit off-kilter here, let’s try to repair this and make it better.’ These epigenetic switches keep this up for the rest of the animal’s life,” Dillin stated.

 

Two Conserved Histone Demethylases Regulate Mitochondrial Stress-Induced Longevity

Carsten Merkwirth6, Virginija Jovaisaite6, Jenni Durieux,…., Reuben J. Shaw, Johan Auwerx, Andrew Dillin

Highlights
  • H3K27 demethylases jmjd-1.2 and jmjd-3.1 are required for ETC-mediated longevity
  • jmjd-1.2 and jmjd-3.1 extend lifespan and are sufficient for UPRmt activation
  • UPRmt is required for increased lifespan due to jmjd-1.2 or jmjd-3.1 overexpression
  • JMJD expression is correlated with UPRmt and murine lifespan in inbred BXD lines

Across eukaryotic species, mild mitochondrial stress can have beneficial effects on the lifespan of organisms. Mitochondrial dysfunction activates an unfolded protein response (UPRmt), a stress signaling mechanism designed to ensure mitochondrial homeostasis. Perturbation of mitochondria during larval development in C. elegans not only delays aging but also maintains UPRmt signaling, suggesting an epigenetic mechanism that modulates both longevity and mitochondrial proteostasis throughout life. We identify the conserved histone lysine demethylases jmjd-1.2/PHF8 and jmjd-3.1/JMJD3 as positive regulators of lifespan in response to mitochondrial dysfunction across species. Reduction of function of the demethylases potently suppresses longevity and UPRmt induction, while gain of function is sufficient to extend lifespan in a UPRmt-dependent manner. A systems genetics approach in the BXD mouse reference population further indicates conserved roles of the mammalian orthologs in longevity and UPRmt signaling. These findings illustrate an evolutionary conserved epigenetic mechanism that determines the rate of aging downstream of mitochondrial perturbations.

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Mitochondrial Stress Induces Chromatin Reorganization to Promote Longevity and UPRmt
Ye Tian, Gilberto Garcia, Qian Bian, Kristan K. Steffen, Larry Joe, Suzanne Wolff, Barbara J. Meyer, Andrew Dillincorrespondence
http://dx.doi.org/10.1016/j.cell.2016.04.011             Publication stage: In Press Corrected Proof
Highlights
  • LIN-65 accumulates in the nucleus in response to mitochondrial stress
  • Mitochondrial stress-induced chromatin changes depend on MET-2 and LIN-65
  • LIN-65 and DVE-1 exhibit interdependence in nuclear accumulation
  • met-2 and atfs-1 act in parallel to affect mitochondrial stress-induced longevity

Organisms respond to mitochondrial stress through the upregulation of an array of protective genes, often perpetuating an early response to metabolic dysfunction across a lifetime. We find that mitochondrial stress causes widespread changes in chromatin structure through histone H3K9 di-methylation marks traditionally associated with gene silencing. Mitochondrial stress response activation requires the di-methylation of histone H3K9 through the activity of the histone methyltransferase met-2 and the nuclear co-factor lin-65. While globally the chromatin becomes silenced by these marks, remaining portions of the chromatin open up, at which point the binding of canonical stress responsive factors such as DVE-1 occurs. Thus, a metabolic stress response is established and propagated into adulthood of animals through specific epigenetic modifications that allow for selective gene expression and lifespan extension

 Siddharta Mukherjee’s Writing Career Just Got Dealt a Sucker Punch
Author: Theral Timpson

Siddharha Mukherjee won the 2011 Pulitzer Prize in non-fiction for his book, The Emperor of All Maladies.  The book has received widespread acclaim among lay audience, physicians, and scientists alike.  Last year the book was turned into a special PBS series.  But, according to a slew of scientists, we should all be skeptical of his next book scheduled to hit book shelves this month, The Gene, An Intimate History.

Publishing an article on epigenetics in the New Yorker this week–perhaps a selection from his new book–Mukherjee has waltzed into one of the most active scientific debates in all of biology: that of gene regulation, or epigenetics.

Jerry Coyne, the evolutionary biologist known for keeping journalists honest, has published a two part critique of Mukherjee’s New Yorker piece.  The first part–wildly tweeted yesterday–is a list of quotes from Coyne’s colleagues and those who have written in to the New Yorker, including two Nobel prize winners, Wally Gilbert and Sidney Altman, offering some very unfriendly sentences.

Wally Gilbert: “The New Yorker article is so wildly wrong that it defies rational analysis.”

Sidney Altman:  “I am not aware that there is such a thing as an epigenetic code.  It is unfortunate to inflict this article, without proper scientific review, on the audience of the New Yorker.”

The second part is a thorough scientific rebuttal of the Mukherjee piece.  It all serves as a great drama about one of the most contested ideas in biology and also as a cautionary tale to journalists, even experienced writers such as Mukherjee, about the dangers of wading into scientific arguments.  Readers may remember that a few years ago, science writer, David Dobbs, similarly skated into the same topic with his piece, Die, Selfish Gene, Die, and which raised a similar shitstorm, much of it from Coyne.

Mukherjee’s mistake is in giving credence to only one side of a very fierce debate–that the environment causes changes in the genome which can be passed on; another kind of evolution–as though it were settled science.   Either Mukherjee, a physicisan coming off from a successful book and PBS miniseries on cancer, is setting himself up as a scientist, or he has been a truly naive science reporter.   If he got this chapter so wrong, what does it mean about an entire book on the gene?

Coyne quotes one of his colleagues who raised some questions about the New Yorker’s science reporting, one particular question we’ve been asking here at Mendelspod.  How do we know what we know?  Does science now have an edge on any other discipline for being able to create knowledge?

Coyne’s colleague is troubled by science coverage in the New Yorker, and goes so far as to write that the New Yorker has been waging a “war on behalf of cultural critics and literary intellectuals against scientists and technologists.”

From my experience, it’s not quite that tidy.  First of all, the New Yorker is the best writing I read each week.  Period.  Second, I haven’t found their science writing to have the slant claimed in the quote above.  For example, most other mainstream outlets–including the New York Times with the Amy Harmon pieces–have given the anti-GMO crowd an equal say in the mistaken search for a “balance” on whether GMOs are harmful.  (Remember John Stewart’s criticism of Fox News?  That they give a false equivalent between two sides even when there is no equivalent on the other side?)

But the New Yorker has not fallen into this trap on GMOs and most of their pieces on the topic–mainly by Michael Specter–have been decidedly pro science and therefore decided pro GMO.

So what led Mukherjee to play scientist as well as journalist?  There’s no question about whether I enjoy his prose.  His writing beautifully whisks me away so that I don’t feel that I’m really working to understand.  There is a poetic complexity that constantly brings different threads effortlessly together, weaving them into the same light.  At one point he uses the metaphor of a web for the genome, with the epigenome being the stuff that sticks to the web.  He borrows the metaphor from the Hindu notion of “being”, or jaal.

“Genes form the threads of the web; the detritus that adheres to it transforms every web into a singular being.”

There have been a few writers on Twitter defending Mukherjee’s piece.  Tech Review’s Antonio Regalado called Coyne and his colleagues “tedious literalists” who have an “issue with epigenetic poetry.”

At his best, Mukherjee can take us down the sweet alleys of his metaphors and family stories with a new curiosity for the scientific truth.  He can hold a mirror up to scientists, or put the spotlight on their work.   At their worst, Coyne and his scientific colleagues can reek of a fear of language and therefore metaphor.  The always outspoken scientist and author, Richard Dawkins, who made his name by personifying the gene, was quick to personify epigentics in a tweet:   “It’s high time the 15 minutes of underserved fame for “epigenetics” came to an overdue end.”  Dawkins is that rare scientist who has consistently been as comfortable with rhetoric and language as he is with data.

Hats off to Coyne who reminds us that a metaphor–however lovely–does not some science make. If Mukherjee wants to play scientist, let him create and gather data. If it’s the role of science journalist he wants, let him collect all the science he can before he begins to pour it into his poetry.

 

Same but Different  

How epigenetics can blur the line between nature and nurture.

Annals of Science MAY 2, 2016 ISSUE     BY

The author’s mother (right) and her twin are a study in difference and identity. CREDIT: PHOTOGRAPH BY DAYANITA SINGH FOR THE NEW YORKER

October 6, 1942, my mother was born twice in Delhi. Bulu, her identical twin, came first, placid and beautiful. My mother, Tulu, emerged several minutes later, squirming and squalling. The midwife must have known enough about infants to recognize that the beautiful are often the damned: the quiet twin, on the edge of listlessness, was severely undernourished and had to be swaddled in blankets and revived.

The first few days of my aunt’s life were the most tenuous. She could not suckle at the breast, the story runs, and there were no infant bottles to be found in Delhi in the forties, so she was fed through a cotton wick dipped in milk, and then from a cowrie shell shaped like a spoon. When the breast milk began to run dry, at seven months, my mother was quickly weaned so that her sister could have the last remnants.
Tulu and Bulu grew up looking strikingly similar: they had the same freckled skin, almond-shaped face, and high cheekbones, unusual among Bengalis, and a slight downward tilt of the outer edge of the eye, something that Italian painters used to make Madonnas exude a mysterious empathy. They shared an inner language, as so often happens with twins; they had jokes that only the other twin understood. They even smelled the same: when I was four or five and Bulu came to visit us, my mother, in a bait-and-switch trick that amused her endlessly, would send her sister to put me to bed; eventually, searching in the half-light for identity and difference—for the precise map of freckles on her face—I would realize that I had been fooled.

But the differences were striking, too. My mother was boisterous. She had a mercurial temper that rose fast and died suddenly, like a gust of wind in a tunnel. Bulu was physically timid yet intellectually more adventurous. Her mind was more agile, her tongue sharper, her wit more lancing. Tulu was gregarious. She made friends easily. She was impervious to insults. Bulu was reserved, quieter, and more brittle. Tulu liked theatre and dancing. Bulu was a poet, a writer, a dreamer.

….. more

Why are identical twins alike? In the late nineteen-seventies, a team of scientists in Minnesota set out to determine how much these similarities arose from genes, rather than environments—from “nature,” rather than “nurture.” Scouring thousands of adoption records and news clips, the researchers gleaned a rare cohort of fifty-six identical twins who had been separated at birth. Reared in different families and different cities, often in vastly dissimilar circumstances, these twins shared only their genomes. Yet on tests designed to measure personality, attitudes, temperaments, and anxieties, they converged astonishingly. Social and political attitudes were powerfully correlated: liberals clustered with liberals, and orthodoxy was twinned with orthodoxy. The same went for religiosity (or its absence), even for the ability to be transported by an aesthetic experience. Two brothers, separated by geographic and economic continents, might be brought to tears by the same Chopin nocturne, as if responding to some subtle, common chord struck by their genomes.

One pair of twins both suffered crippling migraines, owned dogs that they had named Toy, married women named Linda, and had sons named James Allan (although one spelled the middle name with a single “l”). Another pair—one brought up Jewish, in Trinidad, and the other Catholic, in Nazi Germany, where he joined the Hitler Youth—wore blue shirts with epaulets and four pockets, and shared peculiar obsessive behaviors, such as flushing the toilet before using it. Both had invented fake sneezes to diffuse tense moments. Two sisters—separated long before the development of language—had invented the same word to describe the way they scrunched up their noses: “squidging.” Another pair confessed that they had been haunted by nightmares of being suffocated by various metallic objects—doorknobs, fishhooks, and the like.

The Minnesota twin study raised questions about the depth and pervasiveness of qualities specified by genes: Where in the genome, exactly, might one find the locus of recurrent nightmares or of fake sneezes? Yet it provoked an equally puzzling converse question: Why are identical twins different? Because, you might answer, fate impinges differently on their bodies. One twin falls down the crumbling stairs of her Calcutta house and breaks her ankle; the other scalds her thigh on a tipped cup of coffee in a European station. Each acquires the wounds, calluses, and memories of chance and fate. But how are these changes recorded, so that they persist over the years? We know that the genome can manufacture identity; the trickier question is how it gives rise to difference.

….. more

But what turns those genes on and off, and keeps them turned on or off? Why doesn’t a liver cell wake up one morning and find itself transformed into a neuron? Allis unpacked the problem further: suppose he could find an organism with two distinct sets of genes—an active set and an inactive set—between which it regularly toggled. If he could identify the molecular switches that maintain one state, or toggle between the two states, he might be able to identify the mechanism responsible for cellular memory. “What I really needed, then, was a cell with these properties,” he recalled when we spoke at his office a few weeks ago. “Two sets of genes, turned ‘on’ or ‘off’ by some signal.”

more…

“Histones had been known as part of the inner scaffold for DNA for decades,” Allis went on. “But most biologists thought of these proteins merely as packaging, or stuffing, for genes.” When Allis gave scientific seminars in the early nineties, he recalled, skeptics asked him why he was so obsessed with the packing material, the stuff in between the DNA.  …. A skein of silk tangled into a ball has very different properties from that same skein extended; might the coiling or uncoiling of DNA change the activity of genes?

In 1996, Allis and his research group deepened this theory with a seminal discovery. “We became interested in the process of histone modification,” he said. “What is the signal that changes the structure of the histone so that DNA can be packed into such radically different states? We finally found a protein that makes a specific chemical change in the histone, possibly forcing the DNA coil to open. And when we studied the properties of this protein it became quite clear that it was also changing the activity of genes.” The coils of DNA seemed to open and close in response to histone modifications—inhaling, exhaling, inhaling, like life.

Allis walked me to his lab, a fluorescent-lit space overlooking the East River, divided by wide, polished-stone benches. A mechanical stirrer, whirring in a corner, clinked on the edge of a glass beaker. “Two features of histone modifications are notable,” Allis said. “First, changing histones can change the activity of a gene without affecting the sequence of the DNA.” It is, in short, formally epi-genetic, just as Waddington had imagined. “And, second, the histone modifications are passed from a parent cell to its daughter cells when cells divide. A cell can thus record ‘memory,’ and not just for itself but for all its daughter cells.”

…..

 

 

The New Yorker screws up big time with science: researchers criticize the Mukherjee piece on epigenetics

Jerry Coyne
https://whyevolutionistrue.wordpress.com/2016/05/05/the-new-yorker-screws-up-big-time-with-science-researchers-criticize-the-mukherjee-piece-on-epigenetics/

Abstract: This is a two part-post about a science piece on gene regulation that just appeared in the New Yorker. Today I give quotes from scientists criticizing that piece; tomorrow I’ll present a semi-formal critique of the piece by two experts in the field.

esterday I gave readers an assignment: read the new New Yorkerpiece by Siddhartha Mukherjee about epigenetics. The piece, called “Same but different” (subtitle: “How epigenetics can blur the line between nature and nurture”) was brought to my attention by two readers, both of whom praised it.  Mukherjee, a physician, is well known for writing the Pulitzer-Prize-winning book (2011) The Emperor of All Maladies: A Biography of Cancer. (I haven’t read it yet, but it’s on my list.)  Mukherjee has a new book that will be published in May: The Gene: An Intimate History. As I haven’t seen it, the New Yorker piece may be an excerpt from this book.

Everyone I know who has read The Emperor of All Maladies gives it high praise. I wish I could say the same for Mukherjee’s New Yorker piece. When I read it at the behest of the two readers, I found his analysis of gene regulation incomplete and superficial. Although I’m not an expert in that area, I knew that there was a lot of evidence that regulatory proteins called “transcription factors”, and not “epigenetic markers” (see discussion of this term tomorrow) or modified histones—the factors emphasized by Mukherjee—played hugely important roles in gene regulation. The speculations at the end of the piece about “Lamarckian evolution” via environmentally induced epigenetic changes in the genome were also unfounded, for we have no evidence for that kind of adaptive evolution. Mukherjee does, however, mention that lack of evidence, though I wish he’d done so more strongly given that environmental modification of DNA bases is constantly touted as an important and neglected factor in evolution.

Unbeknownst to me, there was a bit of a kerfuffle going on in the community of scientists who study gene regulation, with many of them finding serious mistakes and omissions in Mukherjee’s piece.  There appears to have been some back-and-forth emailing among them, and several wrote letters to the New Yorker, urging them to correct the misconceptions, omissions, and scientific errors in “Same but different.” As I understand it, both Mukherjee and the New Yorker simply batted these criticisms away, and, as far as I know, will not publish any corrections.  So today and tomorrow I’ll present the criticisms here, just so they’ll be on the record.

Because Mukherjee writes very well, and because even educated laypeople won’t know the story of gene regulation revealed over the last few decades,  they may not see the big lacunae in his piece. It is, then,  important to set matters straight, for at least we should know what science has told us about how genes are turned on and off. The criticism of Mukherjee’s piece, coming from scientists who really are experts in gene regulation, shows a lack of care on the part of Mukherjee and theNew Yorker: both a superficial and misleading treatment of the state of the science, and a failure of the magazine to properly vet this piece (I have no idea whether they had it “refereed” not just by editors but by scientists not mentioned in the piece).

Let me add one thing about science and the New Yorker. I believe I’ve said this before, but the way the New Yorker treats science is symptomatic of the “two cultures” problem. This is summarized in an email sent me a while back by a colleague, which I quote with permission:

The New Yorker is fine with science that either serves a literary purpose (doctors’ portraits of interesting patients) or a political purpose (environmental writing with its implicit critique of modern technology and capitalism). But the subtext of most of its coverage (there are exceptions) is that scientists are just a self-interested tribe with their own narrative and no claim to finding the truth, and that science must concede the supremacy of literary culture when it comes to anything human, and never try to submit human affairs to quantification or consilience with biology. Because the magazine is undoubtedly sophisticated in its writing and editing they don’t flaunt their postmodernism or their literary-intellectual proprietariness, but once you notice it you can make sense of a lot of their material.

. . . Obviously there are exceptions – Atul Gawande is consistently superb – but as soon as you notice it, their guild war on behalf of cultural critics and literary intellectuals against scientists, technologists, and analytic scholars becomes apparent.

…. more

Researchers criticize the Mukherjee piece on epigenetics: Part 2

Trigger warning: Long science post!

Yesterday I provided a bunch of scientists’ reactions—and these were big names in the field of gene regulation—to Siddhartha Mukherjee’s ill-informed piece in The New Yorker, “Same but different” (subtitle: “How epigenetics can blur the line between nature and nurture”). Today, in part 2, I provide a sentence-by-sentence analysis and reaction by two renowned researchers in that area. We’ll start with a set of definitions (provided by the authors) that we need to understand the debate, and then proceed to the critique.

Let me add one thing to avoid confusion: everything below the line, including the definition (except for my one comment at the end) was written by Ptashne and Greally.

by Mark Ptashne and John Greally

Introduction

Ptashne is The Ludwig Professor of Molecular Biology at the Memorial Sloan Kettering Cancer Center in New York. He wrote A Genetic Switch, now in its third edition, which describes the principles of gene regulation and the workings of a ‘switch’; and, with Alex Gann, Genes and Signals, which extends these principles and ideas to higher organisms and to other cellular processes as well.  John Greally is the Director of the Center for Epigenomics at the Albert Einstein College of Medicine in New York.

 

The New Yorker  (May 2, 2016) published an article entitled “Same But Different” written by Siddhartha Mukherjee.  As readers will have gathered from the letters posted yesterday, there is a concern that the article is misleading, especially for a non-scientific audience. The issue concerns our current understanding of “gene regulation” and how that understanding has been arrived at.

First some definitions/concepts:

Gene regulation refers to the “turning on and off of genes”.  The primary event in turning a gene “on” is to transcribe (copy) it into messenger RNA (mRNA). That mRNA is then decoded, usually, into a specific protein.  Genes are transcribed by the enzyme called RNA polymerase.

Development:  the process in which a fertilized egg (e.g., a human egg) divides many times and eventually forms an organism.  During this process, many of the roughly 23,000 genes of a human are turned “on” or “off” in different combinations, at different times and places in the developing organism. The process produces many different cell types in different organs (e.g. liver and brain), but all retain the original set of genes.

Transcription factors: proteins that bind to specific DNA sequences near specific genes and turn transcription of those genes on and off. A transcriptional ‘activator’, for example, bears two surfaces: one binds a specific sequence in DNA, and the other binds to, and thereby recruits to the gene, protein complexes that include RNA polymerase. It is widely acknowledged that the identity of a cell in the body depends on the array of transcription factors present in the cell, and the cell’s history.  RNA molecules can also recognize specific genomic sequences, and they too sometimes work as regulators.  Neither transcription factors nor these kinds of RNA molecules – the fundamental regulators of gene expression and development – are mentioned in the New Yorker article.

Signals:  these come in many forms (small molecules like estrogen, larger molecules (often proteins such as cytokines) that determine the ability of transcription factors to work.  For example, estrogen binds directly to a transcription factor (the estrogen receptor) and, by changing its shape, permits it to bind DNA and activate transcription.

Memory”:  a dividing cell can (often does) produce daughters that are identical, and that express identical genes as does the mother cell.  This occurs because the transcription factors present in the mother cell are passively transmitted to the daughters as the cell divides, and they go to work in their new contexts as before.  To make two different daughters, the cell must distribute its transcription factors asymmetrically.

Positive Feedback: An activator can maintain its own expression by  positive feedback.  This requires, simply, that a copy of the DNA sequence to which the activator binds is  present  near its own gene. Expression of the activator  then becomes self-perpetuating.  The activator (of which there now are many copies in the cell) activates  other target genes as it maintains its own expression. This kind of ‘memory circuit’, first described  in  bacteria, is found in higher organisms as well.  Positive feedback can explain how a fully differentiated cell (that is, a cell that has reached its developmental endpoint) maintains its identity.

Nucleosomes:  DNA in higher organisms (eukaryotes) is wrapped, like beads on a string, around certain proteins (called histones), to form nucleosomes.  The histones are subject to enzymatic modifications: e.g., acetyl, methyl, phosphate, etc. groups can be added to these structures. In bacteria there are no nucleosomes, and the DNA is more or less ‘naked’.

“Epigenetic modifications: please don’t worry about the word ”epigenetic”; it is misused in any case. What Mukherjee refers to by this term are the histone modifications mentioned above, and a modification to DNA itself: the addition of methyl groups. Keep in mind that the organisms that have taught us the most about development – flies (Drosophila) and worms (C. elegans)—do not have the enzymes required for DNA methylation. That does not mean that DNA methylation cannot do interesting things in humans, for example, but it is obviously not at the heart of gene regulation.

Specificity Development requires the highly specific sequential turning on and off of sets of genes.  Transcription factors and RNA supply this specificity, but   enzymes that impart modifications to histones  cannot: every nucleosome (and hence every gene) appears the same to the enzyme.  Thus such enzymes cannot pick out particular nucleosomes associated with particular genes to modify.  Histone modifications might be imagined to convey ‘memory’ as cells divide – but there are no convincing indications that this happens, nor are there molecular models that might explain why they would have the imputed effects.

Analysis and critique of Mukherjee’s article

The picture we have just sketched has taken the combined efforts of many scientists over 50 years to develop.  So what, then, is the problem with the New Yorker article?

There are two: first, the picture we have just sketched, emphasizing the primary role of transcription factors and RNA, is absent.  Second, that picture is replaced by highly dubious speculations, some of which don’t make sense, and none of which has been shown to work as imagined in the article.

(Quotes from the Mukherjee article are indented and in plain text; they are followed by comments, flush left and in bold, by Ptashne and Greally.)

In 1978, having obtained a Ph.D. in biology at Indiana University, Allis began to tackle a problem that had long troubled geneticists and cell biologists: if all the cells in the body have the same genome, how does one become a nerve cell, say, and another a blood cell, which looks and functions very differently?

The problems referred to were recognized long before 1978.  In fact, these were exactly the problems that the great French scientists François Jacob and Jacques Monod took on in the 1950s-60s.  In a series of brilliant experiments, Jacob and Monod showed that in bacteria, certain genes encode products that regulate (turn on and off) specific other genes.  Those regulatory molecules turned out to be proteins, some of which respond to signals from the environment.  Much of the story of modern biology has been figuring out how these proteins – in bacteria and in higher organisms  – bind to and regulate specific genes.  Of note is that in higher organisms, the regulatory proteins look and act like those in bacteria, despite the fact that eukaryotic DNA is wrapped in nucleosomes  whereas bacterial DNA is not.   We have also learned that certain RNA molecules can play a regulatory role, a phenomenon made possible by the fact that RNA molecules, like regulatory proteins, can recognize specific genomic sequences.

In the nineteen-forties, Conrad Waddington, an English embryologist, had proposed an ingenious answer: cells acquired their identities just as humans do—by letting nurture (environmental signals) modify nature (genes). For that to happen, Waddington concluded, an additional layer of information must exist within a cell—a layer that hovered, ghostlike, above the genome. This layer would carry the “memory” of the cell, recording its past and establishing its future, marking its identity and its destiny but permitting that identity to be changed, if needed. He termed the phenomenon “epigenetics”—“above genetics.”

This description greatly misrepresents the original concept.  Waddington argued that development proceeds not by the loss (or gain) of genes, which would be a “genetic” process, but rather that some genes would be selectively expressed in specific and complex cellular patterns as development proceeds.  He referred to this intersection of embryology (then called “epigenesis”) and genetics as “epigenetic”.We now understand that regulatory proteins work in combinations to turn on and off genes, including their own genes, and that sometimes the regulatory proteins respond to signals sent by other cells.  It should be emphasized that Waddington never proposed any “ghost-like” layer of additional information hovering above the gene.  This is a later misinterpretation of a literal translation of the term epigenetics, with “epi-“ meaning “above/upon” the genetic information encoded in DNA sequence.  Unfortunately, this new and pervasive definition encompasses all of transcriptional regulation and is of no practical value.

…..more

By 2000, Allis and his colleagues around the world had identified a gamut of proteins that could modify histones, and so modulate the activity of genes. Other systems, too, that could scratch different kinds of code on the genome were identified (some of these discoveries predating the identification of histone modifications). One involved the addition of a chemical side chain, called a methyl group, to DNA. The methyl groups hang off the DNA string like Christmas ornaments, and specific proteins add and remove the ornaments, in effect “decorating” the genome. The most heavily methylated parts of the genome tend to be dampened in their activity.

It is true that enzymes that modify histones have been found—lots of them.  A striking problem is that, after all this time, it is not at all clear what the vast majority of these modifications do.  When these enzymatic activities are eliminated by mutation of their active sites (a task substantially easier to accomplish in yeast than in higher organisms) they mostly have little or no effect on transcription.  It is not even clear that histones are the biologically relevant substrates of most of these enzymes.  

 In the ensuing decade, Allis wrote enormous, magisterial papers in which a rich cast of histone-modifying proteins appear and reappear through various roles, mapping out a hatchwork of complexity. . . These protein systems, overlaying information on the genome, interacted with one another, reinforcing or attenuating their signals. Together, they generated the bewildering intricacy necessary for a cell to build a constellation of other cells out of the same genes, and for the cells to add “memories” to their genomes and transmit these memories to their progeny. “There’s an epigenetic code, just like there’s a genetic code,” Allis said. “There are codes to make parts of the genome more active, and codes to make them inactive.”

By ‘epigenetic code’ the author seems to mean specific arrays of nucleosome modifications, imparted over time and cell divisions, marking genes for expression.  This idea has been tested in many experiments and has been found not to hold.

….. and more

 

Larry H. Bernstein, MD, FCAP

I hope that this piece brings greater clarity to the discussion.  I have heard the use of the term “epigenetics” for over a decade.  The term was never so clear.  I think that the New Yorker article was a reasonable article for the intended audience.  It was not intended to clarify debates about a mechanism for epigenetic based changes in evolutionary science.  I think it actually punctures the “classic model” of the cell depending only on double stranded DNA and transcription, which deflates our concept of the living cell.  The concept of epigenetics was never really formulated as far as I have seen, and I have done serious work in enzymology and proteins at a time that we did not have the technology that exists today.  I have considered with the critics that protein folding, protein misfolding, protein interactions with proximity of polar and nonpolar groups, and the regulatory role of microRNAs that are not involved in translation, and the evolving concept of what is “dark (noncoding) DNA” lend credence to the complexity of this discussion.  Even more interesting is the fact that enzymes (and isoforms of enzymes) have a huge role in cellular metabolic differences and in the function of metabolic pathways.  What is less understood is the extremely fast reactions involved in these cellular reactions.  These reactions are in my view critical drivers.  This is brought out by Erwin Schroedinger in the book What is Life? which infers that there can be no mathematical expression of life processes.

 

 

 

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What about PDL-1 in oncotherapy diagnostics for NSCLC?

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Questions on PD-L1 Diagnostics for Immunotherapy in NSCLC
Alexander M. Castellino, PhD
http://www.medscape.com/viewarticle/862275

Two immunotherapies that target the cell programmed death (PD) pathway are now available, and both nivolumab (Opdivo, Bristol-Myers Squibb Company) and pembrolizumab (Keytruda, Merck Sharp & Dohme Corp) are approved for treating advanced, refractory, non–small cell lung cancer (NSCLC). Across several studies in patients with NSCLC, response to these agents has been correlated with PD-L1 staining, which determines PD-L1 levels in the tumor tissue. How do the available assays for PD-L1 compare?

The linear correlation between three commercially available assays is good across a range of cutoff points, concluded a presentation at the 2016 American Association for Clinical Research Annual Meeting.

Cutoffs are defined as the percentage of cells expressing PD-L1 when analyzed histochemically. “The dataset builds confidence that the assays may be used according to the cutoff clinically validated for the drug in question,” Marianne J. Radcliffe, MD, diagnostic associate director at AstraZeneca, toldMedscape Medical News.

“The correlation is good between the assays across the range examined,” she added.

However, a recently published study showed a high rate of discordance between another set of PD-L1 assays that were tested.

Dr Marianne Radcliffe

“Different diagnostic tests yield different results, depending on the cutoff for each assay. We need to harmonize the assays so clinicians are talking about the same thing,” Brendon Stiles, MD, associate professor of cardiothoracic surgery at Weill Cornell Medicine and New York-Presbyterian Hospital, New York City, told Medscape Medical News.

For Dr Stiles, these studies raise the issue that it is difficult to compare results of diagnostic testing across the different drugs and even with the same drug that are derived from different assays. “More importantly, it raises confusion in clinical practice when a patient’s sample stains positive for PD-L1 with one assay and negative with another,” he said.

“The commercial strategy for developing companion diagnostics for each drug is not in the best interests of the patients. It generates confusion among both clinicians and patients,” Dr Stiles commented. “We need to know if these assays can be used interchangeably,” he said.

As new agents come into the clinic, Dr Stiles believes there should be a universal yes-or-no answer, so that clinicians can use the assay to help decide on the use of immunotherapy.

Three Assays Tested

The study presented by Dr Radcliffe and colleagues investigated three commercially available assays, Ventana SP263, Dako 22C3, and Dako 28-8, with regard to how they compare at different cutoffs. Different studies use different cutoffs to express positivity.

Ventana SP263 was developed as a companion diagnostic for durvalumab (under development by AstraZeneca) using a rabbit monoclonal antibody. Positivity is defined as ≥25% staining of tumor cells.

Dako 22C3 was developed, and is approved, as a companion diagnostic for pembrolizumab. It uses a mouse monoclonal antibody. Positivity is defined as ≥1% and ≥50% staining of tumor cells.

Dako 28-8 was developed as a companion diagnostic for nivolumab and uses a rabbit monoclonal antibody (different from the one used in the Ventana SP263). In clinical practice, this assay is used as a complementary diagnostic for nivolumab, but the drug is approved for use regardless of PD-L1 expression. Positivity is defined as ≥1%, ≥5%, or ≥10% staining of tumor cells.

Ventana SP142 was not included in the study because it is not commercially available, Dr Ratcliffe indicated.
The three assays were used on consecutive sections of 500 archival NSCLC tumor samples obtained from commercial vendors. A single pathologist trained by the manufacturer read all samples in batches on an assay-by-assay basis. Samples were assessed per package inserts provided by Ventana and Dako in a Clinical Laboratory Improvement Amendments program-certified laboratory.

Dr Ratcliffe indicated that between reads of samples from the same patient, there was a washout period for the pathologist to remove bias.

The NSCLC samples included patients with stage I (38%), II (39%), III (20%), and IV (<1%) disease. Histologies included nonsquamous (54%) and squamous (43%) cancers.

All three PD-L1 assays showed similar patterns of staining in the range of 0% to 100%, Dr Ratcliffe indicated.

 

The correlation between any two of the assays was determined from tumor cell membrane staining. The correlation was linear with Spearman correlation of 0.911 for Ventana SP263 vs Dako 22C3; 0.935 for Ventana SP263 vs Dako 28-8; and 0.954 for Dako 28-8 vs Dako 22C3.

“With an overall predictive value of >90%, the assays have closely aligned dynamic ranges, but more work is needed,” Dr Ratcliffe said. “In general, scoring of immunohistochemical assays can be more variable between 1% and 10%, and we plan to look at this in more detail,” she said. These samples need to be reviewed by an independent pathologist, she added.

Dr Radcliffe said that currently, “Direct clinical efficacy data supporting a specific diagnostic test should still be considered as the highest standard of proof for diagnostic clinical utility.”

Why Correlations Are Needed

Pembrolizumab is approved for use only in patients with PD-L1-positive, previously treated NSCLC. A similar patient profile is being considered for nivolumab, for which testing for PD-L1 expression is not required.

For new PD-immunotherapy agents in clinical development, it is not clear whether PD-L1 testing will be mandated.

However, in clinical practice, it is clear that some patients respond to therapy, even if they are PD-L1 negative, as defined from the study. “Is it a failure of the assay, tumor heterogeneity, or is there another time point when PD-L1 expression is turned on?” Dr Stiles asked.

Dr Stiles also pointed out that a recent publication from Yale researchers showed a high a rate of discordance. In this study, PD-L1 expression was determined using two rabbit monoclonal antibodies. Both of these were different from the ones used in the Ventana SP263 and Dako 28-8 assays.

In this study, whole-tissue sections from 49 NSCLC samples were used, and a corresponding tissue microarray was also used with the same 49 samples. Researchers showed that in 49 NSCLC tissue samples, there was intra-assay variability, with results showing fair to poor concordance with the two antibodies. “Assessment of 588 serial section fields of view from whole tissue showed discordant expression at a frequency of 25%.

“Objective determination of PD-L1 protein levels in NSCLC reveals heterogeneity within tumors and prominent interassay variability or discordance. This could be due to different antibody affinities, limited specificity, or distinct target epitopes. Efforts to determine the clinical value of these observations are under way,” the study authors conclude.

The Blueprint Proposal

Coincidentally, a blueprint proposal was announced here at the AACR meeting at a workshop entitled FDA-AACR-ASCO Complexities in Personalized Medicine: Harmonizing Companion Diagnostics across a Class of Targeted Therapies.

The blueprint proposal was developed by four pharmaceutical giants (Bristol-Myers Squibb Company, Merck & Co, Inc, AstraZeneca PLC, and Genentech, Inc) and two diagnostic companies (Agilent Technologies, Inc/Dako Corp and Roche/Ventana Medical Systems, Inc).

In this proposal, the development of an evidence base for PD-1/PD-L1 companion diagnostic characterization for NSCLC would be built into studies conducted in the preapproval stage. Once the tests are approved, the information will lay the foundation for postapproval studies to inform stakeholders (eg, patients, physicians, pathologists) on how the test results can best be used to make treatment decisions.

The blueprint proposal is available online.

Dr Ratcliffe is an employee and shareholder of AstraZeneca. Dr Stiles has disclosed no relevant financial relationships.

 American Association for Cancer Research (AACR) 2016 Annual Meeting: Abstract LB-094, presented April 18, 2016.
Quantitative Assessment of the Heterogeneity of PD-L1 Expression in Non–Small-Cell Lung Cancer
Joseph McLaughlin, 1,2; Gang Han, 3; Kurt A. Schalper, 2; ….,  Roy Herbst, 1; Patricia LoRusso, 1; David L. Rimm, 2

JAMA Oncol. 2016;2(1):46-54.       http://dx.doi.org:/10.1001/jamaoncol.2015.3638.

Importance  Early-phase trials with monoclonal antibodies targeting PD-1 (programmed cell death protein 1) and PD-L1 (programmed cell death 1 ligand 1) have demonstrated durable clinical responses in patients with non–small-cell lung cancer (NSCLC). However, current assays for the prognostic and/or predictive role of tumor PD-L1 expression are not standardized with respect to either quantity or distribution of expression.

Objective  To demonstrate PD-L1 protein distribution in NSCLC tumors using both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (QIF) and compare results obtained using 2 different PD-L1 antibodies.

Design, Setting, and Participants  PD-L1 was measured using E1L3N and SP142, 2 rabbit monoclonal antibodies, in 49 NSCLC whole-tissue sections and a corresponding tissue microarray with the same 49 cases. Non–small-cell lung cancer biopsy specimens from 2011 to 2012 were collected retrospectively from the Yale Thoracic Oncology Program Tissue Bank. Human melanoma Mel 624 cells stably transfected with PD-L1 as well as Mel 624 parental cells, and human term placenta whole tissue sections were used as controls and for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using chromogenic IHC and the AQUA (Automated Quantitative Analysis) method of QIF. Tumor-infiltrating lymphocytes (TILs) were scored in hematoxylin-eosin slides using current consensus guidelines. The association between PD-L1 protein expression, TILs, and clinicopathological features were determined.

Main Outcomes and Measures  PD-L1 expression discordance or heterogeneity using the diaminobenzidine chromogen and QIF was the main outcome measure selected prior to performing the study.

Results  Using chromogenic IHC, both antibodies showed fair to poor concordance. The PD-L1 antibodies showed poor concordance (Cohen κ range, 0.124-0.340) using conventional chromogenic IHC and showed intra-assay heterogeneity (E1L3N coefficient of variation [CV], 6.75%-75.24%; SP142 CV, 12.17%-109.61%) and significant interassay discordance using QIF (26.6%). Quantitative immunofluorescence showed that PD-L1 expression using both PD-L1 antibodies was heterogeneous. Using QIF, the scores obtained with E1L3N and SP142 for each tumor were significantly different according to nonparametric paired test (P < .001). Assessment of 588 serial section fields of view from whole tissue showed discordant expression at a frequency of 25%. Expression of PD-L1 was correlated with high TILs using both E1L3N (P = .007) and SP142 (P = .02).

Conclusions and Relevance  Objective determination of PD-L1 protein levels in NSCLC reveals heterogeneity within tumors and prominent interassay variability or discordance. This could be due to different antibody affinities, limited specificity, or distinct target epitopes. Efforts to determine the clinical value of these observations are under way.

 

 
Introduction We are in an era of rapid incorporation of basic scientific discoveries into the drug development pipeline. Currently, numerous sponsors are developing therapeutic products that may use similar or identical biomarkers for therapeutic selection, measured or detected by an in vitro companion diagnostic device. The current practice is to independently develop a companion diagnostic for each therapeutic. Thus, the matrix of therapeutics and companion diagnostics, if each therapeutic were approved in conjunction with a companion diagnostic, may present a complex challenge for testing and decision making in the clinic, potentially putting patients at risk if inappropriate diagnostic tests were used to make treatment decisions. To address this challenge, there is a desire to understand assay comparability and/or standardize analytical and clinical performance characteristics supporting claims that are shared across companion diagnostic devices. Pathologists and oncologists also need clarity on how to interpret test results to inform downstream treatment options for their patients.
Clearly using each of the companion diagnostics to select one of the several available targeted therapies in the same class is not practical and may be impossible. Likewise, having a single test or assay as a sole companion test for all of the multiple therapeutic options within a class is also impractical since the individual therapies have differing modes of action, intended use populations, specificities, safety and efficacy outcomes. Thus, a single assay or test may not adequately capture the appropriate patient population that may benefit (or not) from each individual therapeutic option within a class of therapies. Furthermore, aligning multiple sponsors’ study designs and timelines in order that they all adopt a single companion test may inadvertently slow down development of critical therapeutic products and delay patient access to these life-saving products.
Any solution to this challenge will be multifaceted and will, by necessity, involve multiple stakeholders. Thus, the US Food and Drug Administration (FDA), the American Association for Cancer Research (AACR) and American Society of Clinical Oncology (ASCO) convened a workshop titled “Complexities in Personalized Medicine: Harmonizing Companion Diagnostics Across a Class of Targeted Therapies” to draw out and assess possible solutions. Recognizing that the complex scientific, regulatory and market forces at play here require a collaborative effort, an industry workgroup volunteered to develop a blueprint proposal of potential solutions using nonsmall cell lung cancer (NSCLC) as the use case indication.
Goal and Scope of Blueprint The imminent arrival to the market of multiple PD1 / PD-L1 compounds and the possibility of one or more associated companion diagnostics is unprecedented in the field of oncology. Some may assume that since these products target the same biological pathway, they are interchangeable; however, each PD1/PD-L1 compound is unique with respect to its clinical pharmacology and each compound is being developed in the context of a unique biological scientific hypothesis and registration strategy. Similarly, each companion diagnostic has been optimized within the individual therapeutic development programs to meet specific development goals, e.g., 1) validation for patient selection, 2) subgroup analysis as a prognostic variable, or 3) enrichment.
Further, each companion diagnostic test is optimized for its specific therapy and with its own unique performance characteristics and scoring/interpretation guidelines.
The blueprint development group recognizes that to assume that any one of the available tests could be used for guiding the treatment decision with any one or all of the drugs available in this class presents a potential risk to patients that must be addressed.
The goal of this proposal is to agree and deliver, via cross industry collaboration, a package of information /data upon which analytic comparison of the various diagnostic assays may be conducted, potentially paving the way for post-market standardization and/or practice guideline development as appropriate.
A comparative study of PD-L1 diagnostic assays and the classification of patients as PD-L1 positive and PD-L1 negative
Presentation Time: Monday, Apr 18, 2016, 8:00 AM -12:00 PM
Location: Section 10
Poster Board Number: 18
Author Block: Marianne J. Ratcliffe1, Alan Sharpe2, Anita Midha1, Craig Barker2, Paul Scorer2, Jill Walker2. 1AstraZeneca, Alderley Park, United Kingdom; 2AstraZeneca, Cambridge, United Kingdom
Abstract Body: Background: PD-1/PD-L1 directed antibodies are emerging as effective therapeutics in multiple oncology settings. Keynote 001 and Checkmate 057 have shown more frequent response to PD-1 targeted therapies in NSCLC patients with high tumour PD-L1 expression than patients with low or no PD-L1 expression. Multiple diagnostic PD-L1 tests are available using different antibody clones, different staining protocols and diverse scoring algorithms. It is vital to compare these assays to allow appropriate interpretation of clinical outcomes. Such understanding will promote harmonization of PD-L1 testing in clinical practice.
Methods: Approximately 500 tumour biopsy samples from NSCLC patients, including squamous and non-squamous histologies, will be assessed using three leading PD-L1 diagnostics assays. PD-L1 assessment by the Ventana SP263 assay that is currently being used in Durvalumab clinical trials (positivity cut off: ≥25% tumour cells with membrane staining) will be compared with the Dako 28-8 assay (used in the Nivolumab Checkmate 057 trial at the 1%, 5% and 10% tumour membrane positivity cut offs), and the Dako 22C3 assay (used in the Pembrolizumab Keynote 001 trial) at the 1% and 50% cut offs).
Results: Preliminary data from 81 non-squamous patients indicated good concordance between the Ventana SP263 and Dako 28-8 assays. Optimal overall percent agreement (OPA) was observed between Dako 28-8 at the 10% cut off and the Ventana SP263 assay (OPA; 96%, Positive percent agreement (PPA); 91%, Negative percent agreement (NPA); 98%), where the Ventana SP263 assay was set as the reference. Data on the full cohort will be presented for all three assays, and a lower 95% confidence interval calculated using the Clopper-Pearson method.
Conclusions: This study indicates that the patient population defined by Ventana SP263 at the 25% cut off is similar to that identified by the Dako-28-8 assay at the 10% tumour membrane cut off. This, together with data on the 22C3 assay, will enable cross comparison of studies using different PD-L1 tests, and widen options for harmonization of PD-L1 diagnostic testing.

http://www.abstractsonline.com/Plan/ViewAbstract.aspx

Table 1
Reference: Ventana SP-263 (≥25% tumour membrane staining)
Dako 28-8 assay cut off PPA
(%)
NPA
(%)
OPA
(%)
>1% 58 100 81
>5% 72 100 90
>10% 91 98 96

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New Insights into mtDNA, mitochondrial proteins, aging, and metabolic control

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Newly discovered proteins may protect against age-related illnesses  

The proteins could play a key role in the aging process and the onset of diseases linked to older age

BY Beth Newcomb   APRIL 13, 2016

http://news.usc.edu/98412/newly-discovered-proteins-may-protect-against-agings-illnesses/

http://news.usc.edu/files/2016/04/20160208_PinchasCohen_web-824×549.jpg

Pinchas Cohen led a team that identified tiny proteins that appear to play a role in controlling how the body ages. (Photo/Beth Newcomb)

A group of six newly discovered proteins may help to divulge secrets of how we age, potentially unlocking insights into diabetes, Alzheimer’s, cancer and other aging-related diseases.

The tiny proteins appear to play several big roles in our bodies’ cells, from decreasing the amount of damaging free radicals and controlling the rate at which cells die to boosting metabolism and helping tissues throughout the body respond better to insulin. The naturally occurring amounts of each protein decrease with age, leading researchers to believe that they play an important role in the aging process and the onset of diseases linked to older age.

The research team led by Pinchas Cohen, dean of the USC Davis School of Gerontology, identified the tiny proteins for the first time and observed their surprising origin from organelles in the cell called mitochondria and their game-changing roles in metabolism and cell survival. This latest finding builds upon prior research by Cohen and his team that uncovered two significant proteins, humanin and MOTS-c, hormones that appear to have significant roles in metabolism and diseases of aging.

Unlike most other proteins, humanin and MOTS-c are encoded in mitochondria, the structure within cells that produces energy from food, instead of in the cell’s nucleus where most genes are contained.

Key functions

Mitochondria have their own small collection of genes, which were once thought to play only minor roles within cells but now appear to have important functions throughout the body. Cohen’s team used computer analysis to see if the part of the mitochondrial genome that provides the code for humanin was coding for other proteins as well. The analysis uncovered the genes for six new proteins, which were dubbed small humanin-like peptides, or SHLPs, 1 through 6 (the name of this hardworking group of proteins is appropriately pronounced “schlep”).

After identifying the six SHLPs and successfully developing antibodies to test for several of them, the team examined both mouse tissues and human cells to determine their abundance in different organs as well as their functions. The proteins were distributed quite differently among organs, which suggests that the proteins have varying functions based on where they are in the body.

Of particular interest is SHLP 2, Cohen said. The protein appears to have profound insulin-sensitizing, anti-diabetic effects as well as potent neuro-protective activity that may emerge as a strategy to combat Alzheimer’s disease. He added that SHLP 6 is also intriguing, with a unique ability to promote cancer cell death and thus potentially target malignant diseases.

“Together with the previously identified mitochondrial peptides, the newly recognized SHLP family expands the understanding of the mitochondria as an intracellular signaling organelle that communicates with the rest of the body to regulate metabolism and cell fate,” Cohen said. “The findings are an important advance that will be ripe for rapid translation into drug development for diseases of aging.”

The study first appeared online in the journal Aging on April 10. Cohen’s research team included collaborators from the Albert Einstein College of Medicine; the findings have been licensed to the biotechnology company CohBar for possible drug development.

The research was supported by a Glenn Foundation Award and National Institutes of Health grants to Cohen (1P01AG034906, 1R01AG 034430, 1R01GM 090311, 1R01ES 020812) and an Ellison/AFAR postdoctoral fellowship to Kelvin Yen. Study authors Laura Cobb, Changhan Lee, Nir Barzilai and Pinchas Cohen are consultants and stockholders of CohBar Inc.

Feature: The man who wants to beat back aging

By Stephen S. Hall Sep. 16, 201

Nir Barzilai hopes to persuade FDA to bless the proposed anti-aging trial, which is unconventional in its goals and design.
http://www.sciencemag.org/news/2015/09/feature-man-who-wants-beat-back-aging

On a blazingly hot morning this past June, a half-dozen scientists convened in a hotel conference room in suburban Maryland for the dress rehearsal of what they saw as a landmark event in the history of aging research. In a few hours, the group would meet with officials at the U.S. Food and Drug Administration (FDA), a few kilometers away, to pitch an unprecedented clinical trial—nothing less than the first test of a drug to specifically target the process of human aging.

“We think this is a groundbreaking, perhaps paradigm-shifting trial,” said Steven Austad, chairman of biology at the University of Alabama, Birmingham, and scientific director of the American Federation for Aging Research (AFAR). After Austad’s brief introductory remarks, a scientist named Nir Barzilai tuned up his PowerPoint and launched into a practice run of the main presentation.

Barzilai is a former Israeli army medical officer and head of a well-known study of centenarians based at the Albert Einstein College of Medicine in the Bronx, New York. To anyone who has seen the ebullient scientist in his natural laboratory habitat, often in a short-sleeved shirt and always cracking jokes, he looked uncharacteristically kempt in a blue blazer and dress khakis. But his practice run kept hitting a historical speed bump. He had barely begun to explain the rationale for the trial when he mentioned, in passing, “lots of unproven, untested treatments under the category of anti-aging.” His colleagues pounced.

“Nir,” interrupted S. Jay Olshansky, a biodemographer of aging from the University of Illinois, Chicago. The phrase “anti-aging … has an association that is negative.”

“I wouldn’t dignify them by calling them ‘treatments,’” added Michael Pollak, director of cancer prevention at McGill University in Montreal, Canada. “They’re products.”

Barzilai, a 59-year-old with a boyish mop of gray hair, wore a contrite grin. “We know the FDA is concerned about this,” he conceded, and deleted the offensive phrase.

Then he proceeded to lay out the details of an ambitious clinical trial. The group—academics all—wanted to conduct a double-blind study of roughly 3000 elderly people; half would get a placebo and half would get an old (indeed, ancient) drug for type 2 diabetes called metformin, which has been shown to modify aging in some animal studies. Because there is still no accepted biomarker for aging, the drug’s success would be judged by an unusual standard—whether it could delay the development of several diseases whose incidence increases dramatically with age: cardiovascular disease, cancer, and cognitive decline, along with mortality. When it comes to these diseases, Barzilai is fond of saying, “aging is a bigger risk factor than all of the other factors combined.”

But the phrase “anti-aging” kept creeping into the rehearsal, and critics kept jumping in. “Okay,” Barzilai said with a laugh when it came up again. “Third time, the death penalty.”

The group’s paranoia about the term “anti-aging” captured both the audacity of the proposed trial and the cultural challenge of venturing into medical territory historically associated with charlatans and quacks. The metformin initiative, which Barzilai is generally credited with spearheading, is unusual by almost any standard of drug development. The people pushing for the trial are all academics, none from industry (although Barzilai is co-founder of a biotech company, CohBar Inc., that is working to develop drugs targeting age-related diseases). The trial would be sponsored by the nonprofit AFAR, not a pharmaceutical company. No one stood to make money if the drug worked, the scientists all claimed; indeed, metformin is not only generic, costing just a few cents a dose, but belongs to a class of drugs that has been part of the human apothecary for 500 years. Patient safety was unlikely to be an issue; millions of diabetics have taken metformin since the 1960s, and its generally mild side effects are well-known.

Finally, the metformin group insisted they didn’t need a cent of federal money to proceed (although they do intend to ask for some). Nor did they need formal approval from FDA to proceed. But they very much wanted the agency’s blessing. By recognizing the merit of such a trial, Barzilai believes,  FDA would make aging itself a legitimate target for drug development.

By the time the scientists were done, the rehearsal—which was being filmed for a television documentary—had the feel of a pep rally. They spoke with unguarded optimism. “What we’re talking about here,” Olshansky said, “is a fundamental sea change in how we look at aging and disease.” To Austad, it is “the key, potentially, to saving the health care system.”

As the group piled into a van for the drive to FDA headquarters, there was more talk about setting precedents and opening doors. So it was a little disconcerting when Austad led the delegation up to the main entrance of FDA—and couldn’t get the door open.   ……

Mitochondrial Peptides Found in a Preclinical Study Seen to Control Cell Metabolism

APRIL 13, 2016 BY MAGDALENA KEGEL  IN NEWS.
http://mitochondrialdiseasenews.com/2016/04/13/newly-discovered-mitochondrial-factors-might-give-insights-into-aging/

 

CohBar, a developer of mitochondria-based therapeutics, announced that preclinical research by its academic collaborators has found small humanin-like peptides (SHLPs) that can control metabolism and cell survival. The findings have implications for age-related diseases such as Alzheimer’s and cancer.

The study, “Naturally Occurring Mitochondrial-derived Peptides are Age-dependent Regulators of Apoptosis, Insulin Sensitivity, and Inflammatory Markers,” was the result of a joint effort between researchers at the University of Southern California (USC) and theInstitute for Aging Research at the Albert Einstein College of Medicine of Yeshiva University. The study was published in the journal Aging.

Researchers discovered the SHLPs by examining the genome of mitochondria with the help of a bioinformatics approach, which identified six peptides. The team then verified the presence of the factors and explored their function in laboratory animals.

CohBar, who have the exclusive license to develop SHLPs into therapeutics, works closely with its academic partners to explore the peptides in preclinical models.

While it was previously believed that mitochondria only have 37 genes, research has revealed that the mitochondrial genome is far more versatile, potentially harboring a multitude of new genes, which can encode peptides acting as cellular signaling factors. The peptides, it has turned out, have shown neuroprotective and anti-inflammatory effects, and act to protect cells in disease-modifying ways in preclinical models of aging.

CohBar’s goal is to bring these peptides to the market as therapies for age-related diseases, such as obesity, type 2 diabetes, cancer, atherosclerosis and neurodegenerative disorders.

“Together with the previously described mitochondrial-derived peptides humanin and MOTS-c, the SHLP family expands our understanding of the role that these peptides play in intracellular signaling throughout the body to regulate both metabolism and cell survival,” Pinchas Cohen, dean of the USC Leonard Davis School of Gerontology, founder and director of CohBar, and the study’s senior author, said in a press release. “These findings further illustrate the enormous potential that mitochondria-based therapeutics could have on treating age-associated diseases like Alzheimer’s and cancer.”

“The pre-clinical evidence continues to confirm that these peptides represent a new class of naturally occurring metabolic regulators,” added Simon Allen, CohBar’s CEO. “They form the foundation of our pipeline of first-in-class treatments for age-related diseases, and we are committed to rapidly advancing them through pre-clinical and clinical activities as we move forward.”

Naturally occurring mitochondrial-derived peptides are age-dependent regulators of apoptosis, insulin sensitivity, and inflammatory markers

Laura J. Cobb1,5, Changhan Lee2, Jialin Xiao2, Kelvin Yen2, Richard G. Wong2, Hiromi K. Nakamura1, ….., Derek M. Huffman4, Junxiang Wan2, Radhika Muzumdar3, Nir Barzilai4 , and Pinchas Cohen2
http://www.impactaging.com/papers/v8/n4/full/100943.html

Mitochondria are key players in aging and in the pathogenesis of age-related diseases. Recent mitochondrial transcriptome analyses revealed the existence of multiple small mRNAs transcribed from mitochondrial DNA (mtDNA). Humanin (HN), a peptide encoded in the mtDNA 16S ribosomal RNA region, is a neuroprotective factor. An in silico search revealed six additional peptides in the same region of mtDNA as humanin; we named these peptides small humanin-like peptides (SHLPs). We identified the functional roles for these peptides and the potential mechanisms of action. The SHLPs differed in their ability to regulate cell viability in vitro. We focused on SHLP2 and SHLP3 because they shared similar protective effects with HN. Specifically, they significantly reduced apoptosis and the generation of reactive oxygen species, and improved mitochondrial metabolism in vitro. SHLP2 and SHLP3 also enhanced 3T3-L1 pre-adipocyte differentiation. Systemic hyperinsulinemic-euglycemic clamp studies showed that intracerebrally infused SHLP2 increased glucose uptake and suppressed hepatic glucose production, suggesting that it functions as an insulin sensitizer both peripherally and centrally. Similar to HN, the levels of circulating SHLP2 were found to decrease with age. These results suggest that mitochondria play critical roles in metabolism and survival through the synthesis of mitochondrial peptides, and provide new insights into mitochondrial biology with relevance to aging and human biology.

Human mitochondrial DNA (mtDNA) is a double-stranded, circular molecule of 16,569 bp and contains 37 genes encoding 13 proteins, 22 tRNAs, and 2 rRNAs. Recent mitochondrial transcriptome analyses revealed the existence of small RNAs derived from mtDNA [1]. In 2001, Nishimoto and colleagues identified humanin (HN), a 24-amino-acid peptide encoded from the 16S ribosomal RNA (rRNA) region of mtDNA. HN is a potent neuroprotective factor capable of antagonizing Alzheimer’s disease (AD)-related cellular insults [2]. HN is a component of a novel retrograde signaling pathway from the mitochondria to the nucleus, which is distinct from mitochondrial signaling pathways, such as the SIRT4-AMPK pathway [3]. HN-dependent cellular protection is mediated in part by interacting with and antagonizing pro-apoptotic Bax-related peptides [4] and IGFBP-3 (IGF binding protein 3) [5].

Because of their involvement in energy production and free radical generation, mitochondria likely play a major role in aging and age-related diseases [68]. In fact, improvement of mitochondrial function has been shown to ameliorate age-related memory loss in aged mice [9]. Recent studies have shown that HN levels decrease with age, suggesting that HN could play a role in aging and age-related diseases, such as Alzheimer’s disease (AD), atherosclerosis, and diabetes. Along with lower HN levels in the hypothalamus, skeletal muscle, and cortex of older rodents, the circulating levels of HN were found to decline with age in both humans and mice [10]. Notably, circulating HN levels were found to be (i) significantly higher in long-lived Ames dwarf mice but lower in short-lived growth hormone (GH) transgenic mice, (ii) significantly higher in a GH-deficient cohort of patients with Laron syndrome, and (iii) reduced in mice and humans treated with GH or IGF-1 (insulin-like growth factor 1) [11]. Age-dependent declines in the circulating HN levels may be due to higher levels of reactive oxygen species (ROS) that contribute to atherosclerosis development. Using mouse models of atherosclerosis, it was found that HN-treated mice had a reduced disease burden and significant health improvements [12,13]. In addition, HN improved insulin sensitivity, suggesting clinical potential for mitochondrial peptides in diseases of aging [10]. The discovery of HN represents a unique addition to the spectrum of roles that mitochondria play in the cell [14,15]. A second mitochondrial-derived peptide (MDP), MOTS-c (mitochondrial open reading frame of the 12S rRNA-c), has also been shown to have metabolic effects on muscle and may also play a role in aging [16].

We further investigated mtDNA for the presence of other MDPs. Recent technological advances have led to the identification of small open reading frames (sORFs) in the nuclear genomes ofDrosophila[17,18] and mammals [19,20]. Therefore, we attempted to identify novel sORFs using the following approaches: 1) in silico identification of potential sORFs; 2) determination of mRNA expression levels; 3) development of specific antibodies against these novel peptides to allow for peptide detection in cells, organs, and plasma; 4) elucidating the actions of these peptides by performing cell-based assays for mitochondrial function, signaling, viability, and differentiation; and 5) delivering these peptides in vivo to determine their systemic metabolic effects. Focusing on the 16S rRNA region of the mtDNA where the humanin gene is located, we identified six sORFs and named them small humanin-like peptides (SHLPs) 1-6. While surveying the biological effects of SHLPs, we found that SHLP2 and SHLP3 were cytoprotective; therefore, we investigated their effects on apoptosis and metabolism in greater detail. Further, we showed that circulating SHLP2 levels declined with age, similar to HN, suggesting that SHLP2 is involved in aging and age-related disease progression.

SHLP2 and SHLP3 regulate the expression of metabolic and inflammatory markers

Epidemiological studies have demonstrated that increased levels of mediators of inflammation and acute-phase reactants, such as fibrinogen, C-reactive protein (CRP), and IL-6, correlate with the incidence of type 2 diabetes mellitus (T2DM) [3436]. In humans, anti-inflammatory drugs, such as aspirin and sodium salicylate, reduce fasting plasma glucose levels and ameliorate the symptoms of T2DM. In addition, anti-diabetic drugs, such as fibrates [37] and thiazolidinediones [38], have been found to lower some markers of inflammation. SHLP2 increased the levels of leptin, which is known to improve insulin sensitivity, but had no effect on the levels of the pro-inflammatory cytokines IL-6 and MCP-1. SHLP3 significantly increased the leptin levels, but also elevated IL-6 and MCP-1 levels, which could explain the lack of an in vivo insulin-sensitizing effect of SHLP3. The mechanism by which SHLPs regulate the expression of metabolic and inflammatory markers remains unclear and needs to be further investigated. Furthermore, SHLPs have different effects on inflammatory marker expression, suggesting differential regulation and function of individual SHLPs.

SHLP2 in aging

Mitochondria have been implicated in increased lifespan in several life-extending treatments [39,40]; however, it is not known whether the relationship is correlative or causative [40]. Additionally, it is well known that hormone levels change with aging. For example, levels of aldosterone, calcitonin, growth hormone, and IGF-I decrease with age. Circulating HN levels decline with age in humans and rodents, specifically in the hypothalamus and skeletal muscle of older rats. These changes parallel increases in the incidence of age-associated diseases such as AD and T2DM. The decline in circulating SHLP2 levels with age (Fig. 6), the anti-oxidative stress function of SHLP2 (Fig. 3C), and its neuroprotective effect (Fig. 6B) indicate that SHLP2 has a role in the regulation of aging and age-related diseases.

Conclusion

By analyzing the mitochondrial transcriptome, we found that sORFs from mitochondrial DNA encode functional peptides. We identified many mRNA transcripts within 13 protein-coding mitochondrial genes [1]. Such previously underappreciated sORFs have also been described in the nuclear genome [41]. The MDPs we describe here may represent retrograde communication signals from the mitochondria to the nucleus and may explain important aspects of mitochondrial biology that are implicated in health and longevity.

Larry, John Walker is working on mt proteins dynamics. His rotor – stator mechanism in ATPase synthase, a ‘complex’ that biologist accepted as energy generator is likely wrong. I was suppose to have met him in Germany few years ago. Energy in biological systems has nothing to do with heat. Heat is an outcome of a reaction, meaning that IR spectra accordingly to wave theory is a source of information memorized in water interference with carbon open systems within protein and glyo-proteins complexes as well as genome space-time outcomes. Physically speaking from a pure perspective of science ATP is highly unstable form of phosphate ‘chains’. It cannot hold energy, it is actually in contrary, it is like a resonator, trapping negativity, thus functioning as space propeller by expanding carbon skeleton of protein ‘machines’ Now, we don’t know what is ‘aging’ in a pure physical sense, except that we observe structural changes in what we call complexes. We we know is that proteins are not stationary structures, but highly dynamic forms of matter, seemingly occupying discrete and relative spaces. A piece of mt ATP ase could be discovered in the nucleus as transcription factor. Our notion of operational space in terms of electro dynamics from a motor – stator perspective is now translated toward defining semi conducting and supracoductive strings. The reality of which is so much more fascinating and beautiful as time progresses overally. There are spaces where time does not change, and there are spaces where time walks, and there are spaces, where time flies, and there are spaces where time runs. Amazing, indeed! The story of aging gets a lot deeper that science could even imagine, probably to roots of immortal energy- spaces. We know that matter is transient, that is nearly all living matter, replenishes of about 3 to 7 weeks.

Take a glass full of some kind of liquid, you know the mass of the glass and the mass of the liquid (say wine, beer, water, or milk) You also know to an approximate reality the composition of both. Now lift the glass full of liquid and let it break on a surface of your choice. Depending on the surface pieces of the glass would travel differential from a center projected by the vertical axis of your hand. What technology does today is recollecting those pieces and modelling them to fit in a form again that would resemble a holding device, a glass. The liquid we don’t know exactly how it spilled due the nature of its absorbancy of both surface physics and physical ‘state’ properties. Thus we can say how much approximate energy we have held thinking of m/z as time flight objectives. Each technology can read 1D and approximate the 2D, absolutely lacking computational methodology for 3D dynamic reality. Many scientists confuse space and volume. Volume is a one dimensional characteristic! So is crystalography! BY taking quantum chemical method computing principles following imaginative rules we could approach 2D, however , that is not enough to define 3D. Time we use as a reference frame of clocks we have invented in order to keep track of a sense to observable ‘change’ . But remember, time is absolute and parallel in continuity while energy is discrete , coming in quantum packages, realization of accumulated information. Information is highly redundant we see, so annotating information is an objective to modern days simulations that could predict outcomes of possible parallel realities we call worlds. One could ‘jump’ from one reality to another through guidance of light and water, but what remains unsolved is why people make mistakes, constantly by accusing in name of greed and power , or disobedience of commandments of the Lord!

On Thu, Apr 21, 2016 at 3:41 AM, Leaders in Pharmaceutical Business Intelligence (LPBI) Group wrote:

> larryhbern posted: “New Insights into mtDNA, mitochondrial proteins, > aging, and metabolic control Larry H. Bernstein, MD, FCAP, Curator LPBI > Newly discovered proteins may protect against age-related illnesses The > proteins could play a key role in the ” >

 

Metabolic features of the cell danger response
– Mitochondria in Health and Disease

Mitochondrion  Volume 16, May 2014, Pages 7–17     doi:10.1016/j.mito.2013.08.006

 

Highlights

  •  The Cell Danger Response (CDR) is defined in terms of an ancient metabolic response to threat.
  •  The CDR encompasses inflammation, innate immunity, oxidative stress, and the ER stress response.
  •  The CDR is maintained by extracellular nucleotide (purinergic) signaling.
  •  Abnormal persistence of the CDR lies at the heart of many chronic diseases.
  •  Antipurinergic therapy (APT) has proven effective in many chronic disorders in animal models

The cell danger response (CDR) is the evolutionarily conserved metabolic response that protects cells and hosts from harm. It is triggered by encounters with chemical, physical, or biological threats that exceed the cellular capacity for homeostasis. The resulting metabolic mismatch between available resources and functional capacity produces a cascade of changes in cellular electron flow, oxygen consumption, redox, membrane fluidity, lipid dynamics, bioenergetics, carbon and sulfur resource allocation, protein folding and aggregation, vitamin availability, metal homeostasis, indole, pterin, 1-carbon and polyamine metabolism, and polymer formation. The first wave of danger signals consists of the release of metabolic intermediates like ATP and ADP, Krebs cycle intermediates, oxygen, and reactive oxygen species (ROS), and is sustained by purinergic signaling. After the danger has been eliminated or neutralized, a choreographed sequence of anti-inflammatory and regenerative pathways is activated to reverse the CDR and to heal. When the CDR persists abnormally, whole body metabolism and the gut microbiome are disturbed, the collective performance of multiple organ systems is impaired, behavior is changed, and chronic disease results. Metabolic memory of past stress encounters is stored in the form of altered mitochondrial and cellular macromolecule content, resulting in an increase in functional reserve capacity through a process known as mitocellular hormesis. The systemic form of the CDR, and its magnified form, the purinergic life-threat response (PLTR), are under direct control by ancient pathways in the brain that are ultimately coordinated by centers in the brainstem. Chemosensory integration of whole body metabolism occurs in the brainstem and is a prerequisite for normal brain, motor, vestibular, sensory, social, and speech development. An understanding of the CDR permits us to reframe old concepts of pathogenesis for a broad array of chronic, developmental, autoimmune, and degenerative disorders. These disorders include autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD), asthma, atopy, gluten and many other food and chemical sensitivity syndromes, emphysema, Tourette’s syndrome, bipolar disorder, schizophrenia, post-traumatic stress disorder (PTSD), chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), epilepsy, suicidal ideation, organ transplant biology, diabetes, kidney, liver, and heart disease, cancer, Alzheimer and Parkinson disease, and autoimmune disorders like lupus, rheumatoid arthritis, multiple sclerosis, and primary sclerosing cholangitis.

The double face of mitochondrial dysfunction

Dmitry Knorre, Anna Zyrina, and Fedor Severin

pp 420-420

Full text | PDF

 

 

Flawed Mitochondrial DNA Could Undermine Stem Cell Therapies

http://www.genengnews.com/gen-news-highlights/flawed-mitochondrial-dna-could-undermine-stem-cell-therapies/81252622/

http://www.genengnews.com/Media/images/GENHighlight/thumb_Apr18_2016_OHSU_ConfocalMicroscopyImage7543773219.jpg

This is a confocal microscopy image of human fibroblasts derived from embryonic stem cells. The nuclei appear in blue, while smaller and more numerous mitochondria appear in red. [Shoukhrat Mitalipov]

Mutations in our mitochondrial DNA tend to be inconspicuous, but they can become more prevalent as we age. They can even vary in frequency from cell to cell. Naturally, some cells will be relatively compromised because they happen to have a higher percentage of mutated mitochondrial DNA. Such cells make a poor basis for stem cell lines. They should be excluded. But how?

To answer this question, a team of scientists scrutinized skin fibroblasts, blood cells, and induced pluripotent stem cells (iPSCs) for mitochondrial genome integrity. When the scientists tested the samples for mitochondrial DNA mutations, the levels of mutations appeared low. But when the scientists sequenced the iPS cell lines, they found higher numbers of mitochondrial DNA mutations, particularly in cells from patients over 60.

The scientists were led by Shoukhrat Mitalipov, Ph.D., director of the Center for Embryonic Cell and Gene Therapy at Oregon Health & Science University, and Taosheng Huang, M.D., a medical geneticist and director of the Mitochondrial Medicine Program at Cincinnati Children’s Hospital. The Mitalipov/Huang-led team also found higher percentages of mitochondria containing mutations within a cell. The higher the load of mutated mitochondrial DNA in a cell, the more compromised the cell’s function.

Since each iPSC line is created from a different cell, each line may contain different types of mitochondrial DNA mutations and mutation loads. To choose the least damaged line, the authors recommend screening multiple lines per patient. “It’s a good idea to check the iPS clones for mitochondrial DNA mutations and make sure you pick a good cell line,” said Dr. Huang.

This recommendation appeared April 14 in the journal Cell Stem Cell, in an article entitled, “Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs.” This article holds that mitochondrial genome integrity is a vital readout in assessing the proficiency of patient-derived regenerative products destined for clinical applications.

“We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but are not detectable in whole tissues,” wrote the article’s authors. “The frequency of mtDNA defects in iPSCs increased with age, and many mutations were nonsynonymous or resided in RNA coding genes and thus can lead to respiratory defects.”

Potential therapies using stem cells hold tremendous promise for treating human disease. However, defects in the mitochondria could undermine the iPS cells’ ability to repair damaged tissue or organs.

“If you want to use iPS cells in a human, you must check for mutations in the mitochondrial genome,” declared Dr. Huang. “Every single cell can be different. Two cells next to each other could have different mutations or different percentages of mutations.”

Prior to the creation of a therapeutic iPS cell line, a collection of cells is taken from the patient. These cells will be tested for mutations. If the tester uses Sanger sequencing, older technology that is not as sensitive as newer next-generation sequencing, any mutation that occurs in less than 20% of the sample will go undetected. But mitochondrial DNA mutations might occur in less than 20% of mitochondria in the pooled cells. As a result, mutation rates have not been well understood. “These mitochondrial mutations are actually hidden,” explained Dr. Mitalipov.

The mitochondrial genome is relatively small, containing just 37 genes, so screening should be feasible using next generation sequencing, Dr. Mitalipov added. “It should be relatively cheap and do-able.”

Dr. Mitalipov also commented on a more general point, the implications of the current study on illuminating the mechanisms of age-related disease: “Pathogenic mutations in our mitochondrial DNA have long been thought to be a driving force in aging and age-onset diseases, though clear evidence was missing. This foundational knowledge of how cells are damaged in the natural process of aging may help to illuminate the role of mutated mitochondria in degenerative disease.”

New Mitalipov paper on stem cell mitochondria: challenge for IPS cell field?

http://www.ipscell.com/tag/age-related-accumulation-of-somatic-mitochondrial-dna-mutations-in-adult-derived-human-ipscs/

A new paper from Shoukhrat Mitalipov’s lab on stem cell mitochondria points to a pattern whereby induced pluripotent stem (IPS) cells tend to have more problems if they are from older patients.

What does this paper mean for the stem cell field and could it impact more specifically the clinical applications of IPS cells?

http://i0.wp.com/www.ipscell.com/wp-content/uploads/2016/04/Mitalipov-mitochondrial-mutations.jpg?resize=300%2C295

Graphical Abstract, Kang, et al. 2016

The new paper Kang, et al is entitled “Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs”.

This paper reminds us of the very important realities that mitochondria are key players in stem cell function and that mitochondria have their own genomes that impact that function. A lot of us don’t think about mitochondria and their genome as often as we should.

The paper came to three major scientific conclusions (this from the Highlights section of the paper and also see the graphical abstract for a visual sense of the results overall):

  • Human iPSC clones derived from elderly adults show accumulation of mtDNA mutations
  • Fewer mtDNA mutations are present in ESCs and iPSCs derived from younger adults
  • Accumulated mtDNA mutations can impact metabolic function in iPSCs

Importantly the team looked at IPS cells derived from both blood and skin cells and found that the former were less likely to have mitochondrial mutations.

This study suggests that those teams producing or working with human IPS cells (hIPSCs) should be screening the different lines for mitochondrial mutations. This excellent piece from Sara Reardon on the Mitalipov paper quotes IPS cell expert Jeanne Loring on this very point:

“It’s one of those things most of us don’t think about,” says Jeanne Loring, a stem-cell biologist at the Scripps Research Institute in La Jolla, California. Her lab is working towards using iPS cells to treat Parkinson’s disease, and Loring now plans to go back and examine the mitochondria in her cell lines. She suspects that it will be fairly easy for researchers to screen cells for use in therapies.”

Mitalipov goes further and suggests that his team’s new findings could support the use of human embryonic stem cells (hESC) derived by somatic cell nuclear transfer (SCNT) which would be expected to have mitochondria with fewer mutations. However, as Loring points out in the Reardon article, SCNT is really difficult to successfully perform and only a few labs in the world can do it at present. In that context, working with hIPSC and adding on the additional layer of mitochondrial DNA mutation screening could be more practical.

New York stem cell researcher Dieter Egli, however, is quoted that hIPSC have other differences with hESC as well such as epigenetic differences and he’s quoted in the Reardon piece, “It’s going to be very hard to find a cell line that’s perfect.”

One might reasonably ask both Egli and oneself, “What is a perfect cell line”?

In the end the best approach for use of human pluripotent stem cells of any kind is going to involve a balance between practicality of production and the potentially positive or negative traits of those cells as determined by rigorous validation screening.

With this new paper we’ve just learned more about another layer of screening that is needed. An interesting question is whether adult stem cells such as mesenchymal stromal/stem cells (MSC) also should be screened for mitochondrial mutations. They are often produced from patients who are getting up there in years. I hope that someone will publish on that too.

As to pluripotent cells, I expect that sometimes the best lines, meaning those most perfect for a given clinical application, will be hIPSC (autologous or allogeneic in some instances) and in other cases they may be hESC made from leftover IVF embryos. If SCNT-derived hESC can be more widely produced in an affordable manner and they pass validation as well then those (sometimes called NT-hESC) may also come into play clinically. So far that hasn’t happened for the SCNT cells, but it may over time.   …..

 Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs

Eunju Kang, Xinjian Wang, Rebecca Tippner-Hedges, …, Don P. Wolf, Taosheng Huang, Shoukhrat Mitalipov
http://www.cell.com/cell-stem-cell/pdfExtended/S1934-5909(16)00067-9

In Brief Mitalipov, Huang, and colleagues show that human iPSCs derived from older adults carry more mitochondrial DNA mutations than those derived from younger individuals. Defects in metabolic function caused by mtDNA mutations suggest careful screening of hiPSC clones for mutational load before clinical application.

Highlights

  1. Human iPSC clones derived from elderly adults show accumulation of mtDNA mutations
  2. Fewer mtDNA mutations are present in ESCs and iPSCs derived from younger adults
  3. Accumulated mtDNA mutations can impact metabolic function in iPSCs

Kang et al., 2016, Cell Stem Cell 18, 1–12 May 5, 2016 ª2016 Elsevier Inc. http://dx.doi.org/10.1016/j.stem.2016.02.005

The genetic integrity of iPSCs is an important consideration for therapeutic application. In this study, we examine the accumulation of somatic mitochondrial genome (mtDNA) mutations in skin fibroblasts, blood, and iPSCs derived from young and elderly subjects (24–72 years). We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but are not detectable in whole tissues. The frequency of mtDNA defects in iPSCs increased with age, and many mutations were non-synonymous or resided in RNA coding genes and thus can lead to respiratory defects. Our results highlight a need to monitor mtDNA mutations in iPSCs, especially those generated from older patients, and to examine the metabolic status of iPSCs destined for clinical applications.

Induced pluripotent stem cells (iPSCs) offer an unlimited source for autologous cell replacement therapies to treat age-associated degenerative diseases. Aging is generally characterized by increased DNA damage and genomic instability (Garinis et al., 2008; Lombard et al., 2005); thus, iPSCs derived from elderly subjects may harbor point mutations and larger genomic rearrangements. Indeed, iPSCs display increased chromosome aberrations (Mayshar et al., 2010), subchromosomal copy number variations (CNVs) (Abyzov et al., 2012; Laurent et al., 2011), and exome mutations (Johannesson et al., 2014), compared to natural embryonic stem cell (ESC) counterparts (Ma et al., 2014). The rate of mtDNA mutations is believed to be at least 10- to 20-fold higher than that observed in the nuclear genome (Wallace, 1994), and often both mutated and wild-type mtDNA (heteroplasmy) can coexist in the same cell (Rossignol et al., 2003). Large deletions are most frequently observed mtDNA abnormalities in aged post-mitotic tissues such as brain, heart, and muscle (Bender et al., 2006; Bua et al., 2006; Corral-Debrinski et al., 1992; Cortopassi et al., 1992; Mohamed et al., 2006) and have been implicated in aging and diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and diabetes (Larsson, 2010; Lin and Beal, 2006; Petersen et al., 2003; Wallace, 2005). In addition, mtDNA point mutations were reported in some tumors and replicating tissues (Chatterjee et al., 2006; Ju et al., 2014; Michikawa et al., 1999; Taylor et al., 2003). However, the extent of mtDNA defects in proliferating peripheral tissues commonly used for iPSC induction, such as skin and blood, is thought to be low and limited to common non-coding variants (Schon et al., 2012; Yao et al., 2015). Accumulation of mtDNA variants in these tissues with age was insignificant (Greaves et al., 2010; Hashizume et al., 2015). Several point mutations were identified in iPSCs generated from the newborn foreskin fibroblasts, although most of these variants were non-coding, common for the general population, and did not affect their metabolic activity (Prigione et al., 2011). Somatic mtDNA mutations may be under-reported secondary to the level of sample interrogation. …..

Figure 2. mtDNA Mutations in Skin Fibroblasts, Blood, and the iPSCs of a 72-YearOld B Subject (A) Sixteen mutations at low heteroplasmy levels were detected in the DNA of PF, while a panel of ten FiPSC lines carried nine mutations, including four that were homoplasmic. Gray rectangles define the mutations shared between PF and FiPSCs. (B) Venn diagram showing only one mutation in FiPSCs shared with PF. (C) All ten FiPSC lines carried between one and five high-heteroplasmy (>15%) mutations. (D) Mutation distribution in whole blood and BiPSCs was similar to that in PF and FiPSCs. Six mutations at low-heteroplasmy levels were observed in blood, while BiPSC lines displayed 21 mutations, including four over the 80% heteroplasmy level. (E) Venn diagram showing four mutations in BiPSCs shared with whole blood and the 17 novel variants. (F) Distribution of mutations in individual BiPSC lines. See also Figures S2 and S3; Table S1; Table S3, sheet 2; and Table S4, sheet 1   ….

Figure 4. Transmission and Distribution of Somatic mtDNA Mutations to iPSCs (A) A total of 112 mtDNA mutations were discovered in parental cells (PF, CF, and blood) from 11 subjects. Of these, 39 variants (35%) were found in corresponding 130 iPSC lines. Among non-transmitted, transmitted, and novel mutations in iPSCs, comparable percentages of variants (68%, 69%, and 79%, respectively) were coding mutations in protein, rRNA, or tRNA genes. This suggests that most pathogenic mutations do not affect iPSC induction. However, certain coding mutations including in ND3, ND4L, and 14 tRNA genes were not detected in iPSCs, suggesting possible pathogenicity. n, the number of mtDNA mutations. Blue font genes were detected in parental cells. (B–D) A total of 80 high heteroplasmic (>15%) variants were detected in the present study in 130 FiPSC or BiPSC lines from 11 subjects. (B) The majority of these variants (76%) were non-synonymous or frame-shift mutations in protein-coding genes or affected rRNA and tRNA genes. (C) More than half of the mutations (56%) were never reported in a database containing whole mtDNA sequences from 26,850 healthy subjects representing the general human population (http://www.mitomap.org/MITOMAP). (D) Most mutations (90%) were never reported in a database containing sequences from healthy subjects with corresponding mtDNA haplotypes. freq., frequent. See also Figure S5 and Tables S3 and S4. ….

sjwilliamspa

Mutations will accumulate over age in mitochondrial DNA, however the current study has the difficulty that the authors could not use patient-age-matched controls, in essence they could only compare induced pluripotent stem cells derived from different patients. This could confound the results but the result with higher frequency of mutation in mtDNA in cells reprogrammed from younger patients is interesting but might limit the ability of autologous regenerative therapy in older patients. However reprogramming, although the method not mentioned here although I am assuming by transfection with lentivirus is a rough procedure, involving multiple dedifferentiation steps. Therefore it is very understandable that cells obtained from elderly patients would respond less favorably to such a rough reprogramming regimen, especially if it produced a higher degree of ROS, which has been shown to alter mtDNA. This is why I feel it is more advantageous to obtain a stem cell population from fat cells and forgo the Oct4, htert, reprogramming with lentiviral vectors.

 

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CRISPR/Cas9, Familial Amyloid Polyneuropathy ( FAP) and Neurodegenerative Disease


CRISPR/Cas9, Familial Amyloid Polyneuropathy ( FAP) and Neurodegenerative Disease

Curator: Larry H. Bernstein, MD, FCAP

 

CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology

https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology

The development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a long-standing goal for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement (1). This follows several attempts over the years to manipulate gene function, including homologous recombination (2) and RNA interference (RNAi) (3). RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function (4, 5), but it is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects (6). Other recent approaches to targeted genome modification – zinc-finger nucleases [ZFNs, (7)] and transcription-activator like effector nucleases [TALENs (8)]– enable researchers to generate permanent mutations by introducing doublestranded breaks to activate repair pathways. These approaches are costly and time-consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.

The Biology of Cas9

The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. These repeats were initially discovered in the 1980s in E. coli (9), but their function wasn’t confirmed until 2007 by Barrangou and colleagues, who demonstrated that S. thermophilus can acquire resistance against a bacteriophage by integrating a genome fragment of an infectious virus into its CRISPR locus (10).

Three types of CRISPR mechanisms have been identified, of which type II is the most studied. In this case, invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA – CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity (Figure 1) (11).

Figure 1. Cas9 in vivo: Bacterial Adaptive Immunity

https://www.neb.com/~/media/NebUs/Files/Feature%20Articles/Images/FA_Cas9_Fig1_Cas9InVivo.png

In the acquisition phase, foreign DNA is incorporated into the bacterial genome at the CRISPR loci. CRISPR loci is then transcribed and processed into crRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence. (Figure not drawn to scale.)

https://www.neb.com/~/media/NebUs/Files/Feature%20Articles/Images/FA_Cas9_GenomeEditingGlossary.png

One Cas protein, Cas9 (also known as Csn1), has been shown, through knockdown and rescue experiments to be a key player in certain CRISPR mechanisms (specifically type II CRISPR systems). The type II CRISPR mechanism is unique compared to other CRISPR systems, as only one Cas protein (Cas9) is required for gene silencing (12). In type II systems, Cas9 participates in the processing of crRNAs (12), and is responsible for the destruction of the target DNA (11). Cas9’s function in both of these steps relies on the presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus and a HNH-like nuclease domain that resides in the mid-region of the protein (13).

To achieve site-specific DNA recognition and cleavage, Cas9 must be complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA (11). The tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9 (12).

During the destruction of target DNA, the HNH and RuvC-like nuclease domains cut both DNA strands, generating double-stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript (11, 14). The HNH domain cleaves the complementary strand, while the RuvC domain cleaves the noncomplementary strand.

The double-stranded endonuclease activity of Cas9 also requires that a short conserved sequence, (2–5 nts) known as protospacer-associated motif (PAM), follows immediately 3´- of the crRNA complementary sequence (15). In fact, even fully complementary sequences are ignored by Cas9-RNA in the absence of a PAM sequence (16).

Cas9 and CRISPR as a New Tool in Molecular Biology

The simplicity of the type II CRISPR nuclease, with only three required components (Cas9 along with the crRNA and trRNA) makes this system amenable to adaptation for genome editing. This potential was realized in 2012 by the Doudna and Charpentier labs (11). Based on the type II CRISPR system described previously, the authors developed a simplified two-component system by combining trRNA and crRNA into a single synthetic single guide RNA (sgRNA). sgRNAprogrammed Cas9 was shown to be as effective as Cas9 programmed with separate trRNA and crRNA in guiding targeted gene alterations (Figure 2A).

To date, three different variants of the Cas9 nuclease have been adopted in genome-editing protocols. The first is wild-type Cas9, which can site-specifically cleave double-stranded DNA, resulting in the activation of the doublestrand break (DSB) repair machinery. DSBs can be repaired by the cellular Non-Homologous End Joining (NHEJ) pathway (17), resulting in insertions and/or deletions (indels) which disrupt the targeted locus. Alternatively, if a donor template with homology to the targeted locus is supplied, the DSB may be repaired by the homology-directed repair (HDR) pathway allowing for precise replacement mutations to be made (Figure 2A) (17, 18).

Cong and colleagues (1) took the Cas9 system a step further towards increased precision by developing a mutant form, known as Cas9D10A, with only nickase activity. This means it cleaves only one DNA strand, and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only, resulting in reduced indel mutations (1, 11, 19). Cas9D10A is even more appealing in terms of target specificity when loci are targeted by paired Cas9 complexes designed to generate adjacent DNA nicks (20) (see further details about “paired nickases” in Figure 2B).

The third variant is a nuclease-deficient Cas9 (dCas9, Figure 2C) (21). Mutations H840A in the HNH domain and D10A in the RuvC domain inactivate cleavage activity, but do not prevent DNA binding (11, 22). Therefore, this variant can be used to sequence-specifically target any region of the genome without cleavage. Instead, by fusing with various effector domains, dCas9 can be used either as a gene silencing or activation tool (21, 23–26). Furthermore, it can be used as a visualization tool. For instance, Chen and colleagues used dCas9 fused to Enhanced Green Fluorescent Protein (EGFP) to visualize repetitive DNA sequences with a single sgRNA or nonrepetitive loci using multiple sgRNAs (27).

Figure 2. CRISPR/Cas9 System Applications

https://www.neb.com/~/media/NebUs/Files/Feature%20Articles/Images/FA_Cas9_Fig2_Cas9forGenomeEditing.png?device=modal

  1. Wild-type Cas9 nuclease site specifically cleaves double-stranded DNA activating double-strand break repair machinery. In the absence of a homologous repair template non-homologous end joining can result in indels disrupting the target sequence. Alternatively, precise mutations and knock-ins can be made by providing a homologous repair template and exploiting the homology directed repair pathway.
    B. Mutated Cas9 makes a site specific single-strand nick. Two sgRNA can be used to introduce a staggered double-stranded break which can then undergo homology directed repair.
    C. Nuclease-deficient Cas9 can be fused with various effector domains allowing specific localization. For example, transcriptional activators, repressors, and fluorescent proteins.

Targeting Efficiency and Off-target Mutations

Targeting efficiency, or the percentage of desired mutation achieved, is one of the most important parameters by which to assess a genome-editing tool. The targeting efficiency of Cas9 compares favorably with more established methods, such as TALENs or ZFNs (8). For example, in human cells, custom-designed ZFNs and TALENs could only achieve efficiencies ranging from 1% to 50% (29–31). In contrast, the Cas9 system has been reported to have efficiencies up to >70% in zebrafish (32) and plants (33), and ranging from 2–5% in induced pluripotent stem cells (34). In addition, Zhou and colleagues were able to improve genome targeting up to 78% in one-cell mouse embryos, and achieved effective germline transmission through the use of dual sgRNAs to simultaneously target an individual gene (35).

A widely used method to identify mutations is the T7 Endonuclease I mutation detection assay (36, 37) (Figure 3). This assay detects heteroduplex DNA that results from the annealing of a DNA strand, including desired mutations, with a wildtype DNA strand (37).

Figure 3. T7 Endonuclease I Targeting Efficiency Assay

https://www.neb.com/~/media/NebUs/Files/Feature%20Articles/Images/FA_Cas9_Fig3_T7Assay_TargetEfficiency.png

Genomic DNA is amplified with primers bracketing the modified locus. PCR products are then denatured and re-annealed yielding 3 possible structures. Duplexes containing a mismatch are digested by T7 Endonuclease I. The DNA is then electrophoretically separated and fragment analysis is used to calculate targeting efficiency.

Another important parameter is the incidence of off-target mutations. Such mutations are likely to appear in sites that have differences of only a few nucleotides compared to the original sequence, as long as they are adjacent to a PAM sequence. This occurs as Cas9 can tolerate up to 5 base mismatches within the protospacer region (36) or a single base difference in the PAM sequence (38). Off-target mutations are generally more difficult to detect, requiring whole-genome sequencing to rule them out completely.

Recent improvements to the CRISPR system for reducing off-target mutations have been made through the use of truncated gRNA (truncated within the crRNA-derived sequence) or by adding two extra guanine (G) nucleotides to the 5´ end (28, 37). Another way researchers have attempted to minimize off-target effects is with the use of “paired nickases” (20). This strategy uses D10A Cas9 and two sgRNAs complementary to the adjacent area on opposite strands of the target site (Figure 2B). While this induces DSBs in the target DNA, it is expected to create only single nicks in off-target locations and, therefore, result in minimal off-target mutations.

By leveraging computation to reduce off-target mutations, several groups have developed webbased tools to facilitate the identification of potential CRISPR target sites and assess their potential for off-target cleavage. Examples include the CRISPR Design Tool (38) and the ZiFiT Targeter, Version 4.2 (39, 40).

Applications as a Genome-editing and Genome Targeting Tool

Following its initial demonstration in 2012 (9), the CRISPR/Cas9 system has been widely adopted. This has already been successfully used to target important genes in many cell lines and organisms, including human (34), bacteria (41), zebrafish (32), C. elegans (42), plants (34), Xenopus tropicalis (43), yeast (44), Drosophila (45), monkeys (46), rabbits (47), pigs (42), rats (48) and mice (49). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA (14, 21, 29). Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations (50). A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation (26, 51, 52), epigenetic modification (25), and microscopic visualization of specific genome loci (27).

The CRISPR/Cas9 system requires only the redesign of the crRNA to change target specificity. This contrasts with other genome editing tools, including zinc finger and TALENs, where redesign of the protein-DNA interface is required. Furthermore, CRISPR/Cas9 enables rapid genome-wide interrogation of gene function by generating large gRNA libraries (51, 53) for genomic screening.

The Future of CRISPR/Cas9

The rapid progress in developing Cas9 into a set of tools for cell and molecular biology research has been remarkable, likely due to the simplicity, high efficiency and versatility of the system. Of the designer nuclease systems currently available for precision genome engineering, the CRISPR/Cas system is by far the most user friendly. It is now also clear that Cas9’s potential reaches beyond DNA cleavage, and its usefulness for genome locus-specific recruitment of proteins will likely only be limited by our imagination.

 

Scientists urge caution in using new CRISPR technology to treat human genetic disease

By Robert Sanders, Media relations | MARCH 19, 2015
http://news.berkeley.edu/2015/03/19/scientists-urge-caution-in-using-new-crispr-technology-to-treat-human-genetic-disease/

http://news.berkeley.edu/wp-content/uploads/2015/03/crispr350.jpg

The bacterial enzyme Cas9 is the engine of RNA-programmed genome engineering in human cells. (Graphic by Jennifer Doudna/UC Berkeley)

A group of 18 scientists and ethicists today warned that a revolutionary new tool to cut and splice DNA should be used cautiously when attempting to fix human genetic disease, and strongly discouraged any attempts at making changes to the human genome that could be passed on to offspring.

Among the authors of this warning is Jennifer Doudna, the co-inventor of the technology, called CRISPR-Cas9, which is driving a new interest in gene therapy, or “genome engineering.” She and colleagues co-authored a perspective piece that appears in the March 20 issue of Science, based on discussions at a meeting that took place in Napa on Jan. 24. The same issue of Science features a collection of recent research papers, commentary and news articles on CRISPR and its implications.    …..

A prudent path forward for genomic engineering and germline gene modification

David Baltimore1,  Paul Berg2, …., Jennifer A. Doudna4,10,*, et al.
http://science.sciencemag.org/content/early/2015/03/18/science.aab1028.full
Science  19 Mar 2015.  http://dx.doi.org:/10.1126/science.aab1028

 

Correcting genetic defects

Scientists today are changing DNA sequences to correct genetic defects in animals as well as cultured tissues generated from stem cells, strategies that could eventually be used to treat human disease. The technology can also be used to engineer animals with genetic diseases mimicking human disease, which could lead to new insights into previously enigmatic disorders.

The CRISPR-Cas9 tool is still being refined to ensure that genetic changes are precisely targeted, Doudna said. Nevertheless, the authors met “… to initiate an informed discussion of the uses of genome engineering technology, and to identify proactively those areas where current action is essential to prepare for future developments. We recommend taking immediate steps toward ensuring that the application of genome engineering technology is performed safely and ethically.”

 

Amyloid CRISPR Plasmids and si/shRNA Gene Silencers

http://www.scbt.com/crispr/table-amyloid.html

Santa Cruz Biotechnology, Inc. offers a broad range of gene silencers in the form of siRNAs, shRNA Plasmids and shRNA Lentiviral Particles as well as CRISPR/Cas9 Knockout and CRISPR Double Nickase plasmids. Amyloid gene silencers are available as Amyloid siRNA, Amyloid shRNA Plasmid, Amyloid shRNA Lentiviral Particles and Amyloid CRISPR/Cas9 Knockout plasmids. Amyloid CRISPR/dCas9 Activation Plasmids and CRISPR Lenti Activation Systems for gene activation are also available. Gene silencers and activators are useful for gene studies in combination with antibodies used for protein detection.    Amyloid CRISPR Knockout, HDR and Nickase Knockout Plasmids

 

CRISPR-Cas9-Based Knockout of the Prion Protein and Its Effect on the Proteome


Mehrabian M, Brethour D, MacIsaac S, Kim JK, Gunawardana C.G, Wang H, et al.
PLoS ONE 2014; 9(12): e114594. http://dx.doi.org/10.1371/journal.pone.0114594

The molecular function of the cellular prion protein (PrPC) and the mechanism by which it may contribute to neurotoxicity in prion diseases and Alzheimer’s disease are only partially understood. Mouse neuroblastoma Neuro2a cells and, more recently, C2C12 myocytes and myotubes have emerged as popular models for investigating the cellular biology of PrP. Mouse epithelial NMuMG cells might become attractive models for studying the possible involvement of PrP in a morphogenetic program underlying epithelial-to-mesenchymal transitions. Here we describe the generation of PrP knockout clones from these cell lines using CRISPR-Cas9 knockout technology. More specifically, knockout clones were generated with two separate guide RNAs targeting recognition sites on opposite strands within the first hundred nucleotides of the Prnp coding sequence. Several PrP knockout clones were isolated and genomic insertions and deletions near the CRISPR-target sites were characterized. Subsequently, deep quantitative global proteome analyses that recorded the relative abundance of>3000 proteins (data deposited to ProteomeXchange Consortium) were undertaken to begin to characterize the molecular consequences of PrP deficiency. The levels of ∼120 proteins were shown to reproducibly correlate with the presence or absence of PrP, with most of these proteins belonging to extracellular components, cell junctions or the cytoskeleton.

http://journals.plos.org/plosone/article/figure/image?size=inline&id=info:doi/10.1371/journal.pone.0114594.g001

http://journals.plos.org/plosone/article/figure/image?size=inline&id=info:doi/10.1371/journal.pone.0114594.g003

 

Development and Applications of CRISPR-Cas9 for Genome Engineering

Patrick D. Hsu,1,2,3 Eric S. Lander,1 and Feng Zhang1,2,*
Cell. 2014 Jun 5; 157(6): 1262–1278.   doi:  10.1016/j.cell.2014.05.010

Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine.

The development of recombinant DNA technology in the 1970s marked the beginning of a new era for biology. For the first time, molecular biologists gained the ability to manipulate DNA molecules, making it possible to study genes and harness them to develop novel medicine and biotechnology. Recent advances in genome engineering technologies are sparking a new revolution in biological research. Rather than studying DNA taken out of the context of the genome, researchers can now directly edit or modulate the function of DNA sequences in their endogenous context in virtually any organism of choice, enabling them to elucidate the functional organization of the genome at the systems level, as well as identify causal genetic variations.

Broadly speaking, genome engineering refers to the process of making targeted modifications to the genome, its contexts (e.g., epigenetic marks), or its outputs (e.g., transcripts). The ability to do so easily and efficiently in eukaryotic and especially mammalian cells holds immense promise to transform basic science, biotechnology, and medicine (Figure 1).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/bin/nihms659174f1.jpg

For life sciences research, technologies that can delete, insert, and modify the DNA sequences of cells or organisms enable dissecting the function of specific genes and regulatory elements. Multiplexed editing could further allow the interrogation of gene or protein networks at a larger scale. Similarly, manipulating transcriptional regulation or chromatin states at particular loci can reveal how genetic material is organized and utilized within a cell, illuminating relationships between the architecture of the genome and its functions. In biotechnology, precise manipulation of genetic building blocks and regulatory machinery also facilitates the reverse engineering or reconstruction of useful biological systems, for example, by enhancing biofuel production pathways in industrially relevant organisms or by creating infection-resistant crops. Additionally, genome engineering is stimulating a new generation of drug development processes and medical therapeutics. Perturbation of multiple genes simultaneously could model the additive effects that underlie complex polygenic disorders, leading to new drug targets, while genome editing could directly correct harmful mutations in the context of human gene therapy (Tebas et al., 2014).

Eukaryotic genomes contain billions of DNA bases and are difficult to manipulate. One of the breakthroughs in genome manipulation has been the development of gene targeting by homologous recombination (HR), which integrates exogenous repair templates that contain sequence homology to the donor site (Figure 2A) (Capecchi, 1989). HR-mediated targeting has facilitated the generation of knockin and knockout animal models via manipulation of germline competent stem cells, dramatically advancing many areas of biological research. However, although HR-mediated gene targeting produces highly precise alterations, the desired recombination events occur extremely infrequently (1 in 106–109 cells) (Capecchi, 1989), presenting enormous challenges for large-scale applications of gene-targeting experiments.

Genome Editing Technologies Exploit Endogenous DNA Repair Machinery

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/bin/nihms659174f2.gif

To overcome these challenges, a series of programmable nuclease-based genome editing technologies have been developed in recent years, enabling targeted and efficient modification of a variety of eukaryotic and particularly mammalian species. Of the current generation of genome editing technologies, the most rapidly developing is the class of RNA-guided endonucleases known as Cas9 from the microbial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats), which can be easily targeted to virtually any genomic location of choice by a short RNA guide. Here, we review the development and applications of the CRISPR-associated endonuclease Cas9 as a platform technology for achieving targeted perturbation of endogenous genomic elements and also discuss challenges and future avenues for innovation.   ……

Figure 4   Natural Mechanisms of Microbial CRISPR Systems in Adaptive Immunity

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/bin/nihms659174f4.gif

……  A key turning point came in 2005, when systematic analysis of the spacer sequences separating the individual direct repeats suggested their extrachromosomal and phage-associated origins (Mojica et al., 2005Pourcel et al., 2005Bolotin et al., 2005). This insight was tremendously exciting, especially given previous studies showing that CRISPR loci are transcribed (Tang et al., 2002) and that viruses are unable to infect archaeal cells carrying spacers corresponding to their own genomes (Mojica et al., 2005). Together, these findings led to the speculation that CRISPR arrays serve as an immune memory and defense mechanism, and individual spacers facilitate defense against bacteriophage infection by exploiting Watson-Crick base-pairing between nucleic acids (Mojica et al., 2005Pourcel et al., 2005). Despite these compelling realizations that CRISPR loci might be involved in microbial immunity, the specific mechanism of how the spacers act to mediate viral defense remained a challenging puzzle. Several hypotheses were raised, including thoughts that CRISPR spacers act as small RNA guides to degrade viral transcripts in a RNAi-like mechanism (Makarova et al., 2006) or that CRISPR spacers direct Cas enzymes to cleave viral DNA at spacer-matching regions (Bolotin et al., 2005).   …..

As the pace of CRISPR research accelerated, researchers quickly unraveled many details of each type of CRISPR system (Figure 4). Building on an earlier speculation that protospacer adjacent motifs (PAMs) may direct the type II Cas9 nuclease to cleave DNA (Bolotin et al., 2005), Moineau and colleagues highlighted the importance of PAM sequences by demonstrating that PAM mutations in phage genomes circumvented CRISPR interference (Deveau et al., 2008). Additionally, for types I and II, the lack of PAM within the direct repeat sequence within the CRISPR array prevents self-targeting by the CRISPR system. In type III systems, however, mismatches between the 5′ end of the crRNA and the DNA target are required for plasmid interference (Marraffini and Sontheimer, 2010).  …..

In 2013, a pair of studies simultaneously showed how to successfully engineer type II CRISPR systems from Streptococcus thermophilus (Cong et al., 2013) andStreptococcus pyogenes (Cong et al., 2013Mali et al., 2013a) to accomplish genome editing in mammalian cells. Heterologous expression of mature crRNA-tracrRNA hybrids (Cong et al., 2013) as well as sgRNAs (Cong et al., 2013Mali et al., 2013a) directs Cas9 cleavage within the mammalian cellular genome to stimulate NHEJ or HDR-mediated genome editing. Multiple guide RNAs can also be used to target several genes at once. Since these initial studies, Cas9 has been used by thousands of laboratories for genome editing applications in a variety of experimental model systems (Sander and Joung, 2014). ……

The majority of CRISPR-based technology development has focused on the signature Cas9 nuclease from type II CRISPR systems. However, there remains a wide diversity of CRISPR types and functions. Cas RAMP module (Cmr) proteins identified in Pyrococcus furiosus and Sulfolobus solfataricus (Hale et al., 2012) constitute an RNA-targeting CRISPR immune system, forming a complex guided by small CRISPR RNAs that target and cleave complementary RNA instead of DNA. Cmr protein homologs can be found throughout bacteria and archaea, typically relying on a 5 site tag sequence on the target-matching crRNA for Cmr-directed cleavage.

Unlike RNAi, which is targeted largely by a 6 nt seed region and to a lesser extent 13 other bases, Cmr crRNAs contain 30–40 nt of target complementarity. Cmr-CRISPR technologies for RNA targeting are thus a promising target for orthogonal engineering and minimal off-target modification. Although the modularity of Cmr systems for RNA-targeting in mammalian cells remains to be investigated, Cmr complexes native to P. furiosus have already been engineered to target novel RNA substrates (Hale et al., 20092012).   ……

Although Cas9 has already been widely used as a research tool, a particularly exciting future direction is the development of Cas9 as a therapeutic technology for treating genetic disorders. For a monogenic recessive disorder due to loss-of-function mutations (such as cystic fibrosis, sickle-cell anemia, or Duchenne muscular dystrophy), Cas9 may be used to correct the causative mutation. This has many advantages over traditional methods of gene augmentation that deliver functional genetic copies via viral vector-mediated overexpression—particularly that the newly functional gene is expressed in its natural context. For dominant-negative disorders in which the affected gene is haplosufficient (such as transthyretin-related hereditary amyloidosis or dominant forms of retinitis pigmentosum), it may also be possible to use NHEJ to inactivate the mutated allele to achieve therapeutic benefit. For allele-specific targeting, one could design guide RNAs capable of distinguishing between single-nucleotide polymorphism (SNP) variations in the target gene, such as when the SNP falls within the PAM sequence.

 

 

CRISPR/Cas9: a powerful genetic engineering tool for establishing large animal models of neurodegenerative diseases

Zhuchi Tu, Weili Yang, Sen Yan, Xiangyu Guo and Xiao-Jiang Li

Molecular Neurodegeneration 2015; 10:35  http://dx.doi.org:/10.1186/s13024-015-0031-x

Animal models are extremely valuable to help us understand the pathogenesis of neurodegenerative disorders and to find treatments for them. Since large animals are more like humans than rodents, they make good models to identify the important pathological events that may be seen in humans but not in small animals; large animals are also very important for validating effective treatments or confirming therapeutic targets. Due to the lack of embryonic stem cell lines from large animals, it has been difficult to use traditional gene targeting technology to establish large animal models of neurodegenerative diseases. Recently, CRISPR/Cas9 was used successfully to genetically modify genomes in various species. Here we discuss the use of CRISPR/Cas9 technology to establish large animal models that can more faithfully mimic human neurodegenerative diseases.

Neurodegenerative diseases — Alzheimer’s disease(AD),Parkinson’s disease(PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and frontotemporal dementia (FTD) — are characterized by age-dependent and selective neurodegeneration. As the life expectancy of humans lengthens, there is a greater prevalence of these neurodegenerative diseases; however, the pathogenesis of most of these neurodegenerative diseases remain unclear, and we lack effective treatments for these important brain disorders.

CRISPR/Cas9,  Non-human primates,  Neurodegenerative diseases,  Animal model

There are a number of excellent reviews covering different types of neurodegenerative diseases and their genetic mouse models [812]. Investigations of different mouse models of neurodegenerative diseases have revealed a common pathology shared by these diseases. First, the development of neuropathology and neurological symptoms in genetic mouse models of neurodegenerative diseases is age dependent and progressive. Second, all the mouse models show an accumulation of misfolded or aggregated proteins resulting from the expression of mutant genes. Third, despite the widespread expression of mutant proteins throughout the body and brain, neuronal function appears to be selectively or preferentially affected. All these facts indicate that mouse models of neurodegenerative diseases recapitulate important pathologic features also seen in patients with neurodegenerative diseases.

However, it seems that mouse models can not recapitulate the full range of neuropathology seen in patients with neurodegenerative diseases. Overt neurodegeneration, which is the most important pathological feature in patient brains, is absent in genetic rodent models of AD, PD, and HD. Many rodent models that express transgenic mutant proteins under the control of different promoters do not replicate overt neurodegeneration, which is likely due to their short life spans and the different aging processes of small animals. Also important are the remarkable differences in brain development between rodents and primates. For example, the mouse brain takes 21 days to fully develop, whereas the formation of primate brains requires more than 150 days [13]. The rapid development of the brain in rodents may render neuronal cells resistant to misfolded protein-mediated neurodegeneration. Another difficulty in using rodent models is how to analyze cognitive and emotional abnormalities, which are the early symptoms of most neurodegenerative diseases in humans. Differences in neuronal circuitry, anatomy, and physiology between rodent and primate brains may also account for the behavioral differences between rodent and primate models.

 

Mitochondrial dynamics–fusion, fission, movement, and mitophagy–in neurodegenerative diseases

Hsiuchen Chen and David C. Chan
Human Molec Gen 2009; 18, Review Issue 2 R169–R176
http://dx.doi.org:/10.1093/hmg/ddp326

Neurons are metabolically active cells with high energy demands at locations distant from the cell body. As a result, these cells are particularly dependent on mitochondrial function, as reflected by the observation that diseases of mitochondrial dysfunction often have a neurodegenerative component. Recent discoveries have highlighted that neurons are reliant particularly on the dynamic properties of mitochondria. Mitochondria are dynamic organelles by several criteria. They engage in repeated cycles of fusion and fission, which serve to intermix the lipids and contents of a population of mitochondria. In addition, mitochondria are actively recruited to subcellular sites, such as the axonal and dendritic processes of neurons. Finally, the quality of a mitochondrial population is maintained through mitophagy, a form of autophagy in which defective mitochondria are selectively degraded. We review the general features of mitochondrial dynamics, incorporating recent findings on mitochondrial fusion, fission, transport and mitophagy. Defects in these key features are associated with neurodegenerative disease. Charcot-Marie-Tooth type 2A, a peripheral neuropathy, and dominant optic atrophy, an inherited optic neuropathy, result from a primary deficiency of mitochondrial fusion. Moreover, several major neurodegenerative diseases—including Parkinson’s, Alzheimer’s and Huntington’s disease—involve disruption of mitochondrial dynamics. Remarkably, in several disease models, the manipulation of mitochondrial fusion or fission can partially rescue disease phenotypes. We review how mitochondrial dynamics is altered in these neurodegenerative diseases and discuss the reciprocal interactions between mitochondrial fusion, fission, transport and mitophagy.

 

Applications of CRISPR–Cas systems in Neuroscience

Matthias Heidenreich  & Feng Zhang
Nature Rev Neurosci 2016; 17:36–44   http://dx.doi.org:/10.1038/nrn.2015.2

Genome-editing tools, and in particular those based on CRISPR–Cas (clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein) systems, are accelerating the pace of biological research and enabling targeted genetic interrogation in almost any organism and cell type. These tools have opened the door to the development of new model systems for studying the complexity of the nervous system, including animal models and stem cell-derived in vitro models. Precise and efficient gene editing using CRISPR–Cas systems has the potential to advance both basic and translational neuroscience research.
Cellular neuroscience
, DNA recombination, Genetic engineering, Molecular neuroscience

Figure 3: In vitro applications of Cas9 in human iPSCs.close

http://www.nature.com/nrn/journal/v17/n1/carousel/nrn.2015.2-f3.jpg

a | Evaluation of disease candidate genes from large-population genome-wide association studies (GWASs). Human primary cells, such as neurons, are not easily available and are difficult to expand in culture. By contrast, induced pluripo…

  1. Genome-editing Technologies for Gene and Cell Therapy

Molecular Therapy 12 Jan 2016

  1. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing

Scientific Reports 31 Mar 2016

  1. Controlled delivery of β-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection

Scientific Reports 12 Nov 2015

 

Alzheimer’s Disease: Medicine’s Greatest Challenge in the 21st Century

https://www.physicsforums.com/insights/can-gene-editing-eliminate-alzheimers-disease/

The development of the CRISPR/Cas9 system has made gene editing a relatively simple task.  While CRISPR and other gene editing technologies stand to revolutionize biomedical research and offers many promising therapeutic avenues (such as in the treatment of HIV), a great deal of debate exists over whether CRISPR should be used to modify human embryos. As I discussed in my previous Insight article, we lack enough fundamental biological knowledge to enhance many traits like height or intelligence, so we are not near a future with genetically-enhanced super babies. However, scientists have identified a few rare genetic variants that protect against disease.  One such protective variant is a mutation in the APP gene that protects against Alzheimer’s disease and cognitive decline in old age. If we can perfect gene editing technologies, is this mutation one that we should be regularly introducing into embryos? In this article, I explore the potential for using gene editing as a way to prevent Alzheimer’s disease in future generations. Alzheimer’s Disease: Medicine’s Greatest Challenge in the 21st Century Can gene editing be the missing piece in the battle against Alzheimer’s? (Source: bostonbiotech.org) I chose to assess the benefit of germline gene editing in the context of Alzheimer’s disease because this disease is one of the biggest challenges medicine faces in the 21st century. Alzheimer’s disease is a chronic neurodegenerative disease responsible for the majority of the cases of dementia in the elderly. The disease symptoms begins with short term memory loss and causes more severe symptoms – problems with language, disorientation, mood swings, behavioral issues – as it progresses, eventually leading to the loss of bodily functions and death. Because of the dementia the disease causes, Alzheimer’s patients require a great deal of care, and the world spends ~1% of its total GDP on caring for those with Alzheimer’s and related disorders. Because the prevalence of the disease increases with age, the situation will worsen as life expectancies around the globe increase: worldwide cases of Alzheimer’s are expected to grow from 35 million today to over 115 million by 2050.

Despite much research, the exact causes of Alzheimer’s disease remains poorly understood. The disease seems to be related to the accumulation of plaques made of amyloid-β peptides that form on the outside of neurons, as well as the formation of tangles of the protein tau inside of neurons. Although many efforts have been made to target amyloid-β or the enzymes involved in its formation, we have so far been unsuccessful at finding any treatment that stops the disease or reverses its progress. Some researchers believe that most attempts at treating Alzheimer’s have failed because, by the time a patient shows symptoms, the disease has already progressed past the point of no return.

While research towards a cure continues, researchers have sought effective ways to prevent Alzheimer’s disease. Although some studies show that mental and physical exercise may lower ones risk of Alzheimer’s disease, approximately 60-80% of the risk for Alzheimer’s disease appears to be genetic. Thus, if we’re serious about prevention, we may have to act at the genetic level. And because the brain is difficult to access surgically for gene therapy in adults, this means using gene editing on embryos.

Reference https://www.physicsforums.com/insights/can-gene-editing-eliminate-alzheimers-disease/

 

Utilising CRISPR to Generate Predictive Disease Models: a Case Study in Neurodegenerative Disorders


Dr. Bhuvaneish.T. Selvaraj  – Scottish Centre for Regenerative Medicine

http://www.crisprsummit.com/utilising-crispr-to-generate-predictive-disease-models-a-case-study-in-neurodegenerative-disorders

  • Introducing the latest developments in predictive model generation
  • Discover how CRISPR is being used to develop disease models to study and treat neurodegenerative disorders
  • In depth Q&A session to answer your most pressing questions

 

Turning On Genes, Systematically, with CRISPR/Cas9

http://www.genengnews.com/gen-news-highlights/turning-on-genes-systematically-with-crispr-cas9/81250697/

 

Scientists based at MIT assert that they can reliably turn on any gene of their choosing in living cells. [Feng Zhang and Steve Dixon]  http://www.genengnews.com/media/images/GENHighlight/Dec12_2014_CRISPRCas9GeneActivationSystem7838101231.jpg

With the latest CRISPR/Cas9 advance, the exhortation “turn on, tune in, drop out” comes to mind. The CRISPR/Cas9 gene-editing system was already a well-known means of “tuning in” (inserting new genes) and “dropping out” (knocking out genes). But when it came to “turning on” genes, CRISPR/Cas9 had little potency. That is, it had demonstrated only limited success as a way to activate specific genes.

A new CRISPR/Cas9 approach, however, appears capable of activating genes more effectively than older approaches. The new approach may allow scientists to more easily determine the function of individual genes, according to Feng Zhang, Ph.D., a researcher at MIT and the Broad Institute. Dr. Zhang and colleagues report that the new approach permits multiplexed gene activation and rapid, large-scale studies of gene function.

The new technique was introduced in the December 10 online edition of Nature, in an article entitled, “Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.” The article describes how Dr. Zhang, along with the University of Tokyo’s Osamu Nureki, Ph.D., and Hiroshi Nishimasu, Ph.D., overhauled the CRISPR/Cas9 system. The research team based their work on their analysis (published earlier this year) of the structure formed when Cas9 binds to the guide RNA and its target DNA. Specifically, the team used the structure’s 3D shape to rationally improve the system.

In previous efforts to revamp CRISPR/Cas9 for gene activation purposes, scientists had tried to attach the activation domains to either end of the Cas9 protein, with limited success. From their structural studies, the MIT team realized that two small loops of the RNA guide poke out from the Cas9 complex and could be better points of attachment because they allow the activation domains to have more flexibility in recruiting transcription machinery.

Using their revamped system, the researchers activated about a dozen genes that had proven difficult or impossible to turn on using the previous generation of Cas9 activators. Each gene showed at least a twofold boost in transcription, and for many genes, the researchers found multiple orders of magnitude increase in activation.

After investigating single-guide RNA targeting rules for effective transcriptional activation, demonstrating multiplexed activation of 10 genes simultaneously, and upregulating long intergenic noncoding RNA transcripts, the research team decided to undertake a large-scale screen. This screen was designed to identify genes that confer resistance to a melanoma drug called PLX-4720.

“We … synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor,” wrote the authors of the Nature paper. “The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual [single-guide RNA] and complementary DNA overexpression.”

A gene signature based on the top screening hits, the authors added, correlated with a gene expression signature of BRAF inhibitor resistance in cell lines and patient-derived samples. It was also suggested that large-scale screens such as the one demonstrated in the current study could help researchers discover new cancer drugs that prevent tumors from becoming resistant.

More at –  http://www.genengnews.com/gen-news-highlights/turning-on-genes-systematically-with-crispr-cas9/81250697/

 

Susceptibility and modifier genes in Portuguese transthyretin V30M amyloid polyneuropathy: complexity in a single-gene disease
Miguel L. Soares1,2, Teresa Coelho3,6, Alda Sousa4,5, …, Maria Joa˜o Saraiva2,5 and Joel N. Buxbaum1
Human Molec Gen 2005; 14(4): 543–553   http://dx.doi.org:/10.1093/hmg/ddi051
https://www.researchgate.net/profile/Isabel_Conceicao/publication/8081351_Susceptibility_and_modifier_genes_in_Portuguese_transthyretin_V30M_amyloid_polyneuropathy_complexity_in_a_single-gene_disease/links/53e123d70cf2235f352733b3.pdf

Familial amyloid polyneuropathy type I is an autosomal dominant disorder caused by mutations in the transthyretin (TTR ) gene; however, carriers of the same mutation exhibit variability in penetrance and clinical expression. We analyzed alleles of candidate genes encoding non-fibrillar components of TTR amyloid deposits and a molecule metabolically interacting with TTR [retinol-binding protein (RBP)], for possible associations with age of disease onset and/or susceptibility in a Portuguese population sample with the TTR V30M mutation and unrelated controls. We show that the V30M carriers represent a distinct subset of the Portuguese population. Estimates of genetic distance indicated that the controls and the classical onset group were furthest apart, whereas the late-onset group appeared to differ from both. Importantly, the data also indicate that genetic interactions among the multiple loci evaluated, rather than single-locus effects, are more likely to determine differences in the age of disease onset. Multifactor dimensionality reduction indicated that the best genetic model for classical onset group versus controls involved the APCS gene, whereas for late-onset cases, one APCS variant (APCSv1) and two RBP variants (RBPv1 and RBPv2) are involved. Thus, although the TTR V30M mutation is required for the disease in Portuguese patients, different genetic factors may govern the age of onset, as well as the occurrence of anticipation.

Autosomal dominant disorders may vary in expression even within a given kindred. The basis of this variability is uncertain and can be attributed to epigenetic factors, environment or epistasis. We have studied familial amyloid polyneuropathy (FAP), an autosomal dominant disorder characterized by peripheral sensorimotor and autonomic neuropathy. It exhibits variation in cardiac, renal, gastrointestinal and ocular involvement, as well as age of onset. Over 80 missense mutations in the transthyretin gene (TTR ) result in autosomal dominant disease http://www.ibmc.up.pt/~mjsaraiv/ttrmut.html). The presence of deposits consisting entirely of wild-type TTR molecules in the hearts of 10– 25% of individuals over age 80 reveals its inherent in vivo amyloidogenic potential (1).

FAP was initially described in Portuguese (2) where, until recently, the TTR V30M has been the only pathogenic mutation associated with the disease (3,4). Later reports identified the same mutation in Swedish and Japanese families (5,6). The disorder has since been recognized in other European countries and in North American kindreds in association with V30M, as well as other mutations (7).

TTR V30M produces disease in only 5–10% of Swedish carriers of the allele (8), a much lower degree of penetrance than that seen in Portuguese (80%) (9) or in Japanese with the same mutation. The actual penetrance in Japanese carriers has not been formally established, but appears to resemble that seen in Portuguese. Portuguese and Japanese carriers show considerable variation in the age of clinical onset (10,11). In both populations, the first symptoms had originally been described as typically occurring before age 40 (so-called ‘classical’ or early-onset); however, in recent years, more individuals developing symptoms late in life have been identified (11,12). Hence, present data indicate that the distribution of the age of onset in Portuguese is continuous, but asymmetric with a mean around age 35 and a long tail into the older age group (Fig. 1) (9,13). Further, DNA testing in Portugal has identified asymptomatic carriers over age 70 belonging to a subset of very late-onset kindreds in whose descendants genetic anticipation is frequent. The molecular basis of anticipation in FAP, which is not mediated by trinucleotide repeat expansions in the TTR or any other gene (14), remains elusive.

Variation in penetrance, age of onset and clinical features are hallmarks of many autosomal dominant disorders including the human TTR amyloidoses (7). Some of these clearly reflect specific biological effects of a particular mutation or a class of mutants. However, when such phenotypic variability is seen with a single mutation in the gene encoding the same protein, it suggests an effect of modifying genetic loci and/or environmental factors contributing differentially to the course of disease. We have chosen to examine age of onset as an example of a discrete phenotypic variation in the presence of the particular autosomal dominant disease-associated mutation TTR V30M. Although the role of environmental factors cannot be excluded, the existence of modifier genes involved in TTR amyloidogenesis is an attractive hypothesis to explain the phenotypic variability in FAP. ….

ATTR (TTR amyloid), like all amyloid deposits, contains several molecular components, in addition to the quantitatively dominant fibril-forming amyloid protein, including heparan sulfate proteoglycan 2 (HSPG2 or perlecan), SAP, a plasma glycoprotein of the pentraxin family (encoded by the APCS gene) that undergoes specific calcium-dependent binding to all types of amyloid fibrils, and apolipoprotein E (ApoE), also found in all amyloid deposits (15). The ApoE4 isoform is associated with an increased frequency and earlier onset of Alzheimer’s disease (Ab), the most common form of brain amyloid, whereas the ApoE2 isoform appears to be protective (16). ApoE variants could exert a similar modulatory effect in the onset of FAP, although early studies on a limited number of patients suggested this was not the case (17).

In at least one instance of senile systemic amyloidosis, small amounts of AA-related material were found in TTR deposits (18). These could reflect either a passive co-aggregation or a contributory involvement of protein AA, encoded by the serum amyloid A (SAA ) genes and the main component of secondary (reactive) amyloid fibrils, in the formation of ATTR.

Retinol-binding protein (RBP), the serum carrier of vitamin A, circulates in plasma bound to TTR. Vitamin A-loaded RBP and L-thyroxine, the two natural ligands of TTR, can act alone or synergistically to inhibit the rate and extent of TTR fibrillogenesis in vitro, suggesting that RBP may influence the course of FAP pathology in vivo (19). We have analyzed coding and non-coding sequence polymorphisms in the RBP4 (serum RBP, 10q24), HSPG2 (1p36.1), APCS (1q22), APOE (19q13.2), SAA1 and SAA2 (11p15.1) genes with the goal of identifying chromosomes carrying common and functionally significant variants. At the time these studies were performed, the full human genome sequence was not completed and systematic singlenucleotide polymorphism (SNP) analyses were not available for any of the suspected candidate genes. We identified new SNPs in APCS and RBP4 and utilized polymorphisms in SAA, HSPG2 and APOE that had already been characterized and shown to have potential pathophysiologic significance in other disorders (16,20–22). The genotyping data were analyzed for association with the presence of the V30M amyloidogenic allele (FAP patients versus controls) and with the age of onset (classical- versus late-onset patients). Multilocus analyses were also performed to examine the effects of simultaneous contributions of the six loci for determining the onset of the first symptoms.  …..

The potential for different underlying models for classical and late onset is supported by the MDR analysis, which produces two distinct models when comparing each class with the controls. One could view the two onset classes as unique diseases. If this is the case, then the failure to detect a single predictive genetic model is consistent with two related, but different, diseases. This is exactly what would be expected in such a case of genetic heterogeneity (28). Using this approach, a major gene effect can be viewed as a necessary, but not sufficient, condition to explain the course of the disease. Analyzing the cases but omitting from the analysis of phenotype the necessary allele, in this case TTR V30M, can then reveal a variety of important modifiers that are distinct between the phenotypes.

The significant comparisons obtained in our study cohort indicate that the combined effects mainly result from two and three-locus interactions involving all loci except SAA1 and SAA2 for susceptibility to disease. A considerable number of four-site combinations modulate the age of onset with SAA1 appearing in a majority of significant combinations in late-onset disease, perhaps indicating a greater role of the SAA variants in the age of onset of FAP.

The correlation between genotype and phenotype in socalled simple Mendelian disorders is often incomplete, as only a subset of all mutations can reliably predict specific phenotypes (34). This is because non-allelic genetic variations and/or environmental influences underlie these disorders whose phenotypes behave as complex traits. A few examples include the identification of the role of homozygozity for the SAA1.1 allele in conferring the genetic susceptibility to renal amyloidosis in FMF (20) and the association of an insertion/deletion polymorphism in the ACE gene with disease severity in familial hypertrophic cardiomyopathy (35). In these disorders, the phenotypes arise from mutations in MEFV and b-MHC, but are modulated by independently inherited genetic variation. In this report, we show that interactions among multiple genes, whose products are confirmed or putative constituents of ATTR deposits, or metabolically interact with TTR, modulate the onset of the first symptoms and predispose individuals to disease in the presence of the V30M mutation in TTR. The exact nature of the effects identified here requires further study with potential application in the development of genetic screening with prognostic value pertaining to the onset of disease in the TTR V30M carriers.

If the effects of additional single or interacting genes dictate the heterogeneity of phenotype, as reflected in variability of onset and clinical expression (with the same TTR mutation), the products encoded by alleles at such loci could contribute to the process of wild-type TTR deposition in elderly individuals without a mutation (senile systemic amyloidosis), a phenomenon not readily recognized as having a genetic basis because of the insensitivity of family history in the elderly.

 

Safety and Efficacy of RNAi Therapy for Transthyretin Amyloidosis

Coelho T, Adams D, Silva A, et al.
N Engl J Med 2013;369:819-29.    http://dx.doi.org:/10.1056/NEJMoa1208760

Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart. A therapeutic approach mediated by RNA interference (RNAi) could reduce the production of transthyretin.

Methods We identified a potent antitransthyretin small interfering RNA, which was encapsulated in two distinct first- and second-generation formulations of lipid nanoparticles, generating ALN-TTR01 and ALN-TTR02, respectively. Each formulation was studied in a single-dose, placebo-controlled phase 1 trial to assess safety and effect on transthyretin levels. We first evaluated ALN-TTR01 (at doses of 0.01 to 1.0 mg per kilogram of body weight) in 32 patients with transthyretin amyloidosis and then evaluated ALN-TTR02 (at doses of 0.01 to 0.5 mg per kilogram) in 17 healthy volunteers.

Results Rapid, dose-dependent, and durable lowering of transthyretin levels was observed in the two trials. At a dose of 1.0 mg per kilogram, ALN-TTR01 suppressed transthyretin, with a mean reduction at day 7 of 38%, as compared with placebo (P=0.01); levels of mutant and nonmutant forms of transthyretin were lowered to a similar extent. For ALN-TTR02, the mean reductions in transthyretin levels at doses of 0.15 to 0.3 mg per kilogram ranged from 82.3 to 86.8%, with reductions of 56.6 to 67.1% at 28 days (P<0.001 for all comparisons). These reductions were shown to be RNAi mediated. Mild-to-moderate infusion-related reactions occurred in 20.8% and 7.7% of participants receiving ALN-TTR01 and ALN-TTR02, respectively.

ALN-TTR01 and ALN-TTR02 suppressed the production of both mutant and nonmutant forms of transthyretin, establishing proof of concept for RNAi therapy targeting messenger RNA transcribed from a disease-causing gene.

 

Alnylam May Seek Approval for TTR Amyloidosis Rx in 2017 as Other Programs Advance


https://www.genomeweb.com/rnai/alnylam-may-seek-approval-ttr-amyloidosis-rx-2017-other-programs-advance

Officials from Alnylam Pharmaceuticals last week provided updates on the two drug candidates from the company’s flagship transthyretin-mediated amyloidosis program, stating that the intravenously delivered agent patisiran is proceeding toward a possible market approval in three years, while a subcutaneously administered version called ALN-TTRsc is poised to enter Phase III testing before the end of the year.

Meanwhile, Alnylam is set to advance a handful of preclinical therapies into human studies in short order, including ones for complement-mediated diseases, hypercholesterolemia, and porphyria.

The officials made their comments during a conference call held to discuss Alnylam’s second-quarter financial results.

ATTR is caused by a mutation in the TTR gene, which normally produces a protein that acts as a carrier for retinol binding protein and is characterized by the accumulation of amyloid deposits in various tissues. Alnylam’s drugs are designed to silence both the mutant and wild-type forms of TTR.

Patisiran, which is delivered using lipid nanoparticles developed by Tekmira Pharmaceuticals, is currently in a Phase III study in patients with a form of ATTR called familial amyloid polyneuropathy (FAP) affecting the peripheral nervous system. Running at over 20 sites in nine countries, that study is set to enroll up to 200 patients and compare treatment to placebo based on improvements in neuropathy symptoms.

According to Alnylam Chief Medical Officer Akshay Vaishnaw, Alnylam expects to have final data from the study in two to three years, which would put patisiran on track for a new drug application filing in 2017.

Meanwhile, ALN-TTRsc, which is under development for a version of ATTR that affects cardiac tissue called familial amyloidotic cardiomyopathy (FAC) and uses Alnylam’s proprietary GalNAc conjugate delivery technology, is set to enter Phase III by year-end as Alnylam holds “active discussions” with US and European regulators on the design of that study, CEO John Maraganore noted during the call.

In the interim, Alnylam continues to enroll patients in a pilot Phase II study of ALN-TTRsc, which is designed to test the drug’s efficacy for FAC or senile systemic amyloidosis (SSA), a condition caused by the idiopathic accumulation of wild-type TTR protein in the heart.

Based on “encouraging” data thus far, Vaishnaw said that Alnylam has upped the expected enrollment in this study to 25 patients from 15. Available data from the trial is slated for release in November, he noted, stressing that “any clinical endpoint result needs to be considered exploratory given the small sample size and the very limited duration of treatment of only six weeks” in the trial.

Vaishnaw added that an open-label extension (OLE) study for patients in the ALN-TTRsc study will kick off in the coming weeks, allowing the company to gather long-term dosing tolerability and clinical activity data on the drug.

Enrollment in an OLE study of patisiran has been completed with 27 patients, he said, and, “as of today, with up to nine months of therapy … there have been no study drug discontinuations.” Clinical endpoint data from approximately 20 patients in this study will be presented at the American Neurological Association meeting in October.

As part of its ATTR efforts, Alnylam has also been conducting natural history of disease studies in both FAP and FAC patients. Data from the 283-patient FAP study was presented earlier this year and showed a rapid progression in neuropathy impairment scores and a high correlation of this measurement with disease severity.

During last week’s conference call, Vaishnaw said that clinical endpoint and biomarker data on about 400 patients with either FAC or SSA have already been collected in a nature history study on cardiac ATTR. Maraganore said that these findings would likely be released sometime next year.

Alnylam Presents New Phase II, Preclinical Data from TTR Amyloidosis Programs
https://www.genomeweb.com/rnai/alnylam-presents-new-phase-ii-preclinical-data-ttr-amyloidosis-programs

 

Amyloid disease drug approved

Nature Biotechnology 2012; (3http://dx.doi.org:/10.1038/nbt0212-121b

The first medication for a rare and often fatal protein misfolding disorder has been approved in Europe. On November 16, the E gave a green light to Pfizer’s Vyndaqel (tafamidis) for treating transthyretin amyloidosis in adult patients with stage 1 polyneuropathy symptoms. [Jeffery Kelly, La Jolla]

 

Safety and Efficacy of RNAi Therapy for Transthyretin …

http://www.nejm.org/…/NEJMoa1208760?&#8230;

The New England Journal of Medicine

Aug 29, 2013 – Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart.

 

Alnylam’s RNAi therapy targets amyloid disease

Ken Garber
Nature Biotechnology 2015; 33(577)    http://dx.doi.org:/10.1038/nbt0615-577a

RNA interference’s silencing of target genes could result in potent therapeutics.

http://www.nature.com/nbt/journal/v33/n6/images/nbt0615-577a-I1.jpg

The most clinically advanced RNA interference (RNAi) therapeutic achieved a milestone in April when Alnylam Pharmaceuticals in Cambridge, Massachusetts, reported positive results for patisiran, a small interfering RNA (siRNA) oligonucleotide targeting transthyretin for treating familial amyloidotic polyneuropathy (FAP).  …

  1. Analysis of 589,306 genomes identifies individuals resilient to severe Mendelian childhood diseases

Nature Biotechnology 11 April 2016

  1. CRISPR-Cas systems for editing, regulating and targeting genomes

Nature Biotechnology 02 March 2014

  1. Near-optimal probabilistic RNA-seq quantification

Nature Biotechnology 04 April 2016

 

Translational Neuroscience: Toward New Therapies

https://books.google.com/books?isbn=0262029863

Karoly Nikolich, ‎Steven E. Hyman – 2015 – ‎Medical

Tafamidis for Transthyretin Familial Amyloid Polyneuropathy: A Randomized, Controlled Trial. … Multiplex Genome Engineering Using CRISPR/Cas Systems.

 

Is CRISPR a Solution to Familial Amyloid Polyneuropathy?

Author and Curator: Larry H. Bernstein, MD, FCAP

Originally published as

https://pharmaceuticalintelligence.com/2016/04/13/is-crispr-a-solution-to-familial-amyloid-polyneuropathy/

 

http://scholar.aci.info/view/1492518a054469f0388/15411079e5a00014c3d

FAP is characterized by the systemic deposition of amyloidogenic variants of the transthyretin protein, especially in the peripheral nervous system, causing a progressive sensory and motor polyneuropathy.

FAP is caused by a mutation of the TTR gene, located on human chromosome 18q12.1-11.2.[5] A replacement of valine by methionine at position 30 (TTR V30M) is the mutation most commonly found in FAP.[1] The variant TTR is mostly produced by the liver.[citation needed] The transthyretin protein is a tetramer.    ….

 

 

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