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Removing Alzheimer plaques

Posted in Alzheimer's Disease, Medical Device Therapies for Altzheimer’s Disease, Neurodegenerative Diseases, Neurological Diseases, Neuroscience, tagged Alzheimer plaques, Antibody, Neuroscience on March 27, 2016| Leave a Comment »

Removing Alzheimer plaques

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Transdermal implant releases antibodies to trigger immune system to clear Alzheimer’s plaques March 21, 2016

Test with mice over 39 weeks showed dramatic reduction of amyloid beta plaque load in the brain and reduced phosphorylation of the protein tau, two signs of Alzheimer’s

http://www.kurzweilai.net/transdermal-implant-releases-antibodies-to-trigger-immune-system-to-clear-alzheimers-plaques

An implant that can prevent Alzheimer’s disease. A new capsule can be implanted under the skin to release antibodies that “tag” amyloid beta, signalling the patient’s immune system to clear it before it forms Alzheimer’s plaques. (credit: École polytechnique fédérale de Lausanne

EPFL scientists have developed an implantable capsule containing genetically engineered cells that can recruit a patient’s immune system to combat Alzheimer’s disease.

Placed under the skin, the capsule releases antibody proteins that make their way to the brain and “tag” amyloid beta proteins, signalling the patient’s own immune system to attack and clear the amyloid beta proteins, which are toxic to neurons.

To be most effective, this treatment has to be given as early as possible, before the first signs of cognitive decline. Currently, this requires repeated vaccine injections, which can cause side effects. The new implant can deliver a steady, safe flow of antibodies.

Protection from immune-system rejection

Cell encapsulation device for long-term subcutaneous therapeutic antibody delivery. (B) Macroscopic view of the encapsulation device, composed of a transparent frame supporting polymer permeable membranes and reinforced with an outer polyester mesh. (C) Dense neovascularization develops around a device containing antibody-secreting C2C12 myoblasts, 8 months after implantation in the mouse subcutaneous tissue. (D and E) Representative photomicrographs showing encapsulated antibody-secreting C2C12 myoblasts surviving at high density within the flat sheet device 39 weeks after implantation. (E) Higher magnification: note that the cells produce a collagen-rich matrix stained in blue with Masson’s trichrome protocol. Asterisk: polypropylene porous membrane. Scale bars = 750 mm (B and C),100 mm (D), 50 mm (E). (credit: Aurelien Lathuiliere et al./BRAIN)

The lab of Patrick Aebischer at EPFL designed the “macroencapsulation device” (capsule) with two permeable membranes sealed together with a polypropylene frame, containing a hydrogel that facilitates cell growth. All the materials used are biocompatible and the device is reproducible for large-scale manufacturing.

The cells of choice are taken from muscle tissue, and the permeable membranes let them interact with the surrounding tissue to get all the nutrients and molecules they need. The cells have to be compatible with the patient to avoid triggering the immune system against them, like a transplant can. To do that, the capsule’s membranes shield the cells from being identified and attacked by the immune system. This protection also means that cells from a single donor can be used on multiple patients.

The researchers tested the device mice in a genetic line commonly used to simulate Alzheimer’s disease over a course of 39 weeks, showing dramatic reduction of amyloid beta plaque load in the brain. The treatment also reduced the phosphorylation of the protein tau, another sign of Alzheimer’s observed in these mice.

“The proof-of-concept work demonstrates clearly that encapsulated cell implants can be used successfully and safely to deliver antibodies to treat Alzheimer’s disease and other neurodegenerative disorders that feature defective proteins,” according to the researchers.

The work is published in the journal BRAIN. It involved a collaboration between EPFL’s Neurodegenerative Studies Laboratory (Brain Mind Institute), the Swiss Light Source (Paul Scherrer Institute), and F. Hoffmann-La Roche. It was funded by the Swiss Commission for Technology and Innovation and F. Hoffmann-La Roche Ltd.

Abstract of A subcutaneous cellular implant for passive immunization against amyloid-β reduces brain amyloid and tau pathologies

Passive immunization against misfolded toxic proteins is a promising approach to treat neurodegenerative disorders. For effective immunotherapy against Alzheimer’s disease, recent clinical data indicate that monoclonal antibodies directed against the amyloid-β peptide should be administered before the onset of symptoms associated with irreversible brain damage. It is therefore critical to develop technologies for continuous antibody delivery applicable to disease prevention. Here, we addressed this question using a bioactive cellular implant to deliver recombinant anti-amyloid-β antibodies in the subcutaneous tissue. An encapsulating device permeable to macromolecules supports the long-term survival of myogenic cells over more than 10 months in immunocompetent allogeneic recipients. The encapsulated cells are genetically engineered to secrete high levels of anti-amyloid-β antibodies. Peripheral implantation leads to continuous antibody delivery to reach plasma levels that exceed 50 µg/ml. In a proof-of-concept study, we show that the recombinant antibodies produced by this system penetrate the brain and bind amyloid plaques in two mouse models of the Alzheimer’s pathology. When encapsulated cells are implanted before the onset of amyloid plaque deposition in TauPS2APP mice, chronic exposure to anti-amyloid-β antibodies dramatically reduces amyloid-β40 and amyloid-β42 levels in the brain, decreases amyloid plaque burden, and most notably, prevents phospho-tau pathology in the hippocampus. These results support the use of encapsulated cell implants for passive immunotherapy against the misfolded proteins, which accumulate in Alzheimer’s disease and other neurodegenerative disorders.

references:

Lathuilière A, Laversenne V, Astolfo A, Kopetzki E, Jacobsen H, Stampanoni M, Bohrmann B, Schneider BL, Aebischer P. A subcutaneous cellular implant for passive immunization against amyloid-β reduces brain amyloid and tau pathologies.Brain 08 March 2016. DOI: 10.1093/brain/aww036

A subcutaneous cellular implant for passive immunization against amyloid-β reduces brain amyloid and tau pathologies

Aurélien Lathuilière, Vanessa Laversenne, Alberto Astolfo, Erhard Kopetzki, Helmut Jacobsen, Marco Stampanoni, Bernd Bohrmann,Bernard L. Schneider, Patrick Aebischer

DOI: http://dx.doi.org/10.1093/brain/aww036

Passive immunization using monoclonal antibodies has recently emerged for the treatment of neurological diseases. In particular, monoclonal antibodies can be administered to target the misfolded proteins that progressively aggregate and propagate in the CNS and contribute to the histopathological signature of neurodegenerative diseases. Alzheimer’s disease is the most prevalent proteinopathy, characterized by the deposition of amyloid plaques and neurofibrillary tangles. According to the ‘amyloid cascade hypothesis’, which is supported by strong genetic evidence (Goate and Hardy, 2012), the primary pathogenic event in Alzheimer’s disease is the accumulation and aggregation of amyloid-β into insoluble extracellular plaques in addition to cerebral amyloid angiopathy (Hardy and Selkoe, 2002). High levels of amyloid-β may cause a cascade of deleterious events, including neurofibrillary tangle formation, neuronal dysfunction and death. Anti-amyloid-β antibodies have been developed to interfere with the amyloid-β cascade. Promising data obtained in preclinical studies have validated immunotherapy against Alzheimer’s disease, prompting a series of clinical trials (Bard et al., 2000;Bacskai et al., 2002; Oddo et al., 2004; Wilcock et al., 2004a; Bohrmann et al., 2012). Phase III trials using monoclonal antibodies directed against soluble amyloid-β (bapineuzumab and solanezumab) in patients with mild-to-moderate Alzheimer’s disease showed some effects on biomarkers that are indicative of target engagement. These trials, however, missed the primary endpoints, and it is therefore believed that anti-amyloid-β immunotherapy should be administered at the early presymptomatic stage (secondary prevention) to better potentiate therapeutic effects (Doody et al., 2014; Salloway et al., 2014). For the treatment of Alzheimer’s disease, it is likely that long-term treatment using a high dose of monoclonal antibody will be required. However, bolus administration of anti-amyloid-β antibodies may aggravate dose-dependent adverse effects such as amyloid-related imaging abnormalities (ARIA) (Sperling et al., 2012). In addition, the cost of recombinant antibody production and medical burden associated with repeated subcutaneous or intravenous bolus injections may represent significant constraints, especially in the case of preventive immunotherapy initiated years before the onset of clinical symptoms in patients predisposed to develop Alzheimer’s disease.

Therefore, alternative methods need to be developed for the continuous, long-term administration of antibodies. Here, we used an implant based on a high-capacity encapsulated cell technology (ECT) (Lathuiliere et al., 2014b). The ECT device contains myogenic cells genetically engineered for antibody production. Macromolecules can be exchanged between the implanted cells and the host tissue through a permeable polymer membrane. As the membrane shields the implanted cells from immune rejection in allogeneic conditions, it is possible to use a single donor cell source for multiple recipients. We demonstrate that anti-amyloid immunotherapy using an ECT device implanted in the subcutaneous tissue can achieve therapeutic effects inside the brain. Chronic exposure to anti-amyloid-β monoclonal antibodies produced in vivo using the ECT technology leads to a significant reduction of the amyloid brain pathology in two mouse models of Alzheimer’s disease.

Microglial phagocytosis study

The measurement of antibody-mediated amyloid-β phagocytosis was performed as proposed previously (Webster et al., 2001). In this study, we used either purified preparations of full mAb-11 IgG2a antibody, or a purified Fab antibody fragment. A suspension of 530 µM fluorescent fibrillar amyloid-β₄₂ was prepared in 10 mM HEPES (pH 7.4) by stirring overnight at room temperature. The resulting suspension contained 30 µM fluorescein-conjugated amyloid-β₄₂ and 500 µM unconjugated amyloid-β₄₂ (Bachem). IgG-fibrillar amyloid-β₄₂ immune complexes were obtained by preincubating fluorescent fibrillar amyloid-β₄₂ at a concentration of 50 µM in phosphate-buffered saline (PBS) with various concentrations of purified mAb-11 IgG2a or Fab antibody fragment for 30 min at 37 °C. The immune complexes were washed twice by centrifugation for 5 min at 14 000g and resuspended in the initial volume to obtain a fluorescent fibrillar amyloid-β₄₂ solution (total amyloid-β₄₂ concentration: 530 µM). The day before the experiment, 8 × 10⁴ C8-B4 cells were plated in 24-well plates. The medium was replaced with serum-free DMEM before the addition of the peptides. The cells were incubated for 30 min with fibrillar amyloid-β₄₂ or IgG-fibrillar amyloid-β₄₂ added to the culture medium. Next, the cells were washed twice with Hank’s Balanced Salt Solution (HBSS) and subsequently detached by trypsinization, which also eliminates surface-bound fibrillar amyloid-β₄₂. The cells were fixed for 10 min in 4% paraformaldehyde and finally resuspended in PBS. The cell fluorescence was determined with a flow cytometer (Accuri C6; BD Biosciences), and the data were analysed using the FlowJo software (TreeStar Inc.). To determine the effect of the anti-amyloid-β antibodies on amyloid-β phagocytosis, the concentration of fluorescent fibrillar amyloid-β₄₂ was set at 1.5 µM, which is in the linear region of the dose-response curve depicting fibrillar amyloid-β₄₂ phagocytosis in C8-B4 cells (Fig. 2C). All experiments were performed in duplicate.

The implantation of genetically engineered cells within a retrievable subcutaneous device leads to the continuous production of monoclonal antibodies in vivo. This technology achieves steady therapeutic monoclonal antibody levels in the plasma, offering an effective alternative to bolus injections for passive immunization against chronic diseases. Peripheral delivery of anti-amyloid-β monoclonal antibody by ECT leads to a significant reduction of amyloid burden in two mouse models of Alzheimer’s disease. The effect of the ECT treatment is more pronounced when passive immunization is preventively administered in TauPS2APP mice, most notably decreasing the phospho-tau pathology.

With the recent development of biomarkers to monitor Alzheimer’s pathology, it is recognized that a steady increase in cerebral amyloid over the course of decades precedes the appearance of the first cognitive symptoms (reviewed in Sperling et al., 2011). The current consensus therefore suggests applying anti-amyloid-β immunotherapy during this long asymptomatic phase to avoid the downstream consequences of amyloid deposition and to leverage neuroprotective effects. Several preventive clinical trials have been recently initiated for Alzheimer’s disease. The Alzheimer’s Prevention Initiative (API) and the Dominantly Inherited Alzheimer Network (DIAN) will test antibody candidates in presymptomatic dominant mutation carriers, while the Anti-Amyloid treatment in the Asymptomatic Alzheimer’s disease (A4) trial enrols asymptomatic subjects after risk stratification. If individuals with a high risk of developing Alzheimer’s disease can be identified using current biomarker candidates, these patients are the most likely to benefit from chronic long-term anti-amyloid-β immunotherapy. However, such a treatment may pose a challenge to healthcare systems, as the production capacity of the antibody and its related cost would become a challenging issue (Skoldunger et al., 2012). Therefore, the development of alternative technologies to chronically administer anti-amyloid-β antibody is an important aspect for therapeutic interventions at preclinical disease stages.

Here, we show that the ECT technology for the peripheral delivery of anti-amyloid-β monoclonal antibodies can significantly reduce cerebral amyloid pathology in two mouse models of Alzheimer’s disease. The subcutaneous tissue is a site of implantation easily accessible and therefore well adapted to preventive treatment. It is, however, challenging to reach therapeutic efficacy, as only a small fraction of the produced anti-amyloid-β monoclonal antibodies are expected to cross the blood–brain barrier, although they can next persist in the brain for several months (Wang et al., 2011; Bohrmann et al., 2012). Our results are consistent with previous reports, which have shown that the systemic administration of anti-amyloid-β antibodies can decrease brain amyloid burden in preclinical Alzheimer’s disease models (Bard et al., 2000, 2003; DeMattos et al., 2001; Wilcock et al., 2004a, b; Buttini et al., 2005; Adolfsson et al., 2012).

Remarkably, striking differences exist among therapeutic anti-amyloid-β antibodies in their ability to clear already existing plaques. Soluble amyloid-β species can saturate the small fraction of pan-amyloid-β antibodies entering the CNS and inhibit further target engagement (Demattos et al., 2012). Therefore, antibodies recognizing soluble amyloid-β may fail to bind and clear insoluble amyloid deposits (Das et al., 2001; Racke et al., 2005; Levites et al., 2006; Bohrmann et al., 2012). Furthermore, antibody-amyloid-β complexes are drained towards blood vessels, promoting cerebral amyloid angiopathy (CAA) and subsequent microhaemorrhages. The mAb-11 antibody used in the present study is similar to gantenerumab, which is highly specific for amyloid plaques and reduces amyloid burden in patients with Alzheimer’s disease (Bohrmann et al., 2012; Demattos et al., 2012; Ostrowitzki et al., 2012). We find that the murine IgG2a mAb-11 antibody efficiently enhances the phagocytosis of amyloid-β fibrils by microglial cells. In addition, ECT administration of the mAb-11 F(ab’)₂ fragment lacking the Fc region fails to recruit microglial cells, and leads only to a trend towards clearance of the amyloid plaques. Therefore, our results suggest a pivotal role for microglial cells in the clearance of amyloid plaques following mAb-11 delivery by ECT. Importantly, we do not find any evidence that this treatment may cause microhaemorrhages in the mouse models used in this study. It remains entirely possible that direct binding to amyloid plaques of a F(ab’)₂ fragment lacking effector functionality can contribute to therapeutic efficacy, as suggested by previous studies using antibody fragments (Bacskai et al., 2002;Tamura et al., 2005; Wang et al., 2010; Cattepoel et al., 2011). However, compared to IgG2a, the lower plasma levels achieved with F(ab’)₂ are likely to limit the efficacy of peripheral ECT-mediated immunization. The exact role of the effector domain and its interaction with immune cells expressing Fc receptors, could be determined by comparing the therapeutic effects of a control antibody carrying a mutated Fc portion, similar to a previous study which addressed this question using deglycosylated anti-amyloid-β antibodies (Wilcock et al., 2006a; Fuller et al., 2014).

Remarkably, continuous administration of mAb-11 initiated before plaque deposition had a dramatic effect on the amyloid pathology in TauPS2APP mice, underlining the efficacy of preventive anti-amyloid-β treatments. In this mouse model, where tau hyperphosphorylation is enhanced by amyloid-β (Grueninger et al., 2010), the treatment decreases the number of AT8- and phospho-S422-positive neurons in the hippocampus. Furthermore, the number of MC1-positive hippocampal neurons is significantly reduced, which also indicates an effect of anti-amyloid-β immunotherapy on the accumulation of misfolded tau. These results highlight the effect of amyloid-β clearance on other manifestations of the Alzheimer’s pathology. In line with these findings, previous studies have shown evidence for a decrease in tau hyperphosphorylation following immunization against amyloid-β, both in animal models and in patients with Alzheimer’s disease (Oddo et al., 2004; Wilcock et al., 2009;Boche et al., 2010; Serrano-Pozo et al., 2010; Salloway et al., 2014).

Similar to the subcutaneous injection of recombinant proteins (Schellekens, 2005), ECT implants can elicit significant immune responses against the secreted recombinant antibody. An anti-drug antibody response was detected in half of the mice treated with the mAb-11-releasing devices, in the absence of any anti-CD4 treatment. The glycosylation profile of the mAb-11 synthesized in C2C12 myoblasts is comparable to standard material produced by myeloma or HEK293 cells (Lathuiliere et al., 2014a). Although we cannot exclude that local release by ECT leads to antibody aggregation and denaturation, it is unlikely that this mode of administration further contributes to compound immunogenicity. Because the Fab regions of the chimeric recombinant mAb-11 IgG2a contain human CDRs, it remains to be determined whether the ECT-mediated delivery of antibodies fully matched with the host species would trigger an anti-drug antibody response.

Further developments will be needed to scale up this delivery system to humans. The possibility of using a single allogeneic cell source for all intended recipients is a crucial advantage of the ECT technology to standardize monoclonal antibody delivery. However, the development of renewable cell sources of human origin will be essential to ECT application in the clinic. Although the ARPE-19 cell line has been successfully adapted to ECT and used in clinical trials (Dunn et al., 1996; Zhang et al., 2011), the development of human myogenic cells (Negroni et al., 2009) is an attractive alternative that is worth exploring. Based on the PK analysis of recombinant mAb-11 antibody subcutaneously injected in mice (Supplementary material), we estimate that the flat sheet devices chronically release mAb-11 at a rate of 6.8 and 11.8 µg/h, to reach a plasma level of 50 µg/ml in the implanted animals. In humans, injected IgG1 has a longer half-life (21–25 days), with a volume of distribution of ∼100 ml/kg and an estimated clearance of 0.2 ml/h/kg. These values indicate that the predicted antibody exposure in humans, based on the rate of mAb-11 secretion achieved by ECT in mice, would be only 10 to 20-fold lower than the typical regimens based on monthly bolus injection of 1 mg/kg anti-amyloid-β monoclonal antibody. Hence, it is realistic to consider ECT for therapeutic monoclonal antibody delivery in humans, as the flat sheet device could be scaled up to contain higher amounts of cells. Furthermore, recent progress to engineer antibodies for increased penetration into the brain will enable lowering dosing of biotherapeutics to achieve therapeutic efficacy (Bien-Ly et al., 2014; Niewoehner et al., 2014). For some applications, intrathecal implantation could be preferred to chronically deliver monoclonal antibodies directly inside the CNS (Aebischer et al., 1996; Marroquin Belaunzaran et al., 2011).

Overall, ECT provides a novel approach for the local and systemic delivery of recombinant monoclonal antibodies in the CNS. It will expand the possible therapeutic options for immunotherapy against neurodegenerative disorders associated with the accumulation of misfolded proteins, including Alzheimer’s and Parkinson’s diseases, dementia with Lewy bodies, frontotemporal lobar dementia and amyotrophic lateral sclerosis (Gros-Louis et al., 2010; Bae et al., 2012; Rosenmann, 2013).

Abbreviations
ECT: encapsulated cell technology
mAb: monoclonal antibody

© The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com

sjwilliamspa

The most powerful part of the article is the methodology of the transdermal patch of genetically engineered cells which appear to give a constant stream of antibody therapy. This would be better for the authors to have highlighted because the protein delivery system is great work. However it is a shame that the Alzheimer’s field is still relying on these mouse models which are appearing to lead the filed down a path which leads to failed clinical trials. Lilly has just dropped their Alzheimer’s program and unless the filed shifts to a different hypothesis of disease etilology I feel the whole field will be stuck where it is.

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Schizophrenia genomics

Posted in Genome Biology, Mutant Gene Expression, Neurological Diseases, Neuroscience, Schizophrenia, tagged Akt/PKB, autism spectrum disorders (ASDs), Chromatin, copy number variation (CNV), Epigenetics, histone methylation, Lymphocyte, methyl-CpG-binding domain (MBD), PDK2, PI3K/PTEN, Postmortem Brain, Rett syndrome, Schizophrenia, single nucleotide polymorphism (SNP) on March 15, 2016| Leave a Comment »

Schizophrenia genomics

Larry H. Bernstein, MD, FCAP, Curator

LPBI

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

Kayla A. Chase,¹David P. Gavin,¹Alessandro Guidotti,¹ and Rajiv P. Sharma¹

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

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

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

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

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

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

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

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

3.1. mRNA Levels of HMT Gene Expression

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

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

Fig. 1

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

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

3.2. H3K9me2 levels in the Postmortem Brain

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

Fig. 2

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

3.3. Clinical Correlates with Lymphocyte HMT mRNA Levels

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

Fig. 3

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

4. Discussion

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

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

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

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

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

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

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

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

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Balancing Histone Methylation Activities in Psychiatric Disorders

Cyril Jayakumar Peter¹ and Schahram Akbarian^1,^{^²}

Trends Mol Med. 2011 Jul; 17(7): 372–379. doi: 10.1016/j.molmed.2011.02.003

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

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

Box 1 Schizophrenia and Depression

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

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

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

However, there is evidence that dysregulated gene transcription, indicative of compromised neural circuitry, contributes to disordered brain function in psychosis and mood spectrum disorder [1, 2]. While no single gene transcript is consistently affected, alterations in RNA levels contribute to defects in GABAergic inhibitory neurotransmission and more generally, synapse organization and function, metabolism and mitochondrial functions, and oligodendrocyte pathology [3–5]. While a number of transcriptional and post-transcriptional mechanisms may contribute to these changes, chromatin-associated proteins and epigenetic regulators invoked in sustained alterations of gene expression and function (Box 2) could play a critical role in the pathophysiology, or treatment of mental illness [6,7]. Indeed, there is evidence that changes in acetylation of histone lysine residues, which are broadly associated with active gene expression [8] and considered a potential therapeutic target for cancer and other medical conditions [9], also impact gene expression patterns in the brain and thereby influence emotional and cognitive functions. For example, mice or rats exposed to systemic treatment, or localized intracranial injections of class I/II histone deacetylase inhibitors (HDACi) exhibit behavioral changes reminiscent of those elicited by conventional antidepressant drugs [10–13]. The short chain fatty acid derivative valproic acid, widely prescribed for its mood-stabilizing and anticonvulsant effects, induces brain histone hyperacetylation at a select set of gene promoters when administered to animals at comparatively high doses [14]. Conversely, overexpression of selected HDACs in neuronal structures implicated in the neurobiology of depression, including the hippocampus, elicit a pro-depressant behavioral phenotype [12]. Similarly, animals treated with class I/II HDACi often show improved performance in learning and memory paradigms and furthermore, drug-induced inhibition or activation of class III HDAC (also known as sirtuins) elicits changes in motivational and reward-related behaviors [15]. Therefore, the orderly balance between histone acetyl-transferase and deacetylase activities is critical for cognitive performance and synaptic and behavioral plasticity [16]. Likewise, However, HDACs interfere with acetylation of many non-histone proteins in the nucleus and cytoplasm [16], and moreover, some of these drugs carry a significant side effect burden [9]. Therefore, in light of the emerging role of epigenetic mechanisms in the neurobiology of these and other psychiatric conditions [6], the therapeutic potential of chromatin modifying drugs, other than the HDACi, warrants further investigations. This review will focus on histone lysine methylation, one of the most highly regulated chromatin markings in brain and other tissues. Multiple methyltransferases (KMTs) and demethylases (KDMs) were recently implicated in emotional and cognitive disorders (Fig. 1), and these types of chromatin modifying enzymes could emerge as novel targets in the treatment of mood and psychosis spectrum disorders.

Box 2 Epigenetic regulators and chromatin structure and function

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

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

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

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

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

To date, H3K4, H4K9, H3K27 and H4K20 methylation signals were measured at specific loci and genome-wide in human brain, essentially confirming that each of these epigenetic markings defines the same type of chromatin as in the peripheral tissues or animal brain [26–30]. Interestingly, a subset of psychotherapeutic drugs including the mood-stabilizer valproate, the atypical antipsychotic clozapine and some monoamine oxidase inhibitors and stimulant drugs interfere with brain histone methylation (Table 1).

Molecular mechanisms of histone (lysine) methylation

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

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

KMTs and KDMs with a role in cognition and neuropsychiatric disease

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

H3K4

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

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

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

H3K9

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

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

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

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

H3K27

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

H3K36 and H4K20

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

KMTs and KDMs as Novel Drug Targets

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

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

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

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

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

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

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

…….

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

……

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

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

Holly N. Cukier,¹Joycelyn M. Lee,¹Deqiong Ma,^1,²Juan I. Young,^1,²Vera Mayo,¹ ….,Harry H. Wright,³Ruth K. Abramson,³Jonathan L. Haines,⁴Michael L. Cuccaro,^1,²Margaret A. Pericak-Vance,^1,^2,^❖ and John R. Gilbert^1,²

Autism Res. 2012 Dec; 5(6): 385–397. doi: 10.1002/aur.1251

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

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

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

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

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

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

While there is clinical evidence of MECP2 and MBD5 playing a role in autism, only two studies to date have evaluated patients with ASD for mutations in additional MBD family members (Li et al., 2005; Cukier et al., 2010). Previous work in our laboratory analyzed the coding regions of MBD1, MBD2, MBD3, andMBD4 in over 200 individuals with ASD of African and European ancestry and identified multiple variants that altered the amino acid sequence, were unique to patients with autism, and concordant with disease in multiplex families (Cukier et al., 2010). In contrast, a study by Li and colleagues was restricted to a dataset of 65 Japanese autistic patients and reported only a single variation that might be related to autism (Li et al., 2005). We now expand our initial study of MECP2 to a larger dataset that includes male patients and perform the first study evaluating patients with ASD for alterations in four additional MBD family members: MBD5, MBD6, SETDB1 and SETDB2.

Sequencing across the five MBD genes in 287 patients with ASD and 288 ethnically matched control individuals identified a total of 186 unique variations (Table 1, Supplemental Tables 3–7). These variants included 177 single nucleotide polymorphisms (SNPs), five deletions and four insertions. Ninety (48.4%) of the variations have been previously reported in either the dbSNP 134 database (http://www.ncbi.nlm.nih.gov/projects/SNP/) or RettBASE (http://mecp2.chw.edu.au/), while the remaining 96 variants (51.6%) are novel. Fifty-six variations are predicted to alter the amino acid sequence. Fifty-three of the changes were found solely in patients with ASD and absent from controls. To determine variants most likely to contribute to ASD susceptibility, we prioritized changes that were either unique to affected individuals or that had an increased frequency in cases when compared to controls. The 17 most interesting variants were nonsynonymous and unique to our ASD population (Table 1). We utilized four distinct programs to characterize the variants; GERP (Cooper et al., 2005) and PhastCons (Siepel et al., 2005) to measure the level of amino acid conservation across species and PolyPhen (Adzhubei et al., 2010) and SIFT (Kumar et al., 2009) to predict which alterations might have the damaging consequences to protein function.

Table 1

ASD Unique, Nonsynonymous Variations

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

MBD5

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

Figure 1

Pedigrees of ASD families carrying alterations in MBD5 and MBD6

MBD6

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

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

MECP2

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

Figure 2

Pedigrees of ASD families carrying alterations in MeCP2

SETDB1

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

Figure 3

Pedigrees of ASD families carrying alterations in SETDB1 and SETDB2

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

SETDB2

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

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

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

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

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

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

PI3K/Akt: getting it right matters

T F Franke^{1 Oncogene (2008) 27, 6473–6488; http://dx.doi.org:/10.1038/onc.2008.313}

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

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

Figure 1.

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

Full figure and legend (367K)

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

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

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

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

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

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

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

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

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

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

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

Figure 2.

Akt kinase regulates diverse aspects of neuronal cell function. Akt activation in neuronal cells follows similar mechanisms to those outlined in Figure 1, including activation of PI3K by RTKs including IGF-1/insulin and nerve growth factor (BDNF/NT-3) receptors. Other mechanisms governing Akt activity in neuronal cells include G-coupled receptors for the monoamine neurotransmitters serotonin (5-HT) (for review, see Raymond et al., 2001) and dopamine (DA) (for review, see Beaulieu et al., 2007). Depending on receptor type (D₂-DA and 5-HT_1A receptors vs D₁-DA receptors), binding of 5-HT or DA decreases or increases the activity of adenylyl cyclase (AC), respectively. Changes in cAMP second messenger levels, in turn, alter PKA and PP2A activity. PP2A is inhibited by increased PKA activity, thus maintaining Akt in an activated state after 5-HT_1Areceptor simulation (Hsiung et al., 2008). After binding of DA to D₂-DA receptor, following initial inhibition of AC, a secondary internalization complex is formed between β-arrestin 2, PP2A and Akt leading to the inhibition of Akt. In neuronal cells, activated Akt regulates diverse targets that have been implicated in the regulation of protein translation and cell size (mTORC1), axonal outgrowth (GSK3), apoptosis suppression (BAD) and synaptic plasticity (GABA_A-R). Details regarding functional consequences of Akt regulation for higher brain function are discussed in the text.

Full figure and legend (306K)

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

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

SETDB1 in Early Embryos and Embryonic Stem Cells

Yong-Kook Kang

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

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

Exome sequencing of probands with autism have revealed broadly similar results:de novo mutations in a large set of genes occur in a significant fraction of patients, with relatively high OR’s for damaging mutations in genes expressed in the brain^9,19–21. Most interestingly, CHD8, which like CHD7 reads H3K4me marks, is frequently mutated in autism²², raising the question of whether the H3K4me pathway may play a role in many congenital diseases. Among 249 protein-alteringde novo mutations in CHD (Supplementary Table 4) and 570 such mutations in autism^9,19,20,23, there were two genes, CUL3 and NCKAP1, with damaging mutations in both CHD and autism and none in controls (P = 0.001, Monte Carlo simulation), and several others with mutations in both (e.g., SUV40H1 and CHD7). Similarly, rare copy number variants at 22q11.2, 1q21, and 16p11 are found in patients with autism, CHD or both diseases^24–26. These observations suggest variable expressivity of mutations in key developmental genes. Identification of the complete set of these developmental genes and the full spectrum of the resulting phenotypes will likely be important for patient care and genetic counseling.

Context-specific microRNA function in developmental complexity

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

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

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

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

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

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

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

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

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

Mechanisms of miRNA function

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

miRNA biogenesis

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

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

Figure 1

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

miRISC loading

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

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

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

…….

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

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

Figure 2

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

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

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Alzheimer’s disease, snake venome, amyloid and transthyretin

Posted in Alzheimer's Disease, Etiology, Neurodegenerative Diseases, Neurological Diseases, Neuroscience, Pharmacotherapy and Cell Activity, tagged A beta, Alzheimers Disease, amyloid plaque, infection, Prion diseases, snake venum, Transthyretin (TTR) on March 8, 2016| Leave a Comment »

Alzheimer’s disease, snake venome, amyloid and transthyretin

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Significant points:

Alzheimer’s Disease is characterized by amyloid plaques
The plaques have amyloid beta and tau
Toxic proteins accumulate in AD
snake venome activates enzymes (Endothelin Converting Enzyme-1 and Neprilysin) that break down the plaques that are sufficient in non-AD brain
Aβ peptides derive from proteolytic processing of a large (695/770 amino acids) type 1 transmembrane glycoprotein known as amyloid beta precursor protein (APP)
a natural variant of Amyloid-β (Aβ) carrying the A2V substitution protects heterozygous carriers from AD by its ability to interact with wild-type Aβ, hindering conformational changes and assembly
aggregated Aβ species, particularly oligomeric assemblies, trigger a cascade of events that lead to hyperphosphorylation, misfolding and assembly of the tau protein with formation of neurofibrillary tangles
[Aβ1-6A2VTAT(D)] revealed strong anti-amyloidogenic effects in vitro and protected human neuroblastoma cells from Aβ toxicity
while both Aβ1-6A2V and Aβ1-6WT display a predominant coil configuration, Aβ1-6A2V shows a slightly higher propensity to form secondary structure motifs involving two to three residues
Aβ1-6A2VTAT(D) maintains the in vitro anti-amyloidogenic properties of Aβ1-6A2V(D)
Transthyretin (TTR) influences plasma Aβ by reducing its levels
Transthyretin (TTR) binds Aβ peptide, preventing its deposition and toxicity
TTR facilitated peptide internalization of Aβ1-42 uptake by primary hepatocytes
Brain permeability to TTR
TTR regulates LRP1 levels, suggesting that TTR uses this receptor to promote Aβ clearance

Snake venom may hold key to breaking down plaques that cause Alzheimer’s disease

March 2, 2016 http://medicalxpress.com/news/2016-03-snake-venom-key-plaques-alzheimer.html

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4750079/bin/srep20949-f2.jpg

http://www.ncbi.nlm.nih.gov/pmc/articles/instance/4750079/bin/srep20949-f2.jpg

Alzheimer’s disease, snake venome, amyloid and transthyretin

Snake venom may hold key to breaking down plaques that cause Alzheimer’s disease

http://img.medicalxpress.com/newman/csz/news/800/2016/snakevenomma.jpg

A toxic protein called amyloid beta is thought to play a key role in the onset of Alzheimer’s disease. In healthy people, amyloid beta is degraded by enzymes as it forms. However, in patients with the disease, these enzymes appear unable to adequately perform their actions, causing the toxic protein to accumulate into plaque deposits, which many researchers consider leads to dementia.

One of the Holy Grails of the pharmaceutical industry has been to find a drug that stimulates these enzymes in people, particularly those who are in the early stages of dementia, when amyloid plaques are just starting to accumulate.

Monash researchers have discovered what could well be this elusive drug candidate– a molecule in snake venom that appears to activate the enzymes involved in breaking down the amyloid plaques in the brain that are the hallmark of Alzheimer’s disease. Dr Sanjaya Kuruppu and Professor Ian Smith from Monash University’s Biomedicine Discovery Institute have just published their research in Nature Scientific Reports.

Dr Kuruppu has spent most of his research life studying snake venoms, looking for drug candidates. When he began researching Alzheimer’s disease he says that “snake venom was an obvious place for me to start.”

He was looking for a molecule that would stimulate the enzymes to break down the amyloid plaques. What he found, when screening various snake venoms, was in fact one molecule with the ability to enhance the activity of two plaque degrading enzymes. This molecule was extracted from a venom of a pit viper found in South and Central America. Dr Kuruppu and his team have developed synthetic versions of this molecule. Initial tests done in the laboratory using human cells have shown it to have the same effects as the native version found in the snake venom.

Dr Kuruppu is one of the four researchers in Australia to receive funding from the National Foundation for Medical Research and Innovation to conduct further testing of this newly-identified molecule.

Explore further: Alzheimer protein’s structure may explain its toxicity

More information: A. Ian Smith et al. N-terminal domain of Bothrops asper Myotoxin II Enhances the Activity of Endothelin Converting Enzyme-1 and Neprilysin, Scientific Reports (2016).
http://dx.doi.org:/10.1038/srep22413

N-terminal domain of Bothrops asper Myotoxin II Enhances the Activity of Endothelin Converting Enzyme-1 and Neprilysin

Ian Smith, Niwanthi W. Rajapakse, Oded Kleifeld, Bruno Lomonte,…, Helena C. Parkington, James C. Whisstock & Sanjaya Kuruppu

Scientific Reports 6, Article number: 22413 (2016) http://www.nature.com/articles/srep22413

Neprilysin (NEP) and endothelin converting enzyme-1 (ECE-1) are two enzymes that degrade amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49-P1-20, from the venom of Bothrops asper which directly enhances the activity of both ECE-1 and NEP. This is evidenced by a 2- and 5-fold increase in the Vmax of ECE-1 and NEP respectively. The K49-P1-20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE-1 and NEP was 1.92 ± 0.07 and 1.33 ± 0.12 μM respectively. Using BLITZ biolayer interferometry we have shown that K49-P1-20 interacts directly with each enzyme. Intrinsic fluorescence of the enzymes change in the presence of K49-P1-20 suggesting a change in conformation. ECE-1 mediated reduction in the level of endogenous soluble amyloid beta 42 in cerebrospinal fluid is significantly higher in the presence of K49-P1-20 (31 ± 4% of initial) compared with enzyme alone (11 ± 5% of initial; N = 8, P = 0.005, unpaired t-test). K49-P1-20 could be an excellent research tool to study mechanism(s) of enzyme stimulation, and a potential novel drug lead in the fight against Alzheimer’s disease.

Metalloproteases play a central role in regulating many physiological processes and consequently abnormal activity of these enzymes contribute to a wide range of disease pathologies. These include cardiovascular1 and neurodegenerative disease2 as well as many types of cancers1. Inhibitors of metalloproteases are widely used in research applications with some also approved for use in the clinic. However, molecules which stimulate the activity of these enzymes are rarely encountered, and as such our understanding of the mechanism(s) behind enzyme stimulation remains poor. Stimulators of enzyme activity can provide novel insights into enzyme biology and potentially open up avenues for the design of a novel class of drugs. For instance, ECE-1 and NEP are two metalloproteases that degrade amyloid beta (Aβ), the accumulation of which is a hallmark of Alzheimer’s disease.

Therefore it is of great interest to regulate the production of, and more importantly, the degradation of Aβ by stimulating the activity of these enzymes2. This in turn could reverse, prevent or at least halt the progression of Alzheimer’s disease.

Previous studies using animal models of Alzheimer’s disease have shown that increasing the expression of ECE3 and NEP4 through DNA based techniques can have beneficial effects. However, DNA based approaches can pose challenges for clinical translation. Molecules which can directly stimulate the activity of ECE-1 and NEP, or increase their expression are more attractive alternatives. Several studies have reported on the presence of molecules which increase the expression of or activity of NEP5,6,7. However, there are no reports on molecules which stimulate the activity of ECE-1. For example, polyphenols in green tea have been reported to increase the activity of NEP in cell culture models5, while the neuroprotective hormone humanin has been shown to increase the expression of NEP in a mouse model of Alzheimer’s disease6. In addition, Kynurenic acid elevates NEP expression as well as activity in human neuroblastoma cultures and mouse cortical neurones7. Therefore this study aimed to identify a molecule which stimulates the activity of ECE-1. Here we report on the discovery of K49-P1-20, a 20 amino acid peptide from the venom of B. asper which stimulates the activity of both ECE-1 and NEP. The effect of this peptide on other closely related enzymes was also examined.

Identification of K49-P1-20

We screened venom from species across different geographical regions for their effects on ECE-1 activity. The venom from B. asper was found to stimulate the activity of ECE-1 (624 ± 27% of control; Fig. 1a). Fractionation of venom confirmed that ECE-1 stimulation was mediated by the previously isolated B. aspermyotoxin II (Fig. 1a), a lysine 49 (K49) type phospholipase A2 found in this venom which induces myonecrosis upon envenoming8. Digestion of B. asper myotoxin II with ArgC proteinase indicated that the stimulation of ECE-1 activity was mediated by its N-terminal region (Fig. 1a). The synthetic peptide K49-P1-34 corresponding to the N-terminal region mimicked the stimulator effects of B. asper myotoxin II (Fig. 1a,b). No significant difference in the activation was observed between peptides K49-P1-20 and K49-P1-34 (Fig. 1a). However, the level of stimulation observed in the presence of K49-P9-34 and inverted sequence of K49-P1-20 was significantly less compared with native K49-P1-20 (Fig. 1a). Further digestion of peptide K49-P1-20 resulted in a reduction in its ability to stimulate ECE-1 activity (Fig. 1c) indicating the importance of residues 1-20 for maximal stimulation of ECE-1 activity. Peptide K49-P1-20 failed to inhibit direct twitches of the chick biventer cervicis nerve muscle preparation, confirming its lack of myotoxic effects (Fig. 1d), in agreement with the previous mapping of toxicity determinants of B. asper myotoxin II to its C-terminal region9.

Figure 1

http://www.nature.com/article-assets/npg/srep/2016/160302/srep22413/images_hires/m685/srep22413-f1.jpg

Discovery of K49-P1-20 (a) Comparison of ECE-1 stimulating effects of venom, B. asper myotoxin II, peptides K49-P1-20, K49-P1-34, K49-P9-34 and inverted K49-P1-20 (10 ng/μL); (b) Schematic showing the amino acid sequence of B. asper myotoxin II (ArgC mediated cleavage sites are indicated by arrows). The underlined sections correspond to the sequence of synthetic peptides tested for their effects on ECE-1 activity; (c) trypsin mediated cleavage of K49-P1-20 produces peptides K49-P1-7 and K49-P8-20 (cleavage sites indicated by arrows, top panel); the effect of K49-P1-20, peptides K49-P1-7 and K49-P8-20 on ECE-1 activity (bottom panel); (d) a representative trace showing the effect of K49-P1-20 (25 μg/mL) on direct twitches of the chick biventer cervices muscle. The arrow indicates the point of addition of peptide. *Significantly different than ECE-1 + peptide K49-P1-20, P < 0.05, unpaired t-test, n = 48.

Alanine scan

Alanine substitution of Leu(2) and Ile(9) failed to enhance ECE-1 activity, indicating their importance for stimulating ECE-1 (Fig. 3). Alanine substitution of Leu(2), Phe(3), Glu(4), Leu(10), Glu(12), Thr(13), Lys(15), Lys(19) and Ser(20) failed to enhance NEP activity, indicating their importance for stimulating NEP (Fig. 3).

Figure 3: Alanine scan.

A library of K49-P1-20 analogs were synthesised where each subsequent residue was replaced by an Ala. These analogs were tested for their ability to stimulate ECE-1 and NEP activity. The K49-P1-20 analogs are shown in the middle, with the Ala substitutions indicated in red. Closed bar denotes enzyme alone and the native peptide is indicated in blue *significantly different compared to enzyme alone; P < 0.05; One-way ANOVA; n = 4.

K49-P1-20 and enzyme interaction and conformational changes

BLITZ Biolayer interferometry

N-terminal biotinylation of K49-P1-20 had no significant effect on its ability to stimulate ECE-1 activity (Fig. 4a). Interaction of ECE-1 and NEP with biotinylated K49-P1-20 immobilised on a streptavidin biosensor was indicated by an increase in response units (nm) over time (Fig. 4b). The interaction was rapidly reversible. There was only a minimal interaction between each of the enzymes and biotinylated version of inverted K49-P1-20.

Figure 4: Association between K49-P1-20 and enzymes.

Figure 4

(a) Effect of N-terminal biotinylation of K49-P1-20 on the activity of ECE-1. (b) Representative traces obtained using Biolayer interferometry showing the level of interaction between enzymes and the biotinylated version of native or inverted K49-P1-20; representative traces showing the effect of K49-P1-20 on the intrinsic fluorescence of (c) ECE-1 and (d) NEP. Fluorescence of K49-P1-20 alone, and the sum of fluorescence intensities of K49-P1-20 and enzyme is also indicated.

K49-P1-20 stimulates ECE-1 activity in cerebrospinal fluid

K49-P1-20 (1–30 ng/μL) stimulated the activity of rhECE-1 in cerebrospinal fluid obtained from a patient with Alzheimer’s disease, as evidenced by the enhanced cleavage of bradykinin based QFS (Fig. 7a). Addition of stimulated ECE-1 to cerebrospinal fluid obtained from patients with Alzheimer’s disease (N = 8) resulted in a significant decrease (31 ± 4%) in the levels of endogenous soluble Aβ42 over 4 h, compared with the addition of non-stimulated ECE-1 (11 ± 5%; P = 0.005, unpaired t-testFig. 7b). This decrease was blocked by the ECE-1 specific inhibitor CGS35066 (Fig. 7b).

Figure 7: K49-P1-20 stimulates ECE-1 activity in cerebrospinal fluid

(a) the effect of K49-P1-20 (1–30 ng/μL) on the activity of rhECE-1(0.04–ng/μL) added to cerebrospinal fluid obtained from a patient with Alzheimer’s disease at post mortem. Enzyme activity was measured using the bradykinin based QFS. * & α significantly different compared to ECE-1 alone or K49-P1-20 (1 ng/μL) respectively; P < 0.001; n = 5; one-way ANOVA. (b) The effect of ECE-1 alone (0.04 ng/μL); ECE-1 incubated with K49-P1-20 (300 ng/μL); or ECE-1+ K49-P1-20 + ECE-1 inhibitor CGS35066 (500 nM), on the levels of endogenous Aβ42 in cerebrospinal fluid taken from a patient with Alzheimer’s disease at post-mortem was determined using a commercially available ELISA kit. Significantly different compared to *ECE-1 alone P = 0.005; or **ECE-1 + K49-P1-20, P = 0.009; unpaired t-test, N = 8–11.

Discussion

ECE-1 and NEP are two closely related metalloproteases that play a key role in many physiological and pathophysiological processes2,15,16. A common substrate to both enzymes is Aβ which plays a key role in the pathogenesis of Alzheimer’s disease2,15,16,17,18. Previous studies have reported the discovery of molecules which increase NEP activity5,6,7. However, there are no reports on molecules that increase ECE-1 activity. Here we report on the discovery of a peptide named K49-P1-20 from the venom of B. asper which stimulates the activity of both ECE-1 and NEP. Interaction of K49-P1-20 with ECE-1 or NEP appears to induce a change in its conformation leading to an increase in activity. Unlike the molecules reported in previous studies which increase NEP expression and therefore cellular NEP activity5,6,7, K49-P1-20 appears to allosterically regulate the activity of ECE-1 and NEP.

Animal venoms have long been a source of lead compounds for future pharmaceuticals and research tools19,20. We therefore screened venoms of snakes found in different geographical regions to identify a molecule that modulates the activity of ECE-1, and found that the venom of B. asper stimulated ECE-1 activity. Initial fractionation of venom indicated that this effect was mediated by a toxin known as B. asper myotoxin II which induces myonecrosis following envenoming8. B. aspermyotoxin II belongs to a class of toxins known as Lysine 49 phospholipase A2 myotoxins21. Asp to Lys substitution at position 49 is a key structural feature of these toxins and their toxic effects are independent of the phospholipase A2 activity. Digestion of this toxin with ArgC proteinase indicated that stimulation of ECE-1 activity was mediated by its N-terminal domain. The use of synthetic peptides of varying length corresponding to this region confirmed that these effects were in fact mediated by its first 20 amino acids. Inverted sequence of K49-P1-20 failed to induce an increase in ECE-1 activity (136 ± 12 as % of ECE-1 alone; n = 3-4), indicating that the specific sequence of K49-P1-20 is critical for the observed effects. Further shortening of this peptide resulted in a loss of ECE-1 stimulating effects. K49-P1-20 therefore appears to possess the shortest optimum sequence required for ECE-1 stimulation and was used in all downstream studies. Previous studies have shown that myotoxic effects of B. asper myotoxin II are mediated by is C-terminal domain9. In agreement with this result, K49-P1-20 showed no myotoxicity in chick biventer cervicis muscle.

Compared with enzyme alone, K49-P1-20 also significantly enhanced the activity (expressed as % of control) of closely related enzyme NEP (1606 ± 29), and two other metalloproteases ACE-2 (145 ± 8) and IDE (292 ± 38). The level of ACE-2 and IDE stimulation was however significantly less compared with NEP, therefore indicating degree of specificity towards ECE-1 and NEP. All further studies therefore focused on the effect of K49-P1-20 on ECE-1 and NEP activity. K49-P1-20 increased the activity of ECE-1 and NEP in a concentration dependant manner. The increase in activity of both enzymes become evident at a K49-P1-20 concentration of 0.23 μM, or a peptide: enzyme molar ratio of 1:368. The high level of ECE-1 and NEP stimulation observed in response to K49-P1-20 is most likely the result of a common binding region for K49-P1-20 within these enzymes. ECE-1 and NEP in deed share 40% sequence homology22. However the potential sites of interaction between the enzymes and K49-P1-20 are best identified through structural biology approaches that take into account the secondary and tertiary structure of the enzymes.

Physical interaction between the activating molecule and enzyme is a common characteristic in the mechanisms of enzyme activation23. We used biolayer interferometry to probe possible physical interaction between K49-P1-20 and ECE-1 or NEP. N-terminal biotinylation of K49-P1-20 had no significant impact on its ability to stimulate ECE-1 activity, thus facilitating its use as a tool in research applications. Biotinylated K49-P1-20 immobilised on a streptavidin biosensor interacted directly with both ECE-1 and NEP as evidenced by the increase in response units over time. This interaction however was not observed with the biotinylated version of inverted K49-P1-20.

It is logical to assume that a conformational change that occurs following interaction with K49-P1-20 mediates the increase in enzyme activity. We investigated this by examining the effect of K49-P1-20 on the intrinsic fluorescence of ECE-1 and NEP. Fluorescence spectra of each enzyme in the presence of K49-P1-20 were distinct from that of enzyme alone. In addition, the sum of individual spectra for K49-P1-20 and ECE-1 or NEP failed to overlap with the spectra obtained by incubating K49-P1-20 with enzymes. This suggests that spectral changes that occur in the presence of K49-P1-20 is the likely result of a change in conformation of the enzymes, which in turn is a possible consequence of a direct interaction with K49-P1-20.

Tackling amyloidogenesis in Alzheimer’s disease with A2V variants of Amyloid-β

Giuseppe Di Fede, Marcella Catania, Emanuela Maderna, Michela Morbin,…,,Fabio Moda, Matteo Salvalaglio, Mario Salmona & Fabrizio Tagliavini

Scientific Reports 6, Article number: 20949 (2016) http://dx.doi.org:/10.1038/srep20949

We developed a novel therapeutic strategy for Alzheimer’s disease (AD) exploiting the properties of a natural variant of Amyloid-β (Aβ) carrying the A2V substitution, which protects heterozygous carriers from AD by its ability to interact with wild-type Aβ, hindering conformational changes and assembly thereof. As prototypic compound we designed a six-mer mutated peptide (Aβ1-6A2V), linked to the HIV-related TAT protein, which is widely used for brain delivery and cell membrane penetration of drugs. The resulting molecule [Aβ1-6A2VTAT(D)] revealed strong anti-amyloidogenic effects in vitro and protected human neuroblastoma cells from Aβ toxicity. Preclinical studies in AD mouse models showed that short-term treatment with Aβ1-6A2VTAT(D) inhibits Aβ aggregation and cerebral amyloid deposition, but a long treatment schedule unexpectedly increases amyloid burden, although preventing cognitive deterioration. Our data support the view that the AβA2V-based strategy can be successfully used for the development of treatments for AD, as suggested by the natural protection against the disease in human A2V heterozygous carriers. The undesirable outcome of the prolonged treatment with Aβ1-6A2VTAT(D) was likely due to the TAT intrinsic attitude to increase Aβ production, avidly bind amyloid and boost its seeding activity, warning against the use of the TAT carrier in the design of AD therapeutics.

Alzheimer’s disease (AD) is the most common form of dementia in the elderly. Its clinical course is slow but irreversible since no disease-modifying treatments are currently available. As a result, this illness has a huge socio-sanitary impact and designing of effective therapies is considered a public health priority.

A central pathological feature of AD is the accumulation of misfolded Amyloid-beta (Aβ) peptides in the form of oligomers and amyloid fibrils in the brain1,2,3. It has been advanced that aggregated Aβ species, particularly oligomeric assemblies, trigger a cascade of events that lead to hyperphosphorylation, misfolding and assembly of the tau protein with formation of neurofibrillary tangles and disruption of the neuronal cytoskeleton, widespread synaptic loss and neurodegeneration. According to this view, altered Aβ species are the primary cause of AD and the primary target for therapeutic intervention3,4.

Aβ peptides derive from proteolytic processing of a large (695/770 amino acids) type 1 transmembrane glycoprotein known as amyloid beta precursor protein (APP). APP is cleaved at the N-terminus of the Aβ domain by β-secretase, forming a large, soluble ectodomain (sAPPβ) and a 99-residue, membrane-retained C-terminal fragment (C99). Subsequently, γ-secretase cleaves C99 to release Aβ with different carboxyl termini, including Aβ40, Aβ42 and other minor species5. APP may undergo an alternative, non-amyloidogenic processing where the protein is cleaved within the Aβ domain by α-secretase, forming a soluble ectodomain (sAPPα) and an 83-residue C-terminal fragment (C83)5,6.

We identified a novel mutation in the APP gene resulting in A-to-V substitution at codon 673, corresponding to position 2 in the Aβ sequence7. Studies on biological samples from an A673V homozygous carrier, and cellular and C. elegans models indicated that this mutation shifts APP processing towards the amyloidogenic pathway with increased production of amyloidogenic peptides. Furthermore, the A2V substitution in the Aβ sequence (AβA2V) increases the propensity of the full-length peptides (i.e., Aβ1-40 and Aβ1-42) to adopt a β-sheet structure, boosts the formation of oligomers both in vitroand in vivo and enhances their neurotoxicity8,9,10. Following the observation that humans carrying the mutation in the heterozygous state do not develop AD, we carried out in vitro studies with synthetic peptides that revealed the extraordinary ability of AβA2V to interact with wild-type Aβ (AβWT), interfering with its nucleation or nucleation-dependent polymerization7. This provides grounds for developing a disease-modifying therapy for AD based on modified AβA2V peptides retaining the key functional properties of parental full-length AβA2V.

Following this approach, we generated a mutated six-mer peptide (Aβ1-6A2V), constructed entirely by D-amino acids [Aβ1-6A2V(D)] to increase its stability in vivo, whose interaction with full-length AβWT hinders oligomer production and prevents amyloid fibril formation8.

These results prompted us to develop a prototypic compound by linking Aβ1-6A2V(D) to an all-D form of TAT sequence [TAT(D)], a peptide derived from HIV that powerfully increases virus transmission to neighbour cells11, and is widely used for brain delivery of drugs12,13,14. Here we report that this compound [Aβ1-6A2VTAT(D)] has strong anti-amyloidogenic effects in vitro, leading to inhibition of oligomer, amyloid fibril formation and of Aβ-dependent neurotoxicity. Preclinical studies showed that a short-term treatment with this peptide in an AD mouse model prevents Aβ aggregation and amyloid deposition in the brain but longer treatment unexpectedly increases amyloid burden, most likely due to the TAT intrinsic attitude to enhance Aβ production and to avidly bind amyloid and boost its seeding activity, warning against the use of this carrier in therapeutic approaches for AD.

In silico molecular modeling of AβA2V peptide variants

To predict the structural basis of the anti-amyloidogenic effect of Aβ1-6A2V(D), a comparative conformation analysis of WT and mutated Aβ1-6 was carried out with all-atom classical MD simulations in explicit solvent. Both Aβ1-6WT and Aβ1-6A2V are intrinsically disordered peptides characterized by high flexibility. Nevertheless, the substitution of Ala2 with a Val residue induces significant changes in the appearance of the peptide in solution, resulting in an increase of the apolar character of the solvent accessible surface (SAS) (Fig. 1A) and in a modification of the gyration radius distribution in the Aβ1-6A2V. Figure 1B shows that the probability distribution of the gyration radius is characterized by a global shift to smaller values and by the appearance of a shoulder in the distribution corresponding to gyration radii of 0.5 nm.

Figure 1: Analysis of 1.5 μs explicit solvent MD simulations of the Aβ1-6WT and Aβ1-6A2V peptides.

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(A) Apolar character of the peptide SAS represented as the ratio between SASapolar and the total SAS. (B) Gyration radius distribution. (C) Analysis of secondary structure propensity. “Structure” indicates residues possessing a defined secondary structure, in this case structure indicates residues in a “turn” configuration. “Coil” indicates residues that do not display a defined secondary structure. Analysis of the secondary structure was carried out with DSSP. (D) Typical compact “turn” and elongated “coil” configurations reported for the Aβ1-6A2V and Aβ1-6WT, respectively. (E) Analysis of the most populated structural clusters. Representative structures of the six most probable clusters were reported. The coil configuration has been highlighted in green, the turn in red and a partly folded turn in orange.

An analysis of the secondary structure content displayed by the peptides (Fig. 1C) shows that, while both Aβ1-6A2Vand Aβ1-6WT display a predominant coil configuration, Aβ1-6A2V shows a slightly higher propensity to form secondary structure motifs involving two to three residues. Aβ1-6A2V in fact displays a propensity to form a turn involving the Glu3, Phe4 and Arg5 residues (Fig. 1D). The most populated structural clusters15 (Fig. 1E), in Aβ1-6WT are characterized by an elongated coil structure accounting for 52.6% of the configurations, while the compact “turn” state is only the third most probable cluster, with a population of around 9%. Conversely, in the Aβ1-6A2V, while the most populated structure is still an elongated coil (32%), the “turn” configuration is the second most populated structural cluster (31%).

Both Aβ1-6WT and Aβ1-6A2V under physiological conditions are characterized by intramolecular salt bridges such as those between Asp1 and Arg5 or Glu3-Arg5. In the extended coil configuration (Fig. 1E), salt bridges can be dynamically formed and dissociated without requiring a specific rearrangement of the peptide backbone. However, in the turn configuration salt bridges are typically dissociated; the interaction of the apolar Val2 sidechain with the Arg5 sidechain stabilizes such a dissociated state. The additional sterical hindrance to the rearrangement induced by the Val2 sidechain also contributes to the stabilization of the turn configuration of the A2V peptide.

The propensity of the A2V mutant to adopt a Glu3-Arg5 turn configuration characterized by a significant lifetime can be interpreted as the probable source of the heterotypic interaction of the Aβ1-6A2V with full-length Aβ, which results in hindering its assembly.

Aβ1-6A2V retains the in vitro anti-amyloidogenic features of the parental full-length peptide

We previously showed that Aβ1-6A2V(D) destabilizes the secondary structure of Aβ1-42WT8 and is even more effective than the WT peptide [Aβ1-6WT(D)] and the A2V-mutated L-isomer [Aβ1-6A2V(L)] at preventing the aggregation of full-length AβWT8.

Treatment of SH-SY5Y cells with Aβ1-6WT(D) or Aβ1-6A2V(D) showed that neither is toxic for living cells even at high concentrations (20 μM) (Fig. 2A,B) and that both peptides are able to reduce the toxicity induced by Aβ1-42WT (Fig. 2C,D). However, Aβ1-6A2V(D) showed a stronger effect in counteracting the reduction of cell viability caused by Aβ1-42WT (Fig. 2D), suggesting that the A-to-V substitution actually amplifies the protective effects of the six-mer peptide.

Figure 2: Analysis of the effects of Aβ1-6WT(D), Aβ1-6A2V(D) and Aβ1-6A2VTAT(D) on neurotoxicity in cell models.

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http://www.ncbi.nlm.nih.gov/pmc/articles/instance/4750079/bin/srep20949-f2.jpg

SH-SY5Y cells were differentiated with 10 μM retinoic acid. After 6 days the proper peptide was added to culture medium and cell viability was assessed after 24 h by MTT test. (A,B) Neither Aβ1-6WT(D) nor Aβ1-6A2V(D) are significantly toxic when added to culture medium of differentiated SH-SY5Y cells. Conversely, Aβ1-42WT reduces cell viability by 35%. * Significance vs non-treated cells. (C,D) Both Aβ1-6WT(D) and Aβ1-6A2V(D) are able to counteract the toxic effect of Aβ1-42WT. Aβ1-6A2V(D) showed a stronger effect than Aβ1-6WT(D). (E) Aβ1-6A2VTAT(D) is not toxic when added to culture medium at concentrations ranging between 1 and 5 μM, while it reduces cell viability at higher concentrations. * Significance vs non-treated cells. (F) Aβ1-6A2VTAT(D) showed a dose-dependent effect in reducing Aβ1-42wt toxicity. Comparison of cell viability was performed by Student t-test.

Aβ1-6A2VTAT(D) maintains the in vitro anti-amyloidogenic properties of Aβ1-6A2V(D)

Aβ1-6A2V(D) alone does not efficiently cross either the blood brain barrier (BBB) or cell membranes (data not shown). This is an important feature that would deeply limit its use as an in vivo anti-amyloidogenic drug. So, we linked this peptide to the all-D TAT sequence to improve the translocation of Aβ1-6A2V(D) across the BBB and cell membranes, minimize the degradation of the peptide and reduce the immune response elicited by the molecule. The resulting compound [Aβ1-6A2VTAT(D)] destabilizes the secondary structure of Aβ1-42WT. Indeed, CD spectroscopy studies showed that Aβ1-6A2VTAT(D) inhibits the acquisition of β-sheet conformation by Aβ1-42WT (data not shown), thus affecting the folding of the full-length peptide.

We tested the ability of Aβ1-6A2VTAT(D) to inhibit the fibrillogenic properties of the full-length Aβ in vitro and found that the compound hindered Aβ1-42WT aggregation (Fig. 3). Polarized light and electron microscopy studies on aggregates of Aβ1-42WT formed after 20 days incubation with or without Aβ1-6A2VTAT(D) revealed that the mutated peptide hinders the formation of amyloid structures (Fig. 3B) and reduces the amount of fibrils generated by the full-length peptide (Fig. 3D). Moreover, AFM analysis (Fig. 3E,H) showed that Aβ1-6A2VTAT(D) actually interferes with the oligomerization process of Aβ1-42WT. Indeed, monomeric Aβ1-42WT, incubated alone at a final concentration of 100 μM, formed a family of small oligomers of different size within a range of 6-20 nm in diameter (~ 70%) (Fig. 3E,G). Conversely, the co-incubation with Aβ1-6A2VTAT(D) resulted in the formation of very small globular structures with a range of 5-8 nm in diameter and height of 200-400 pm (~ 70%), large and thin structures, apparently very rich in water (width: 500–700 nm; height: 200–500 pm). Notably, only rare oligomeric structures were detected (Fig. 3F,H).

Figure 3: Inhibition of aggregation of Aβ1-42WT by Aβ1-6A2VTAT(D).

Polarized-light (A,B), electron microscopy (C,D) and atomic force microscopy (AFM) (E–H) studies showing the inhibitory effects of Aβ1-6A2VTAT(D) on amyloid formation, fibril production and oligomerization by Aβ1-42WT. In polarized-light and EM studies, both peptides were used at 0.125 mM, molar ratio = 1:1 or 1:4 respectively, with 20 days incubation. From 5–20 days, 1:1 co-incubation of the two peptides (B,D) displayed a lower amyloid fibril content respect to Aβ1-42WT alone (A,C), showing protofibrils, short fibrils and disaggregated granular material.E,F: Representative Tapping mode of AFM images as determined by amplitude error data of Aβ1-42WT oligomers. Aβ1-42WT peptide 100 μM in phosphate buffer 50 mM, pH 7.4 was incubated at 4 °C for 24 h alone (E) (Z range: -10/ + 10 mV) or in presence of Aβ1-6A2VTAT(D) (F) (Z range: -10/ + 25 mV). The molar ratio of Aβ1-42WT to Aβ1-6A2VTAT(D) was 1:4. Scale bar: 1 μm, inset: 200 nm. (G,H): height plot profiles obtained along different lines traced on the topographic AFM images. Overall, these effects were already evident in the 1:1 mixture of the two peptides (data not shown), suggesting that the inhibition of Aβ1-42WT aggregation by Aβ1-6A2VTAT(D) is a dose-dependent effect.

These effects were observed by incubating Aβ1-42WT and Aβ1-6A2VTAT(D) at a 1:4 molar ratio, but they were also evident at equimolar concentrations of the two peptides.

Moreover, treatment of differentiated SH-SY5Y cells with Aβ1-6A2VTAT(D) showed that the peptide is not toxic when administered at concentrations ranging between 1 and 5 μM (Fig. 2E). When co-incubated with Aβ1-42WT, Aβ1-6A2VTAT(D) displayed a significant dose-dependent reduction of the toxicity induced by full-length Aβ (Fig. 2F).

All these findings indicated that the designed Aβ1-6A2VTAT(D) peptide is particularly efficient at inhibiting Aβ polymerization and toxicity in vitro, and identified it as our lead compound for the subsequent in vivo studies.

During the last few decades, huge efforts have been made to develop disease-modifying therapies for Alzheimer, but the results of these attempts have been frustrating. The anticipated increase of AD patients in the next few decades makes the development of efficient treatments an urgent issue16. In order to prevent the disease and radically change its irreversible course, a long series of experimental strategies against the main molecular actors of the disease (Aβ and tau)17 or novel therapeutic targets18 have been designed based on purely theoretical grounds19 as well as on evidence mainly deriving from preclinical observations in AD animal models20. However, few strategies proved suitable for application in human clinical trials, and none proved to be really effective21.Our approach differs from previous strategies – mainly those involving modified Aβ peptides that have been found to inhibit amyloidogenesis19,22 – since it is based on a natural genetic variant of amyloid-β (AβA2V) that occurs in humans and prevents the development of the disease when present in the heterozygous state7.

In this context, we carried out in vitro and in vivo studies that revealed the extraordinary ability of AβA2V to interact with AβWT, interfering with its aggregation8. These findings were a proof of concept of the validity of therapeutic strategies based on the use of AβA2V variant, and prompted us to develop a new disease-modifying treatment for AD by designing a six-mer mutated D-isomer peptide [Aβ1-6A2V(D)] linked to the short amino acid sequence derived from the HIV TAT peptide, widely used for brain delivery, to make the translocation of Aβ1-6A2V(D) across the BBB feasible.

The use of TAT as a carrier for brain delivery of drugs has been employed in several experimental approaches for the treatment of AD-like pathology in mouse models12,13. Recently, intraperitoneal administration of a TAT-BDNF peptide complex for 1 month was shown to improve the cognitive functions in AD rodent models23.

A previous study showed that, following its peripheral injection, a fluorescein-labelled version of TAT is able to cross the BBB, bind amyloid plaques and activate microglia in the cerebral cortex of APPswe/PS1DE9 transgenic mice24. TAT was then conjugated with a peptide inhibitor (RI-OR2, Ac-rGffvlkGr-NH2) consisting of a retro-inverted version of Aβ16–20 sequence25 that was found to block the formation of Aβ aggregates in vitro and to inhibit the toxicity of Aβ on cultured cells25. Daily i.p. injection of RI-OR2-TAT for 21 days into 10-month-old APPswe/PS1DE9 mice resulted in a reduction in Aβ oligomer levels and amyloid-β burden in cerebral cortex24.

We followed a similar strategy and initially demonstrated that Aβ1-6A2V(D), with or without the TAT sequence, retains in vitro the anti-amyloidogenic properties of the parental full-length mutated Aβ, since it is effective at hindering in vitro the production of oligomers and fibrils, the formation of amyloid and the toxicity induced by Aβ1-42WT peptide on SYSH-5Y cells.

Based on these results, we then decided to test in vivothe anti-amyloidogenic ability of Aβ1-6A2VTAT(D). The compound proved stable in serum after i.p. administration in mice, able to cross the BBB and associated with an immune response that was not found to cause any brain damage.

Short-term treatment with Aβ1-6A2VTAT(D) in the APPswe/PS1DE9 mouse model prevented cognitive deterioration, Aβ aggregation and amyloid deposition in brain. Unexpectedly, a longer treatment schedule, while retaining the results for cognitive impairment, attenuated the effects on Aβ production and increased amyloid burden, most likely due to the intrinsic amyloidogenic properties of TAT.

Indeed, we found that TAT(D), unlike Aβ1-6A2V(D), has a strong ability to bind amyloid deposits. This avidity for amyloid could boost the intrinsic seeding activity of amyloid plaques via a continuous and self-sustained recruitment of Aβ aggregates, leading to an exacerbation of the amyloidogenesis.

A similar effect of TAT was described in a study26reporting that HIV TAT promotes AD-like pathology in an AD mouse model co-expressing human APP bearing the Swedish mutation and TAT peptide (PSAPP/TAT mice). These mice indeed showed more Aβ deposition, neurodegeneration, neuronal apoptotic signalling, and phospho-tau production than PSAPP mice.

Moreover, TAT was found to increase Aβ levels by inhibiting neprilysin27 or enhancing β-secretase cleavage of APP, resulting in increased levels of the C99 APP fragment and 5.5-fold higher levels of Aβ4228. The same study reported that stereotaxic injection of a lentiviral TAT expression construct into the hippocampus of APP/presenilin-1 (PS1) transgenic mice resulted in increased TAT-mediated production of Aβ in vivo as well as an increase in the number and size of Aβ plaques. This is consistent with our findings, indicating a shift in APP processing towards the amyloidogenic processing in vivo at the end of the 5-month treatment with Aβ1-6A2VTAT(D) that was not observed in shorter treatment schedules with the same compound.

Therefore, these data suggest that the final outcome of our in vivo studies with Aβ1-6A2VTAT(D) is the result of side effects of the TAT carrier, whose amyloidogenic intrinsic activity neutralized the anti-amyloidogenic properties of the AβA2V variant. Nevertheless, we believe that the approach based on the use of AβA2V variant can be successfully used in treating AD, because of its potential ability to tackle the main pathogenic events involved in the disease, as suggested by the natural protection against the disease which occurs in human heterozygous A673V carriers.

Transthyretin participates in beta-amyloid transport from the brain to the liver- involvement of the low-density lipoprotein receptor-related protein 1?

Mobina Alemi, Cristiana Gaiteiro, Carlos Alexandre Ribeiro, Luís Miguel Santos,João Rodrigues Gomes,…, Ignacio Romero, Maria João Saraiva & Isabel Cardoso

Scientific Reports 6, Article number: 20164 (2016) http://dx.doi.org:/10.1038/srep20164

Transthyretin (TTR) binds Aβ peptide, preventing its deposition and toxicity. TTR is decreased in Alzheimer’s disease (AD) patients. Additionally, AD transgenic mice with only one copy of the TTR gene show increased brain and plasma Aβ levels when compared to AD mice with both copies of the gene, suggesting TTR involvement in brain Aβ efflux and/or peripheral clearance. Here we showed that TTR promotes Aβ internalization and efflux in a human cerebral microvascular endothelial cell line, hCMEC/D3. TTR also stimulated brain-to-blood but not blood-to-brain Aβ permeability in hCMEC/D3, suggesting that TTR interacts directly with Aβ at the blood-brain-barrier. We also observed that TTR crosses the monolayer of cells only in the brain-to-blood direction, as confirmed by in vivo studies, suggesting that TTR can transport Aβ from, but not into the brain. Furthermore, TTR increased Aβ internalization by SAHep cells and by primary hepatocytes from TTR+/+ mice when compared to TTR−/− animals. We propose that TTR-mediated Aβ clearance is through LRP1, as lower receptor expression was found in brains and livers of TTR−/− mice and in cells incubated without TTR. Our results suggest that TTR acts as a carrier of Aβ at the blood-brain-barrier and liver, using LRP1.

Alzheimer’s disease (AD), described for the first time by Alois Alzheimer in 1906, is characterized by progressive loss of cognitive functions ultimately leading to death1. Pathologically, the disease is characterized by the presence of extraneuronal amyloid plaques consisting of aggregates of amyloid-beta (Aβ) peptide, and neurofibrillary tangles (NFTs) which are intracellular aggregates of abnormally hyperphosphorylated tau protein2. Aβ peptide is generated upon sequential cleavage of the amyloid precursor protein (APP), by beta- and gamma-secretases, and it is believed that an imbalance between Aβ production and clearance results in its accumulation in the brain.

Clearance of Aβ from the brain occurs via active transport at the blood-brain-barrier (BBB) and blood cerebrospinal fluid (CSF) barrier (BCSFB), in addition to the peptidolytic removal of the peptide by several enzymes. The receptors for Aβ at the BBB bind Aβ directly, or bind to one of its carrier proteins, and transport it across the endothelial cell. The low-density lipoprotein receptor-related protein 1 (LRP1) and the receptor for advanced glycation end products (RAGE) are involved in receptor-mediated flux of Aβ across the BBB3. Both LRP1 and RAGE are multi-ligand cell surface receptors that, in addition to Aβ, mediate the clearance of a large number of proteins. While LRP1 appears to mediate the efflux of Aβ from the brain to the periphery, RAGE has been strongly implicated in Aβ influx back into the central nervous system (CNS). With increasing age, the expression of the Aβ efflux transporters is decreased and the Aβ influx transporter expression is increased at the BBB, adding to the amyloid burden in the brain.

Transthyretin (TTR), a 55 kDa homotetrameric protein involved in the transport of thyroid hormones and retinol, has been proposed as a protective protein in AD in the mid-nineties, when Schwarzman and colleagues described this protein as the major Aβ binding protein in CSF. These authors described that TTR was able to inhibit Aβ aggregation and toxicity, suggesting that when TTR fails to sequester Aβ, amyloid formation occurs4,5. Data showing that TTR is decreased in both CSF6 and plasma7,8 of AD patients, strengthen the idea of neuroprotection by TTR. Evidence coming from in vivostudies in AD transgenic mice established in different TTR genetic backgrounds9,10 also suggests that TTR prevents Aβ deposition and protects against neurodegeneration, although the exact mechanism is still unknown. Ribeiro and colleagues reported increased Aβ levels in both brain and plasma of AD mice with only one copy of the TTR gene, when compared to animals with two copies of the gene11, suggesting a role for TTR in Aβ clearance. Growing evidence also suggests a wider role for TTR in CNS neuroprotection, including in ischemia12, regeneration13 and memory14.

The presence of TTR in brain areas other than its site of synthesis and secretion – the choroid plexus (CP) and CSF, respectively–in situations of injury, such as ischemia, has been shown using a mouse model with compromised heat-shock response12. Authors showed that TTR was not being locally synthesized, but instead should derive from CSF TTR. However, other studies demonstrated TTR synthesis by cortical15 or hippocampal neurons both in vitro16, and in vivo17, and some hints on its regulation have already been advanced. Kerridge and colleagues showed that TTR is expressed in SH-SY5Y neuroblastoma cell line, and that it is up-regulated by the AICD fragment of amyloid precursor protein (APP), specifically derived from the APP695 isoform. Induced accumulation of functional AICD resulted in TTR up-regulation and Aβ decreased levels16. Wang and colleagues reported that TTR expression in SH-SY5Y cells, primary hippocampal neurons and hippocampus of APP23 mice is significantly enhanced by heat shock factor 1 (HSF1)17. In any case, TTR is available in the brain and might participate in brain Aβ efflux by promoting BBB permeability to the peptide. With regard to Aβ peripheral elimination, it is known that Aβ bound to ApoE/cholesterol can be incorporated in HDL to be further delivered at the liver for degradation18 and curiously, a fraction of TTR is transported in HDL19. Furthermore, the liver is the major site for TTR degradation and although its hepatic receptor has never been unequivocally identified, it has been reported that it is a RAP-sensitive receptor20. Thus, in this work we assessed the role of TTR in Aβ transport, both from the brain and to the liver.

TTR clearance in vivo

TTR ability to cross the BBB, in both directions, was studied in vivo using TTR −/− mice and injecting h rTTR. To assess the brain-to-blood permeability, immediately before the injection, mice were weighed and anesthetized with intraperitoneal injection of an anesthetic combination of ketamine and medetomidine (7.5 mg/Kg and 0.1 mg/Kg, respectively) and placed in a stereotaxic apparatus (Stoelting Co.). The cranium was exposed using an incision in the skin and one small hole was drilled through the cranium over the right lateral ventricle injection site to the following coordinates: mediolateral −1.0 mm, anterior-posterior −0.22 mm and dorsal-ventral −1.88 mm, from bregma. Then, 10 μg of h rTTR were injected into the brain using a 10 μL motorized syringe (Hamilton Co.) connected to a 30 gauge needle (RN Needle 6 pK, Hamilton Co.) at a rate of 0.75 μL/min (4 μL final volume). After injection, the microsyringe was left in place for 3 minutes to minimize any backflow, and then the incision was closed with sutures (Surgicryl), and the wound was cleaned with 70% ethanol. After surgery, the animals were kept warm, using a warming pad, and blood samples were collected by the tail vein after 20, 40 and 60 minutes, in a capillary tube (previously coated with EDTA). At the time of sacrifice (after 60 minutes), the mice were re-anesthetized with 75 mg/Kg ketamine and 1 mg/Kg medetomidine, and after total absence of reflexes in the paw and tail, mice were perfused through the injection of sterile PBS pH 7.4 via the inferior vena cava until the liver becomes blanched. Then, the brain was rapidly collected and frozen at −80 °C until use.

To assess the blood-to-brain permeability, 10 μg of h rTTR were injected in the tail vein, and blood samples were collected after 20, 40 and 60 minutes. At 60 minutes, and after perfusion as described above, CSF and brain were also collected.

To determine TTR levels, brains were weighted and homogenized in 750 μL of 50 mM TBS pH 7.4 containing protease inhibitor cocktail. After centrifugation for 20 minutes at 14000 rpm at 4 °C, supernatants were collected. TTR concentration in brain, CSF and plasmas was determined by ELISA.

Characterization of the hCMEC/D3 cell line

The hCMEC/D3 cell line represents a valid and powerfulin vitro tool as a BBB model, and presents a less expensive and more logistically feasible alternative to primary hBMEC cells24,25. Thus, our first step was the validation of the hCMEC/D3 model by characterizing this cell line regarding two critical features for our studies: BBB integrity and LRP1 expression.

In the context of endothelial cell tight junctions (TJ), hCMEC/D3 cells were tested for claudin-5 and occludin expression by immunofluorescence. As shown in Fig. 1, hCMEC/D3 cells are positive for TJ structural proteins, claudin-5 and occludin, showing the expected membrane localization (as previously described). These results indicate that the integrity, tightness and structure, as well as the paracellular contact between endothelial cells are guaranteed by these TJ proteins. Along with other TJ proteins expressed by hCMEC/D3, claudin-5 and occludin ensure, with high efficiency, the control of transport across the cells monolayer.

Figure 1: Immunofluorescence localization of TJs components Claudin-5 and Occludin, and of LRP1, in hCMEC/D3.

The expression of the efflux transport receptor LRP1 by the hCMEC/D3 cell line is a key factor when validating this model, both for BBB studies purposes and for Aβ transport research. Thus, we performed immunofluorescence analysis to verify if LRP1 exists in the hCMEC/D3 cells. Our results show that LRP1 is expressed in these cells ensuring the Aβ transport through the cells monolayer (Fig. 1).

Effect of TTR in Aβ1-42 internalization by hCMEC/D3

Aβ1-42 is transported across the BBB, as expected, and is internalized by hCMEC/D3 cells. We firstly investigated FAM-labelled Aβ1-42 (FAM-Aβ1-42, 500 ng/mL)) uptake by these cells in the absence and presence of human recombinant TTR (h rTTR) (7.5 μg/mL), and analysed the results by flow cytometry.

Cells were incubated with FAM-Aβ1-42 at 37 °C producing a rapid uptake of the peptide (Fig. 2A). After 5 minutes of incubation, 35–39% of the cells were fluorescent and after an additional 5 minutes (10 minutes incubation) a significant increase was already measured as over 57% of the cells were fluorescent, although differences between the presence and absence of TTR were not significant. However, after 15 minutes the presence of TTR significantly increased Aβ internalization resulting in about 73% fluorescent cells, in contrast to 61.7% incubated in the absence of TTR (Fig. 2A). Finally after 30 minutes of incubation, and although the difference between internalization levels at 15 and 30 minutes was not statistically significant, FAM-Aβ1-42 internalization was significantly higher in the presence of TTR.

Figure 2: Interaction of FAM-Aβ1-42 with hCMEC/D3 cells in the presence and absence of TTR assessed by flow cytometry:

(A) Internalization levels of FAM-Aβ1-42 by hCMEC/D3 cells in the presence of h rTTR (white columns) was significantly higher than in the absence of the protein (black columns) after 15 and 30 minutes of incubations. (B) Efflux of FAM-Aβ1-42 from hCMEC/D3 measured after 10 minutes of incubation with the peptide was significantly increased at 20 minutes post-replacement with fresh FAM-Aβ1-42-free media, in the presence of h rTTR. N = 3 for each condition and data are expressed as mean±SEM.

Next to investigate the fate of internalized Aβ, we performed an efflux assay. For that, hCMEC/D3 cells were firstly incubated with FAM-Aβ1-42 for 10 minutes, in the absence or presence of h rTTR and then the media were replaced with fresh Aβ-free media. Cells were further incubated at 37 °C and levels of FAM-Aβ1-42 inside cells were measured by flow cytometry, after 10 and 20 minutes. Figure 2B depicts the results showing that in the presence of TTR, FAM-Aβ1-42 effluxes significantly faster than in the absence of this protein, after 20 minutes (45.5% and 67.6% fluorescent cells, respectively).

Effect of TTR in hCMEC/D3 brain-to-blood permeability to Aβ1-42 peptide

In order to investigate the effect of TTR in Aβ1-42 transport across a monolayer of cells, acting as a model of the BBB as previously described, Aβ1-42 transport experiments were performed in hCMEC/D3 cultured in transwells inserts, as shown in Fig. 3A. Cells were grown for 10 days until reaching maximal confluence and allowing TJ formation. Thus, at this point, the cell monolayer should show restricted paracellular permeability, and its confirmation was done using FITC-labelled dextran as a low molecular weight paracellular diffusion marker. In this approach, FITC-labelled dextran 0.25 mg/mL was added to the apical chamber, and then incubated for 1 hour. Wells in which FITC-labelled dextran exceeded 125 ng/mL on the basolateral chamber were considered to have the monolayer disrupted and thus were excluded from the experiment.

Figure 3: Brain-to-blood permeability of hCMEC/D3 cells to Aβ1-42:

(A) Schematic representation of the transwell system used showing the brain and blood sides; Aβ1-42 peptide was always added to the brain side, whereas TTR was added either to the brain or to the blood sides. (B) Brain-to-blood permeability was increased in the presence of h rTTR although without reaching significant differences. However, in the presence of (C) hTTR present in sera, brain-to-blood permeability of hCMEC/D3 cells to Aβ1-42 was significantly increased after 3 hours up to 48 hrs. As a control, Aβ peptide was also added to non-seeded filters to show free passage of the peptide when compared to cell-seeded ones. N = 3 for each condition and data are expressed as mean±SEM. To mimic the absence of TTR, we used TTR-depleted human sera obtained after affinity chromatography, and further analysed by western blot (D) lanes 1- human sera; 2- protein G sepharose beads/anti-human prealbumin antibody; 3-human sera TTR-depleted; 4-Eluted TTR; 5-r hTTR.

We added h rTTR either to the brain or to the blood side, whereas Aβ1-42 was always added to the brain side. Results are displayed in Fig. 3B and show increased permeability of the hCMEC/D3 monolayer to Aβ1-42, when h rTTR is in the brain side, as compared to the levels of Aβ1-42 passage when h rTTR is in the blood side, although the differences were not statistically significant.

To further evaluate the effect of TTR in Aβ1-42 transport across the BBB and in order to obtain a more complex environment in hCMEC/D3 model, we performed the same transwell experiments but using human sera as source of hTTR (TTR concentration 7.5 μg/ml). To mimic the absence of TTR, we used human sera after TTR depletion by affinity chromatography (Fig. 3D). Again, hTTR present in the brain side promoted significant Aβ1-42 transport across the hCMEC/D3, as compared to the situation where hTTR was in the blood side (Fig. 3C). This suggests that TTR participates in Aβ1-42 efflux from the brain through a mechanism that implies TTR/Aβ interaction at the BBB or in its vicinity.

Brain permeability to TTR

Given our evidence in TTR-assisted Aβ transport and to clarify if TTR might be co-transported during such process, we assessed TTR internalization by hCMEC/D3 cells, and as shown in Fig. 4A, TTR was uptaken by these cells.

Figure 4: Permeability of hCMEC/D3 cells to TTR:

(A) hCMEC/D3 cells internalize TTR, as assessed by fluorescence microscopy. (B) hCMEC/D3 cells are permeable to TTR in the brain-to-blood direction but not in the blood-to-brain direction. N = 3 for each condition and data are expressed as mean±SEM.

We next investigated if TTR could cross the hCMEC/D3 monolayer and to assess this, hTTR was added either to the apical or basolateral compartment of the transwells. TTR was then quantified in the media of both chambers and analysed as % TTR that passed to the opposite side. As shown in Fig. 4B, TTR crosses the monolayer in the brain-to-blood direction but not in the blood-to brain direction. This suggests TTR is using a receptor with main expression in the basolateral membrane of the hCMEC/D3 cells.

To confirm these results, we also evaluated TTR clearance in vivo, using TTR−/− mice injected with h rTTR, either intracranially (IC) in the right lateral ventricle or intravenously (IV) in the tail vein. As displayed in Table 1, TTR injected in the brain rapidly reached the periphery as TTR was easily detected in blood, whereas mice injected IV showed negligible levels of the protein in the CSF and brain. Thus, this data corroborates the results obtained in the transwell experiments. This also suggests that TTR can favour Aβ brain efflux but cannot favour its influx, contributing to neuroprotection in AD.

Effect of TTR in Aβ1-42 and Aβ1-40 in AD transgenic mice

Previous work using an AD transgenic model (APPswe/PS1A246E) with different TTR genetic backgrounds (AD/TTR) has demonstrated that Aβ1-42 plasma levels are increased in 7-month old TTR+/− female mice, when compared to TTR+/+ animals11, suggesting a role for TTR in Aβ peripheral clearance.

In this work, to obtain a better knowledge on the effect of TTR in plasma Aβ peptide levels, we extended the study by evaluating not only Aβ1-42 but also Aβ1-40 levels in 3-months old AD/TTR+/+, AD/TTR+/− and AD/TTR−/− female mice. Results are depicted in Fig. 5 and show a negative correlation between TTR and both Aβ1-42 and Aβ1-40. Differences between AD/TTR+/+ and AD/TTR−/− mice were found to be statistical significant for both Aβ peptides. In addition, for Aβ1-42 statistical significant differences were also observed between AD/TTR+/− and AD/TTR−/−.

Figure 5: Effect of TTR genetic reduction in plasma Aβ1-42 and Aβ1-40 levels: Results are shown for 3-month old female mice with three distinct genotypes for TTR: AD/TTR+/+ (N = 5 for Aβ1-42; N = 4 for Aβ1-40), AD/TTR+/− (N = 6 for Aβ1-42; N = 4 for Aβ1-40) and AD/TTR−/− (N = 5 for Aβ1-42; N = 4 for Aβ1-40).

Taken together, our results suggest that TTR influences plasma Aβ by reducing its levels.

Effect of TTR in Aβ1-42 internalization by SAHep cells and primary hepatocytes

Aβ is known to also be delivered at the liver for degradation; therefore, we analysed the effect of TTR in FAM-Aβ1-42 internalization using the SAHep cell line. Uptake of Aβ1-42 peptide increased in the presence of h rTTR showing a positive correlation between Aβ uptake and h rTTR concentration, reaching a maximum of 70% when using 4.5–7.5 μg/mL of TTR in 3 hours (Fig. 6A).

Figure 6: Effect of TTR in Aβ peptide internalization by hepatocytes:

(A) FAM-Aβ1-42 internalization by SAHep cells, in the absence or presence of increasing concentrations of h rTTR, as measured by flow cytometry. TTR concentrations up to 4.5–7.5 μg/mL resulted in increased Aβ internalization by cells. N = 3 for each condition. (B) Flow cytometry of primary cultures of hepatocytes derived from mice with different genetic TTR backgrounds; hepatocytes derived from TTR+/+ mice showed significantly more internalization of FAM-Aβ1-42 than those derived from TTR+/− and from TTR−/−. N = 11, N = 8, N = 14, N = 6 for hepatocytes derived from TTR +/+, TTR +/−, TTR −/− and h rTTR treated TTR −/− mice, respectively. (C) moTTR levels in supernatants of primary hepatocytes measured by ELISA confirmed the genetic reduction in TTR+/− which showed about half of the TTR in TTR+/+, while TTR−/− produced no TTR protein. N = 7 for TTR+/+ and −/− mice and N = 5 for TTR +/−.

To further study the effect of TTR in Aβ1-42 uptake by hepatocytes, and in order to avoid addition of exogenous TTR (since hepatocytes produce TTR), we prepared primary cultures of hepatocytes derived from mice with different TTR genetic backgrounds (TTR+/+, TTR+/− and TTR−/−). TTR secretion was evaluated by ELISA revealing values of approximately 70 and 40 ng/mL for TTR+/+ and TTR+/−, respectively, over a period of 3 hours (Fig. 6C). TTR−/− hepatocytes did not produce TTR, as expected.

As for Aβ1-42 uptake, we observed that TTR facilitated peptide internalization by primary hepatocytes as differences were statistically significant between genetic backgrounds (Fig. 6B). Importantly, addition of h rTTR to TTR−/− hepatocytes partially rescued the phenotype as internalization values equalized those of TTR+/− cells.

Influence of TTR on LRP1 levels

We firstly assessed LRP1 expression by qRT-PCR in total brain extracts of TTR+/+, TTR+/− and TTR−/− mice, and observed significant differences in the expression of this receptor: brains from TTR+/+ mice expressed LRP1 in significantly higher levels than brains from TTR−/− animals (Fig. 7A1). These results were corroborated by measuring LRP1 protein levels by western blot (Fig. 7A2).

Figure 7: LRP1 expression in the brain, liver and cell lines assessed by qRT-PCR, western blot and immunofluorescence: LRP1 levels investigated in the brains from TTR+/+, TTR+/− and TTR−/− mice by

(A1) qRT-PCR (n = 4) and (A2) by western blot (n = 3), showed to correlate directly with TTR levels. hCMEC/D3 cells (n = 3) incubated with TTR showed higher amounts of (B1) mRNA and (B2) protein than cells without TTR. Similarly, livers of TTR+/+ mice expressed more LRP1, both (C1) mRNA (n = 4) and (C2) protein (n = 3), than of TTR−/− mice. (D1) qRT-PCR for LRP1 in SAHep cells incubated with exogenous h rTTR increased their LRP1 mRNA levels (n = 3). (D2) Upon incubation with TTR, SAHep cells increased their LRP1 protein levels.

To further understand the importance of TTR in regulating LRP1 levels in the context of Aβ transport across the BBB, we incubated hCMEC/D3 cells with h rTTR and investigated LRP1 expression by qRT-PCR. As depicted in Fig. 7B1, hCMEC/D3 incubated with TTR displayed higher LRP1 expression, thus confirming the regulation of LRP1 by TTR in these endothelial cells; these results were also corroborated by protein levels, as evaluated by immunocytochemistry (Fig. 7B2)

Similarly to the internalization studies, we also evaluated the ability of TTR to regulate LRP1 levels in hepatocytes by performing qRT-PCR studies in livers from TTR+/+, TTR+/− and TTR−/− mice, as well as in the hepatocyte cell line, SAHep cells. Similarly to the brains, livers from TTR+/+ mice expressed higher levels of LRP1, when compared to the livers from TTR−/− animals (Fig. 7C1). Protein analysis confirmed the effect of TTR at increasing LRP1 and as for the brains, significant differences were observed between TTR+/+ and TTR−/− mice (Fig. 7C2). As for the cell line, SAHep cells analyzed by qRT-PCR (Fig. 7D1) and immunocytochemistry (Fig. 7D2) showed increased LRP1 mRNA and protein levels, respectively, when incubated with TTR.

Altogether, these results indicate that TTR regulates LRP1 levels, suggesting that TTR uses this receptor to promote Aβ clearance.

TTR is a transporter protein mainly synthesized in the liver and in the CP of the brain and secreted into the blood and CSF, respectively. TTR is known to transport several molecules, in particular T4 and retinol through binding to the retinol binding protein (RBP). In the CSF, TTR binds Aβ peptide impeding its deposition in the brain. However, the molecular mechanism underlying this process is not known. Given our earlier evidences that TTR lowers brain and plasma Aβ11, we hypothesized that TTR could function as an Aβ carrier that transports the peptide to its receptor at the brain barriers and at the liver.

Since the cerebral capillaries represent about the double of the total apical surface area of the CP27, we decided to start by studying the effect of TTR in Aβ transport at the BBB. Using the hCMEC/D3 in vitro model of the BBB, we showed that TTR significantly increased Aβ internalization by these cells. Both in the presence and absence of TTR, Aβ internalization levels were high after 15 minutes and no significant increase was measured after 30 minutes. Thus, we assessed efflux by removing media with FAM-Aβ1-42 after a period of incubation to show that TTR was also promoting Aβ efflux from these cells.

To further study the effect of TTR in Aβ transport using the hCMEC/D3 model and given the differential expression of receptors in polarized BBB endothelial cells, we next performed our experiments using transwell cultures. Brain-to-blood transport of Aβ peptide was investigated and we concluded that TTR increased Aβ transport, if added to the brain side but not if added to the blood side. This observation is consistent with a direct TTR/Aβ interaction, as previously demonstrated28. To understand if TTR was also being transported while carrying Aβ, we also evaluated TTR ability to cross the endothelial monolayer to show that this protein can cross in the brain-to-blood direction, but does not cross in the opposite direction. To confirm this, we analyzed in vivo TTR brain permeability using TTR−/− mice injected with h rTTR either into the brain ventricle or into the tail vein. The presence of TTR was then investigated in brain and blood. The results corroborated the in vitroobservations since upon IC administration of TTR, the protein was rapidly found in blood; however, after IV injection of TTR the protein was detected neither in CSF nor in the brain extracts. Our findings are also supported by previous work on TTR turnover and degradation29; in this work authors reported that rat TTR injected intraventricularly into the CSF of rats was mainly degraded in the liver and kidneys (therefore effluxing from the brain), whereas no specific transfer of plasma TTR to the nervous system or degradation of plasma TTR in the nervous system was observed. It is worthy to note that Makover and colleagues injected purified rat TTR in a system containing the same endogenous rat TTR29, and results are similar to the ones we describe now. Therefore, we can conclude that in our system the TTR−/− background did not significantly affected TTR clearance.

The differential brain permeability to TTR indicates the use of a receptor with preferential expression on the basolateral membrane of the endothelial cells forming the BBB, such as LRP1, which in turn is known to internalize Aβ peptide. Whether TTR can cross or not as a complex, namely with Aβ peptide, is not known and needs to be investigated.

TTR gene expression in the brain is usually described as being confined to the CP and meninges, although TTR can be transported to other brain cells. For instance, it is described that in situations of compromised heat-shock response, and as a response to cerebral ischemia, CSF TTR contributes to control neuronal cell death, edema and inflammation12. This implies that TTR is transported from CSF to other brain areas, and thus it is also possible that this protein participates in Aβ transport at the BBB. TTR gene expression has been also attributed to neurons and for instance, SH-SY5Y cells transfected with APP695 isoform showed up-regulation of TTR mRNA expression, with concomitant decrease in Aβ levels16. Other authors showed that the majority of hippocampal neurons from human AD and all those from APP23 mouse brains contain TTR. In addition, quantitative PCR for TTR mRNA and Western blot analysis showed that primary neurons from APP23 mice transcribe TTR mRNA, and that the cells synthesize and secrete TTR protein15. More recently, it has been shown that TTR transcription and protein production can be induced by heat shock factor 1 (HSF1) in hippocampal neurons but not in the liver, both using cell lines and in vivo approaches17.

Importantly, the BCSFB should also be investigated for TTR-assisted Aβ transport, since this protein is the major protein binding Aβ in CSF. In spite of the low TTR levels in CSF (~2 mg/mL), the choroid plexus is presented as the major site of TTR expression, expressed as a ratio of TTR/mass of tissue, corresponding to a ~30-fold higher than that found in plasma30. Interestingly, a recent report describes that in a triple transgenic mouse model of AD only the Aβ1-42 isoform is increased at the epithelial cytosol, and in stroma surrounding choroidal capillaries. Noteworthy, there was increased expression, presumably compensatory, of the choroidal Aβ transporters: LRP1 and RAGE. In addition, authors reported that the expression of TTR was attenuated as compared to non-transgenic mice31.

Previous works indicated that the genetic reduction of TTR in an AD mouse model results in increased Aβ brain levels9,10; another work using 7 month old female mice also showed increased Aβ1-42 plasma levels in AD/TTR+/− mice as compared to age-and gender-matched AD/TTR+/+ animals. In the present work, we extended our study and evaluated both plasma Aβ1-42 and Aβ1-40 isoforms in 3 months old AD/TTR+/+, AD/TTR/+/− and AD/TTR−/− animals, showing that TTR correlates negatively with both isoforms of Aβ. Further, these findings support the idea that plasma may also reflect disease disturbances in AD.

Thus, the following level of our study focused on the effect of TTR in Aβ peptide uptake by the liver. After showing that h rTTR produces a concentration-dependent increase in Aβ internalization by SAHep cells, we worked with primary hepatocytes derived from mice with different TTR backgrounds showing again higher levels of internalization in the presence of TTR.

Interestingly, previous work has shown that TTR is internalized by the liver using a RAP-sensitive receptor20, such as LRP1. Multiple factors influence the function of LRP1-mediated Aβ clearance, such as its expression, shedding, structural modifications and transcriptional regulation by other genes32. Recent studies have clarified how Aβ clearance mechanisms in the CNS are indirectly altered by vascular and metabolism-related genes via the sterol regulatory element binding protein (SREBP2)33. In addition, AD risk genes such as phosphatidylinositol binding clathrin assembly protein (PICALM)34 and apoE isoforms can differentially regulate Aβ clearance from the brain through LRP135.

Consequently, given the importance of this receptor in Aβ clearance both from the brain and at the liver, we evaluated the levels of gene and protein expression in different models. Both LRP1 transcript and protein levels were increased in TTR+/+ brains as compared to TTR−/−. To further confirm the importance of TTR in regulating the levels of LRP1 specifically at the BBB, and contributing to explain the importance of TTR in Aβ clearance, we measured LRP1 in hCMEC/D3 cells with and without incubation with TTR. We observed that the presence of TTR clearly increased the receptor expression, producing significant differences. A similar study was then undertaken for liver and SAHep cells, which again showed regulation of LRP1 expression by TTR. Whether liver TTR regulates liver LRP1 and CSF TTR regulates brain LRP1 is not known and further studies, namely differential silencing of the TTR gene (liver or CP), should be performed.

In a recent study, TTR has been described to regulate insulin-like growth factor receptor I (IGF-IR) expression in mouse hippocampus (but not in choroid plexus) and this effect is due to TTR mainly synthesized by the choroid plexus (and secreted into the CSF) and not by peripheral TTR36. Once more, the possibility for local TTR production has been advanced by some authors16,17, as already mentioned. Finally, it is also known that LRP1 and IGF-IR interact37,38 in a way that the extracellular ligand-binding domain of LRP1 is not involved thus remaining free to bind its ligands. A common link is now established as TTR can regulate the expression of both receptors, albeit in different areas of the brain, opening the possibility for TTR being involved in other processes in the CNS. Moreover, using mice with deleted APP and APLP2, APP has been shown to down-regulate expression of LRP139 via epigenetic events mediated through its intracellular domain (AICD) and to up-regulate TTR, as previously described16. Though it is not known if LRP1 and TTR regulation are part of the same AICD-pathway since TTR levels were not evaluated in the APP and APLP2-deleted mice.

In summary, we show that neuroprotective effects of TTR previously observed in the context of AD are consistent with its role in Aβ clearance at the BBB and liver, and that TTR regulates LRP1 expression, suggesting that TTR is also transported by this receptor. In the future, the TTR-LRP1 cascade should be further investigated for therapeutic targeting.

Summary

TTR decreases in the population of both men and women after age 45 years. This has consequences with respect to AD. TTR is mainly synthesized by the choroid plexus (and secreted into the CSF) and not by peripheral TTR36, but this declines even earlier than that produced by the liver. (Ingenbleek and Bernstein, 2016). This suggests a significant role for these age related changes in the development of AD. Moreover, what has been presented indicates a role for snake venum in increasing the removal of amyloid plaque that develops in AD. TTR is important in A-beta clearance in liver and BBB. There was a shift in APP processing towards the amyloidogenic processing in vivo at the end of the 5-month treatment with Aβ1-6A2VTAT(D) that was not observed in shorter treatment schedules with the same compound

MIT scientists find evidence that Alzheimer’s ‘lost memories’ may one day be recoverable By Ariana Eunjung Cha

https://www.washingtonpost.com/news/to-your-health/wp/2016/03/17/mit-scientists-find-evidence-that-alzheimers-lost-memories-may-one-day-be-recoverable/?tid=pm_national_pop_b

Scientists had assumed for a long time that the disease destroys how those memories are encoded and makes them disappear forever. But what if they weren’t actually gone — just inaccessible?

A new paper published Wednesday by the Massachusetts Institute of Technology’s Nobel Prize-winning Susumu Tonegawa provides the first strong evidence of this possibility and raises the hope of future treatments that could reverse some of the ravages of the disease on memory.

“The important point is, this is a proof of concept,” Tonegawa said. “That is, even if a memory seems to be gone, it is still there. It’s a matter of how to retrieve it.”

[Provocative study raises questions about human transmission of Alzheimer’s protein]

A study published Wednesday in the journal Nature by British researchers John Collinge and Sebastian Brandner provides stunning evidence that Alzheimer’s disease may be transmissible to humans through certain medical or surgical procedures.

Evidence for human transmission of amyloid-β pathology and cerebral amyloid angiopathy

Zane Jaunmuktane, Simon Mead, Matthew Ellis, …., A. Sarah Walker, Peter Rudge, John Collinge & Sebastian Brandner
Nature (10 Sep 2015);525,247–250 doi:10.1038/nature15369

More than two hundred individuals developed Creutzfeldt–Jakob disease (CJD) worldwide as a result of treatment, typically in childhood, with human cadaveric pituitary-derived growth hormone contaminated with prions^{1, 2}. Although such treatment ceased in 1985, iatrogenic CJD (iCJD) continues to emerge because of the prolonged incubation periods seen in human prion infections. Unexpectedly, in an autopsy study of eight individuals with iCJD, aged 36–51 years, in four we found moderate to severe grey matter and vascular amyloid-β (Aβ) pathology. The Aβ deposition in the grey matter was typical of that seen in Alzheimer’s disease and Aβ in the blood vessel walls was characteristic of cerebral amyloid angiopathy³ and did not co-localize with prion protein deposition. None of these patients had pathogenic mutations, APOE ε4 or other high-risk alleles⁴associated with early-onset Alzheimer’s disease. Examination of a series of 116 patients with other prion diseases from a prospective observational cohort study⁵ showed minimal or no Aβ pathology in cases of similar age range, or a decade older, without APOE ε4 risk alleles. We also analysed pituitary glands from individuals with Aβ pathology and found marked Aβ deposition in multiple cases. Experimental seeding of Aβ pathology has been previously demonstrated in primates and transgenic mice by central nervous system or peripheral inoculation with Alzheimer’s disease brain homogenate^{6, 7, 8, 9, 10, 11}. The marked deposition of parenchymal and vascular Aβ in these relatively young patients with iCJD, in contrast with other prion disease patients and population controls, is consistent with iatrogenic transmission of Aβ pathology in addition to CJD and suggests that healthy exposed individuals may also be at risk of iatrogenic Alzheimer’s disease and cerebral amyloid angiopathy. These findings should also prompt investigation of whether other known iatrogenic routes of prion transmission may also be relevant to Aβ and other proteopathic seeds associated with neurodegenerative and other human diseases.

http://www.nih.gov/news-events/news-releases/decoding-molecular-ties-between-vascular-disease-alzheimers

The red-hot debate about transmissible Alzheimer’s: A controversial study suggested that the neurodegenerative disease might be transferred from one person to another.

Figure 3: Early Aβ accumulation in the parenchyma and blood vessels in a subset of eight patients with iCJD aged 36–51 years, but not in controls (stratum aged 36–51 years) of 19 prion diseases of other aetiologies, suggests human transmission

Early A[bgr] accumulation in the parenchyma and blood vessels in a subset of eight patients with iCJD aged 36-51 years, but not in controls (stratum aged 36-51 years) of 19 prion diseases of other aetiologies, suggests human transmission.

a, Widespread, moderate-to-severe early-onset CAA in three, and focal, mild CAA in one iCJD patient but only one focal, mild CAA in 19 controls. b, Significant differences of parenchymal Aβ accumulation (all central nervous system regio…

Human transmission of prion disease has occurred as a result of a range of medical and surgical procedures worldwide as well as by endocannibalism in Papua New Guinea, with incubation periods that can exceed five decades^{12, 13}. A well-recognized iatrogenic route of transmission was by treatment of persons of short stature with preparations of human growth hormone, extracted from large pools of cadaver-sourced pituitary glands, some of which were inadvertently prion-contaminated. Such treatments commenced in 1958 and ceased in 1985 following the reports of the occurrence of CJD amongst recipients. A review of all 1,848 patients who were treated with cadaveric-derived human growth hormone (c-hGH) in the United Kingdom from 1959 through 1985 found that 38 had developed CJD by the year 2000 with a peak incubation period of 20 years¹. Multiple preparations using different extraction methods were used over this period and patients received batches from several preparations. One preparation (Wilhelmi) was common to all patients who developed iCJD and size-exclusion chromatography, used in non-Wilhelmi preparation methods, may have reduced prion contamination¹. As of 2012, a total of 450 cases of iatrogenic CJD have been recognized worldwide after treatment with c-hGH or gonadotropin (226 cases), transplantation of dura mater (228) or cornea (2), and neurosurgery (4) or electroencephalography recording using invasive medical devices (2)². In France, 119/1,880 (6.3%) recipients developed iCJD, in the UK 65/1,800 (3.6%) and in the USA 29/7,700 (0.4%)^{2, 14}.

Since 2008, most UK patients with prion disease have been recruited into the National Prion Monitoring Cohort study⁵, including 22 of 24 recent patients with iatrogenic CJD (iCJD) related to treatment with c-hGH over this period, all of whom necessarily have very long incubation periods. Of this group of patients with iCJD, eight patients (referenced no.s 1, 2, 3, 4, 5, 6, 7, 8,Supplementary Information) aged 36–51 years, with an incubation period from first treatment to onset of 27.9–38.9 years (mean 33 years) and from last treatment to onset of 18.8–30.8 years (mean 25.5 years), underwent autopsy with extensive brain tissue sampling at our hospital. In all eight brain samples we confirmed prion disease with abnormal prion protein labelling of the neuropil, perineuronal network and in most cases microplaques as described previously^{15, 16, 17}. However, four (no.s 4, 5, 6, 8) of the eight patients with iCJD also showed substantial amyloid-β (Aβ) deposition in the central nervous system parenchyma by histology (Fig. 1) and immunoblotting (Fig. 2). A further two brain samples (no.s 1, 3) had focal Aβ pathology in one of the brain regions; one showed Aβ entrapment in PrP plaques and only one was entirely negative for Aβ. Furthermore, there was widespread cortical and leptomeningeal cerebral Aβ angiopathy (CAA)³ in three patients (no.s 4, 6, 8) and focal CAA in one patient (no. 5) (Fig. 1). Such pathology is extremely rare in this age range, 10/290 in the equivalent 36–50 year age strata without CJD¹⁸, P= 0.0002, Fisher’s test. None of our patients with iCJD had pathogenic mutations in the prion protein gene (PRNP). We used a custom next generation sequencing panel⁴ to exclude mutation in any of 16 other genes associated with early-onset Alzheimer’s disease, CAA, or other neurodegenerative disorders, and none carried APOE ε4 or TREM2 R47H alleles (Supplementary Table 2). Although such observations are unprecedented in our wide experience of human prion diseases, we nevertheless considered whether prion disease itself might predispose to, or accelerate, Aβ pathology, for example by cross-seeding of protein aggregation or overload of clearance mechanisms for misfolded proteins. We therefore compared the Aβ pathology in the iCJD cohort with that of a cohort of 116 patients with other prion diseases who had undergone autopsy: sporadic CJD (sCJD) (n = 85, age 42–83), variant CJD (n = 2, age 25 and 36) and inherited prion diseases (IPD) (n = 29, age 29–86). None of the patients in the control cohorts had comparable Aβ pathology (Consortium to Establish a Registry for Alzheimer’s disease (CERAD) score, P = 0.001, CAA, P = 0.005, topographical Aβ score P = 0.02, and cumulative Aβ score P = 0.02 (rank sum test) and digital Aβ quantification P = 0.04 (t-test); all restricted to the strata aged 36–51 years (n = 19)) (Fig. 3, and Extended Data Figs 1 and 2 show similar results in adjusted analyses in the full cohort). Indeed none of 35 prion cases aged 52–60 had significant Aβ pathology, with the exception of two cases at ages 57 and 58 positive for APOE ε4 alleles. Instead, the sCJD cohort shows Aβ pathology in parenchyma and blood vessels to a similar extent/severity as seen in iCJD, only in a much older age group (Extended Data Figs 1 and 2), in keeping with the chance coincidence of late-onset Aβ pathology and sCJD as previously documented in a large study of 110 sCJD patients and 110 age-matched controls aged 27–84 (ref. 19) and a study of 2,661 individuals aged 26–95 (ref. 18). Further, we investigated whether prion and Aβ pathology co-localize in the iCJD cases. In our series there was a distinct absence of overlap of Aβ plaques and PrP (Fig. 1d, e) or Aβ CAA and vascular PrP (Fig. 1b, c), consistent with these pathologies developing independently.

Figure 1: Aβ accumulation in central nervous system parenchyma and blood vessels (CAA) in iCJD.

a, Frontal cortex with widespread diffuse Aβ deposition, formation of plaques, and widespread parenchymal and leptomeningeal CAA (patient no. 4). b, c, Non-colocalized deposition of Aβ and prion protein. Vessels with CAA do not entrap or co-seed prion protein. d, e, Adjacent histological sections stained for Aβ or prion protein show clearly separated plaques of both proteins (no. 5). f, An overlay with colour inversion of prion protein plaques highlights the separation. g, h, Dual labelling, confocal laser microscopy shows no co-localization of parenchymal Aβ plaques (no.s 5, 6) or CAA (no. 6). i, Aβ is detected in pituitary glands in patients with a high Aβ load in the brain. Scale bar corresponds to 200 μm in a, 100 μm in b–h, and 50 μm in i.

Alzheimer’s disease: Lost memories found by Shrestha and Klann

The research, described in the journal Nature, involved two groups of mice. One was a normal control and the other was genetically engineered to have Alzheimer’s-like symptoms. Both groups were given a mild electric shock to their feet. The first group appeared to remember the trauma of the incident by showing fear when placed back in the box where they had been given the shock. The Alzheimer’s mice, on the other hand, seemed to quickly forget what happened and did not have an upset reaction to the box.

Their reaction changed dramatically when the scientists stimulated tagged cells in their brains in the hippocampus — the part of the brain that encodes short-term memories — with a special blue light. When they were put back in the box following the procedure, their memories of the shock appeared to have returned, and they displayed the same fear as their healthy counterparts.

Tonegawa and his colleagues wrote that the treatment appears to have boosted neurons to regrow small buds called dendritic spines that form connections with other cells.

The revelations have “shattered a 20-year paradigm of how we’re thinking about the disease,” Rudy Tanzi, a Harvard neurology professor who is not involved in the research, told the Boston Herald. He said that since the 1980s, researchers believed the memories just weren’t getting stored properly.

The technique used in the study — optical stimulation of brain cells, or “optogenetics” — involves the insertion of a gene into parts of a brain to make them sensitive to blue light and then stimulating them with the light.

In a commentary accompanying the paper, Prerana Shrestha and Eric Klann of the Center for Neural Science at New York University said that the research employed a “clever strategy” and that “the potential to rescue long-term memory in dementia is exciting.”

Doug Brown, director of research at the Alzheimer’s Society, cautioned that the technique is not something that can be translated into a procedure that is safe for the estimated 44 million people worldwide with dementia just yet.

“While interesting,” he told the Guardian, “the practicalities of this approach — using a special blue light to stimulate memory — means that we’re still many years away from knowing if it would be possible to restore lost memories in people.”

Electrical stimulation of the brain may be one alternative scientists can pursue, according to Christine Denny, a neurobiologist at Columbia University. Nature reported that early trials showed that deep-brain stimulation of the hippocampus may improve memory in some Alzheimer’s patients.

Memory retrieval by activating engram cells in mouse models of early Alzheimer’s disease

Dheeraj S. Roy, Autumn Arons, Teryn I. Mitchell, Michele Pignatelli, Tomás J. Ryan & Susumu Tonegawa
Nature(2016) doi:10.1038/nature17172

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive memory decline and subsequent loss of broader cognitive functions¹. Memory decline in the early stages of AD is mostly limited to episodic memory, for which the hippocampus has a crucial role². However, it has been uncertain whether the observed amnesia in the early stages of AD is due to disrupted encoding and consolidation of episodic information, or an impairment in the retrieval of stored memory information. Here we show that in transgenic mouse models of early AD, direct optogenetic activation of hippocampal memory engram cells results in memory retrieval despite the fact that these mice are amnesic in long-term memory tests when natural recall cues are used, revealing a retrieval, rather than a storage impairment. Before amyloid plaque deposition, the amnesia in these mice is age-dependent^{3, 4, 5}, which correlates with a progressive reduction in spine density of hippocampal dentate gyrus engram cells. We show that optogenetic induction of long-term potentiation at perforant path synapses of dentate gyrus engram cells restores both spine density and long-term memory. We also demonstrate that an ablation of dentate gyrus engram cells containing restored spine density prevents the rescue of long-term memory. Thus, selective rescue of spine density in engram cells may lead to an effective strategy for treating memory loss in the early stages of AD.

Figure 1: Optogenetic activation of memory engrams restores fear memory in early AD mice

a–c, Amyloid-β (Aβ) plaques in 9-month-old AD mice (a), in the DG (b), and in the EC (c). d, Plaque counts in HPC sections (n = 4 mice per group). ND, not detected. e, CFC behavioural schedule (n = 10 mice per group). f–i, Freezing leve…

Figure 2: Neural correlates of amnesia in early AD mice.close

a, b, Images showing dendritic spines from DG engram cells of control (a) and AD (b) groups. c, Average spine density showing a decrease in AD mice (n = 7,032 spines) compared with controls (n = 9,437 spines, n = 4 mice per group).

Behavioural rescue and spine restoration by optical LTP is protein-synthesis dependent.

a, Modified behavioural schedule for long-term rescue of memory recall in AD mice in the presence of saline or anisomycin (left). Memory recall 2 days after LTP induction followed by drug administration showed less freezing of AD mice

Turn Off Alzheimer’s Disease

Lomonosov Moscow State University http://www.dddmag.com/news/2016/03/turn-alzheimers-disease

This image shows the three-dimensional structure of the dimer of the metal-binding domain of beta-amyloid peptide having ‘English mutation’. Two peptide molecules connected to each other with the help of zinc ion. Source: This image shows the three-dimensional structure of the dimer of the metal-binding domain of beta-amyloid peptide having ‘English mutation’. Source: Lomonosov Moscow State University

A group of the Lomonosov Moscow State University scientists, together with their colleagues from the Institute of Molecular Biology, Russian Academy of Sciences and the King’s College London, succeeded in sorting out the mechanism of Alzheimer’s disease development and possibly distinguished its key trigger. Their article was published in Scientific Reports.

‘Alzheimer’s disease is a widespread degenerative damage of central nervous system leading to a loss of mental ability.’Until now it was considered incurable,’ tells Vladimir Polshakov, the leading researcher, MSU Faculty of Fundamental Medicine. Though now scientists managed to distinguish the mechanism ‘running’ the disease development, so, a chance appeared to elaborate some new chemical compounds, that may work as an efficient cure.

Several hypotheses are dedicated to the Alzheimer’s disease development. One of the most common is the so-called amyloid hypothesis.

Amyloids (to be precise, beta-amyloid peptides) are molecular constructions of a protein type and in its normal healthy state they provide a protection to the brain cells. They live fast, and having fulfilled their function they fall prey to the work of proteases, the cleaning enzymes that cut all the used protein elements into harmless ‘slags’ that are further reclaimed or removed from a body. However, according to the amyloid hypothesis, at some point something goes wrong, and the cells’ protectors turn to be their killers. Moreover, those peptides start gathering, forming aggregations and hence getting out of the reach of proteases’ cutting blades. Within the amyloid hypothesis this mechanism is more or less precisely described on the later stages of the disease, when the toxic aggregations appeared already and further, when the brain is covered with amyloid plaques. However, the early stage of a beta-amyloid transformation into harmful organic products is highly unexplored.

‘We knew, for example, that a crucial role in initiation of such processes is played by ions of several transition metals, first of all — zinc,’ tells Vladimir Polshakov. ‘Zinc actually conducts a number of useful and healthy functions in a brain, though in this case it was reasonably suspected as a ‘pest’, and particularly as an initiator of a cascade of processes, leading to theAlzheimer’sdisease. However, it remained unclear, what exactly happens during an interaction of zin? ions with peptide molecules, which amino acids bind zinc ions, and how such interaction stipulates a peptide aggregation. We set a goal to clarify at least some of those questions’.

Scientists studied various pathogenic beta-amyloid peptides, their so-called metal binding domains — relatively short peptide regions, capable to bind metal ions. A number of experimental techniques were applied, including nuclear magnetic resonance (NMR) spectroscopy, used to determine the structure of the forming molecular complexes. Some spectra requiring higher sensitivity were additionally measured in London. According to Polshakov, the choice of the studied pathogens was ‘partly a luck’. One of the specimens was the product of so-called ‘English mutation’ — peptide, different from a common beta-amyloid peptide only with one amino acid substitution. Using the NMR spectroscopy scientists managed to sort out chemical processes and structural changes while a peptide molecules interact with zinc ion and undergo further aggregation.

The second pathogen was an isomerized beta-amyloid peptide. It was not different from a normal one in its chemical composition, though one of its amino acid residues, aspartic acid, was in a form with a specific atomic positioning. Such isomerism happens spontaneously, without help of any enzymes, and is related to the ageing processes, another influential factor of the Alzheimer’s disease. Fellow biologists from the Moscow’s Institute of Molecular Biology showed recently, that administration of an isomerized peptide to transgenic mice led to an accelerated formation of amyloid plaques. With the presence of zinc ions, a metal binding domain of the isomerized peptide aggregated so fast that the forming structures were hard to detect. Though scientists managed to distinguish that despite all the differences in processes occurring to the ‘English mutant’ and isomerized peptide in presence of zinc ions, initial stages of these transformations were similar. The trigger happened to be the same — a role of a pathogenic aggregation’s seed was in both cases played by initially formed peptide dimers, i.e. two peptide molecules, connected to each other with help of zinc ion. Such dimers were also detected in normal human peptides, and the difference in all the studied forms could be explained by the speed of formation of corresponding dimer and its proneness to a further aggregation.

Based on their findings, researches proposed the mechanism of zinc-controlled transformation of a peptide-protector into a peptide-killer. That mechanism, scientists notice, explains multiple experimental data, not only gathered by the group, but also collected by their colleagues in other laboratories preoccupied with the Alzheimer’s disease studies. Researchers also hope that thanks to a very certain targeting their discovery would help to produce new medicine capable to block beta-amyloid peptide aggregation stipulated by zinc ions.

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An inconvenient truth about dreams

Posted in Cognition, Neurological Diseases, tagged Cognition, Cognitive Science/Neuroscience, dreams, memory, mood disorder, Obstructive sleep apnea, Schizophrenia on March 6, 2016| Leave a Comment »

An inconvenient truth about dreams

Larry H. Bernstein, MD, FCAP, Curator

LPBI

It is natural to dream. Dreaming is a flashback recording of recent occurrences associated with cleaning out the memory of daily events. There is much written in both literature and in neuroscience as I write this. Dreaming is both natural and perhaps also adaptive, and dreams may be distressing or unintelligible in circumstances that we view as pathological. This is thought to be related to a loss of plasticity of the memory circuits. This occurs with mood disorders, sleep disorders with and without leg movement disorder, and with schizophrenia.

The term given by Martin Luther King, “I had a dream” is out of place under the cirumstances I describe. I am identical twin with a nonidentical triplet sister. Our brother died more than a decade ago, prematurely aged from living with schizophrenia. I and my sister could talk to him and understand what he said, even though it meant nothing to others. What I did not share was the total fragmentation of mental thoughts at an early age. Both I and my sister had guilt over the situation for years. I sought psychiatric assistance that went on for years. But I was not schizophrenic and I had a successful career by any standard, but was burdened trying to make up in achievement what was denied to my immigrant father and to my identical twin brother. It was by no means easy in the 1960s for my parents to deal with the situation, with a societal lack of understanding, a feeling of what have I done wrong, and a serious cost burden.

I went to medical school, which I had decided as a child, when I read Paul de Kruif’s Microbe Hunters.
I had a sister two years older who was a “wunder” kind, who I tried to follow. She had a GM scholarship and set the class curve as an undergraduate in a graduate course in numbers theory. Fortunately, my best friend, who was as brilliant as they come and a Merit Scholar college entry, cautioned not to overburden myself with the chemistry/math major that I never declared. My brother entered the hospital as I entered medical school, and the first year that would be expected to be difficult, certainly was for me.

Only in the last 5 years did I learn from extensive testing that I had a very high intelligence to match my achievement , but that I had Asperger’s. I also learned that I had an uncommon double mutation of the hydroxymethyl-folate reductase gene that is associated nonspecifically with neurological disorders. I take methyl folate for the genetic disorder to give access of folic acid to cross the blood brain barrier.
I’m retired for several years and had enormous difficulty in retiring, and was a workaholic. Work had great meaning and rewards for me.

I am now 74 and had a difficult 3 years with illness and hospitalization for me and my spouse of 45 years. We moved to be near my younger daughter, son-in-law, and grandson. This has brought great satisfaction. All the same, my asthma, sleep apnea, and general condition declined, and the move was more difficult than any I previously experienced. I have vivid dreams that requires clonipin for relief initially.

I have had increased frequency of dreams that can be resolved. However, with my awareness of the suicide of Robin Williams, I was given an awareness of his situation beyond what one would expect who has not seen such patients or has not experienced this. In my situation it was worsened by added depression. In the recent events I thought for the first time how incredible it was for my brother to have experienced this much of his schizophrenic life, even though I am not schizophrenic by any measure.

What’s in a dream?

I have had dreams before that I thought were interesting because of the people who I knew and the situations, that might have been unusual and gave me an inclination to write down. If I collected these, it could perhaps warrant a collection of stories. Those that are very recent have suggested that the one when I entertained my grandson is worthwhile. It was not so noxious, but it does fit the pieces together.
I watched some of the reporting of election returns of republican and democratic candidates. I sort of tossed around and played with the exceptional 6 year old who need not be exposed to such nonsence as we are seeing. It was early evening and to finish his limited allowable screentime, Nanny and Grandpa, and grandchild watched a children,s movie before bedtime. It was … … a takeoff on Red Ridinghood, with good cartoon figures, some recognizable voices, and an interesting storyline. Yes, LRRH does go through the woods to see her grandma, and she meets the wolf, who goes to her grandma’s house. Her grandma is tied up in the closet, and the woodsman, in the role of Paul Bunyan, gives a visit at the time of rescue. The storyline becomes a detective story to cull out the events leading up to a criminal event – who stole grandma’s recipe book, with a long family line of cooking. The grandma was an Olympic skiing champ who beets out the characters who stole her cookbooks. I’ll say no more than that the search comes upon grandma and LRRH escape with a parachute finish and the bad guys, led by a crafty rabbit, slide down on a ski-tram into a waiting police car. So that evening I have a dream that is a cockamaimie replay in which I am driving on the highway and enter a tunnel (like the rail in the movie), and the lane is cluttered with a wolf, and other creatures, making passage quite impossible.

I talked to my sister who called the next day. It was terrific when she said that if I had a pad and wrote them down immediately, they would form a pattern. Again, I have a dream, and I recall there was a pattern of feeling of failure. I am on Gabapentin for the restless leg. This time I have my brother (impossible) in it, I left my coat in a conference room and can’t get it immediately, and I have to return home with an exam the next day. In a recurring pattern, my brother is to drive. I can’t drive because of now having a diplopia from thyroid eye disease related to Grave’s disease. The exam has two questions about plasma from unclotted blood that is spun down and serum from clotted blood. This is very basic. The pattern is related to systemic notions of failure. My sister had a repeated pattern of rushing to get to the classes she teaches and not getting there on time (consistent with her rush rush).

I go back to bed and get another few hours of sleep. We had watched a number of Miss Marple movies recently. In the move I had the stressful experience of going through 40 years of save photographic equipment and photography, research literature, computer stuff, ya da, ya da, ya da. Very thorough, and tiring. The old lady in RRH and Miss Marple were merged into a character in a story related to the corroded pipes in Flint, and a criminal search for the cause of this problem (having watched the debate). Incredibly, this character was going through the material so rapidly, uncovering clues, and I was amazed.
I was struggling to keep up. Then I woke up. So my spouses assurances were correct. This is actually normal dreaming.

It is disturbing, consistent with a recent New York Times article on how the brain cleans out the garbage. I have too much garbage. My medication does have to be adjusted. It is perhaps not the same as my late brother’s experience. My sister’s observations have been helpful. My brother’s dreams were recognizable to me, but not to others, but they also had patterns, but patterns that were more distorted. If mine have been “normal”, but more frequent, this suggests a failure in the brain’s plasticity as I am aging, perhaps from from the stress in a major move. It is perhaps to be viewed as distressing at best compared with the worst case (my brother, or Robin Williams).

This is substantiated by my remembrance of driving on Woodward avenue or the expressway in Detroit, Michigan. I grew up on 2967 Sturtevant off of Dexter Ave. My elementary school no longer exists. We moved to the Northwest section and I graduated from Mumford High School in 1961. I lived in Trumbull, adjacent to Bridgeport, CT for 33 years, where my children grew up. The bizarreness of my recurring dream pattern has to do with a repeated driving and confusion between Detroit and Connecticut. I drove from Connecticut to New York for the last five years before retirement, but I failed to record these experiences. I had two car accidents related to narcolepsy in asbout 7 years related to my sleep apnea prior to getting it treated. In the last, I went to New Jersey to see an associate and driving back to Trumbull I veered off the highway and managed to veer into a tree in the snow. Fortunately I was able to control the car at the last minute. Fortunately, this could be much worse.

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Brain Cancer Vaccine in Development and other considerations

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Clinical & Translational, Clinical Diagnostics, Curation, Diagnostic Immunology, Diagnostics and Lab Tests, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Human Immune System in Health and in Disease, Image Processing/Computing, Imaging-based Cancer Patient Management, Immuno-Oncology & Genomics, Immunodiagnostics, Immunology, Lasers and photonics, Neurological Diseases, Neuroscience, Nuclear Medicine, tagged "inhibiting" RNAs, antitumor immunity, Brain tumors, dendritic cell vaccines, glioblastoma multiforme, Glioma, high resolution microscopy, immunogenic cell death, immunogenicity, Immunotherapy, metabolic labelled radioimaging, tumor lysate on March 5, 2016| Leave a Comment »

Brain Cancer Vaccine in Development and other considerations

Larry H. Bernstein, MD, FCAP, Curator

LPBI

GEN News Highlights Mar 3, 2016

Advanced Immunotherapeutic Method Shows Promise against Brain Cancer

http://www.genengnews.com/gen-news-highlights/advanced-immunotherapeutic-method-shows-promise-against-brain-cancer/81252433/

http://www.genengnews.com/Media/images/GENHighlight/Mar3_2016_LeuvenLab_CellDeathMouseBrain6232214015.jpg

The researchers induced a specific type of cell death in brain cancer cells from mice. The dying cancer cells were then incubated together with dendritic cells, which play a vital role in the immune system. The researchers discovered that this type of cancer cell killing releases “danger signals” that fully activate the dendritic cells. “We re-injected the activated dendritic cells into the mice as a therapeutic vaccine,” Professor Patrizia Agostinis explains. “That vaccine alerted the immune system to the presence of dangerous cancer cells in the body. As a result, the immune system could recognize them and start attacking the brain tumor.” [©KU Leuven Laboratory of Cell Death Research & Therapy, Dr. Abhishek D. Garg]

Scientists from KU Leuven in Belgium say they have shown that next-generation cell-based immunotherapy may offer new hope in the fight against brain cancer.

Cell-based immunotherapy involves the injection of a therapeutic anticancer vaccine that stimulates the patient’s immune system to attack the tumor. Thus far, the results of this type of immunotherapy have been mildly promising. However, Abhishek D. Garg and Professor Patrizia Agostinis from the KU Leuven department of cellular and molecular medicine believe they have found a novel way to produce more effective cell-based anticancer vaccines.

“We re-injected the activated dendritic cells into the mice as a therapeutic vaccine,” explains Prof. Agostinis. “That vaccine alerted the immune system to the presence of dangerous cancer cells in the body. As a result, the immune system could recognize them and start attacking the brain tumor.”

Combined with chemotherapy, this novel cell-based immunotherapy drastically increased the survival rates of mice afflicted with brain tumors. Almost 50% of the mice were completely cured. None of the mice treated with chemotherapy alone became long-term survivors.

“The major goal of any anticancer treatment is to kill all cancer cells and prevent any remaining malignant cells from growing or spreading again,” says Professor Agostinis. “This goal, however, is rarely achieved with current chemotherapies, and many patients relapse. That’s why the co-stimulation of the immune system is so important for cancer treatments. Scientists have to look for ways to kill cancer cells in a manner that stimulates the immune system. With an eye on clinical studies, our findings offer a feasible way to improve the production of vaccines against brain tumors.”

The team published its study (“Dendritic Cell Vaccines Based on Immunogenic Cell Death Elicit Danger Signals and T Cell–Driven Rejection of High-Grade Glioma”) in Science Translational Medicine.

Dendritic cell vaccines based on immunogenic cell death elicit danger signals and T cell–driven rejection of high-grade glioma

Abhishek D. Garg¹,*, Lien Vandenberk²,*, Carolien Koks², Tina Verschuere³, Louis Boon⁴, Stefaan W. Van Gool²,†,‡ and Patrizia Agostinis¹,†,‡
Science Translational Medicine 02 Mar 2016: Vol. 8, Issue 328, pp. 328ra27 DOI: 10.1126/scitranslmed.aae0105

DAMPening Glioma

Dendritic cell (DC)–based vaccines have shown promise for treating high-grade glioma (HGG), but efficacy has been limited by antigenic heterogeneity of the tumors. Now, Garg et al. combine DC vaccines with hypericin-based photodynamic therapy–induced immunogenic cell death (ICD) to treat HGG in an animal model. They found that ICD-based DC vaccines improved survival, and that this effect was dependent on the cell-associated reactive oxygen species and release of damage-associated molecular patterns (DAMPs) acting as danger signals. These ICD-based DC vaccines synergized with standard-of-care therapy to further improve survival in HGG-bearing mice and shifted the tumor immune signature from regulatory to T_H1/T_H17, which is associated with positive outcome in patients.

The promise of dendritic cell (DC)–based immunotherapy has been established by two decades of translational research. Of the four malignancies most targeted with clinical DC immunotherapy, high-grade glioma (HGG) has shown the highest susceptibility. HGG-induced immunosuppression is a roadblock to immunotherapy, but may be overcome by the application of T helper 1 (T_H1) immunity–biased, next-generation, DC immunotherapy. To this end, we combined DC immunotherapy with immunogenic cell death (ICD; a modality shown to induce T_H1 immunity) induced by hypericin-based photodynamic therapy. In an orthotopic HGG mouse model involving prophylactic/curative setups, both biologically and clinically relevant versions of ICD-based DC vaccines provided strong anti-HGG survival benefit. We found that the ability of DC vaccines to elicit HGG rejection was significantly blunted if cancer cell–associated reactive oxygen species and emanating danger signals were blocked either singly or concomitantly, showing hierarchical effect on immunogenicity, or if DCs, DC-associated MyD88 signal, or the adaptive immune system (especially CD8⁺ T cells) were depleted. In a curative setting, ICD-based DC vaccines synergized with standard-of-care chemotherapy (temozolomide) to increase survival of HGG-bearing mice by ~300%, resulting in ~50% long-term survivors. Additionally, DC vaccines also induced an immunostimulatory shift in the brain immune contexture from regulatory T cells to T_H1/cytotoxic T lymphocyte/T_H17 cells. Analysis of the The Cancer Genome Atlas glioblastoma cohort confirmed that increased intratumor prevalence of T_H1/cytotoxic T lymphocyte/T_H17 cells linked genetic signatures was associated with good patient prognosis. Therefore, pending final preclinical checks, ICD-based vaccines can be clinically translated for glioma treatment.

SLC7A11 expression is associated with seizures and predicts poor survival in patients with malignant glioma

Stephanie M. Robert¹, Susan C. Buckingham¹, Susan L. Campbell¹, Stefanie Robel¹, ….., Adrienne C. Lahti³, Louis B. Nabors² and Harald Sontheimer¹,*
Science Translational Medicine 27 May 2015: Vol. 7, Issue 289, pp. 289ra86 DOI: 10.1126/scitranslmed.aaa8103

Glioma is the most common malignant primary brain tumor. Its rapid growth is aided by tumor-mediated glutamate release, creating peritumoral excitotoxic cell death and vacating space for tumor expansion. Glioma glutamate release may also be responsible for seizures, which complicate the clinical course for many patients and are often the presenting symptom. A hypothesized glutamate release pathway is the cystine/glutamate transporter System x_c⁻ (SXC), responsible for the cellular synthesis of glutathione (GSH). However, the relationship of SXC-mediated glutamate release, seizures, and tumor growth remains unclear. Probing expression of SLC7A11/xCT, the catalytic subunit of SXC, in patient and mouse-propagated tissues, we found that ~50% of patient tumors have elevated SLC7A11 expression. Compared with tumors lacking this transporter, in vivo propagated and intracranially implanted SLC7A11-expressing tumors grew faster, produced pronounced peritumoral glutamate excitotoxicity, induced seizures, and shortened overall survival. In agreement with animal data, increased SLC7A11 expression predicted shorter patient survival according to genomic data in the REMBRANDT (National Institutes of Health Repository for Molecular Brain Neoplasia Data) database. In a clinical pilot study, we used magnetic resonance spectroscopy to determine SXC-mediated glutamate release by measuring acute changes in glutamate after administration of the U.S. Food and Drug Administration–approved SXC inhibitor, sulfasalazine (SAS). In nine glioma patients with biopsy-confirmed SXC expression, we found that expression positively correlates with glutamate release, which is acutely inhibited with oral SAS. These data suggest that SXC is the major pathway for glutamate release from gliomas and that SLC7A11 expression predicts accelerated growth and tumor-associated seizures.

Cortical GABAergic excitation contributes to epileptic activities around human glioma

Johan Pallud¹,²,³,⁴, Michel Le Van Quyen¹,²,*, Franck Bielle¹,⁵,*, Christophe Pellegrino⁶,⁷,*, …., Richard Miles¹,², Laurent Capelle¹,¹¹ and Gilles Huberfeld¹,²,⁹,¹²,¹³,†
Science Translational Medicine 09 Jul 2014: Vol. 6, Issue 244, pp. 244ra89 DOI: 10.1126/scitranslmed.3008065

Brain gliomas are highly epileptogenic. Excitatory glutamatergic mechanisms are involved in the generation of epileptic activities in the neocortex surrounding gliomas. However, chloride homeostasis is known to be perturbed in glioma cells. Thus, the contribution of γ-aminobutyric acidergic (GABAergic) mechanisms that depend on intracellular chloride merits closer study. We studied the occurrence, networks, cells, and signaling basis of epileptic activities in neocortical slices from the peritumoral surgical margin resected around human brain gliomas. Postoperative glioma tissue from 69% of patients spontaneously generated interictal-like discharges, synchronized, with a high-frequency oscillation signature, in superficial layers of neocortex around areas of glioma infiltration. Interictal-like events depended both on glutamatergic AMPA receptor–mediated transmission and on depolarizing GABAergic signaling. GABA released by interneurons depolarized 65% of pyramidal cells, in which chloride homeostasis was perturbed because of changes in expression of neuronal chloride cotransporters: KCC2 (K-Cl cotransporter 2) was reduced by 42% and expression of NKCC1 (Na-K-2Cl cotransporter 1) increased by 144%. Ictal-like activities were initiated by convulsant stimuli exclusively in these epileptogenic areas. This study shows that epileptic activities are sustained by excitatory effects of GABA in human peritumoral neocortex, as reported in temporal lobe epilepsies, suggesting that both glutamate and GABA signaling and cellular chloride regulation processes, all also involved in oncogenesis as already shown, induce an imbalance between synaptic excitation and inhibition underlying epileptic discharges in glioma patients. Thus, the control of chloride in neurons and glioma cells may provide a therapeutic target for patients with epileptogenic gliomas.

Spherical Nucleic Acid Nanoparticle Conjugates as an RNAi-Based Therapy for Glioblastoma

Samuel A. Jensen¹,*, Emily S. Day²,³,*, Caroline H. Ko²,³,*, Lisa A. Hurley¹, …, David A. Giljohann²,³, Chad A. Mirkin²,³,† and Alexander H. Stegh¹,³,†
Science Translational Medicine 30 Oct 2013: Vol. 5, Issue 209, pp. 209ra152 DOI: 10.1126/scitranslmed.3006839

Glioblastoma multiforme (GBM) is a neurologically debilitating disease that culminates in death 14 to 16 months after diagnosis. An incomplete understanding of how cataloged genetic aberrations promote therapy resistance, combined with ineffective drug delivery to the central nervous system, has rendered GBM incurable. Functional genomics efforts have implicated several oncogenes in GBM pathogenesis but have rarely led to the implementation of targeted therapies. This is partly because many “undruggable” oncogenes cannot be targeted by small molecules or antibodies. We preclinically evaluate an RNA interference (RNAi)–based nanomedicine platform, based on spherical nucleic acid (SNA) nanoparticle conjugates, to neutralize oncogene expression in GBM. SNAs consist of gold nanoparticles covalently functionalized with densely packed, highly oriented small interfering RNA duplexes. In the absence of auxiliary transfection strategies or chemical modifications, SNAs efficiently entered primary and transformed glial cells in vitro. In vivo, the SNAs penetrated the blood-brain barrier and blood-tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2Like12 (Bcl2L12)—an effector caspase and p53 inhibitor overexpressed in GBM relative to normal brain and low-grade astrocytomas—were effective in knocking down endogenous Bcl2L12 mRNA and protein levels, and sensitized glioma cells toward therapy-induced apoptosis by enhancing effector caspase and p53 activity. Further, systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice, without adverse side effects. Thus, silencing antiapoptotic signaling using SNAs represents a new approach for systemic RNAi therapy for GBM and possibly other lethal malignancies.

Rapid, Label-Free Detection of Brain Tumors with Stimulated Raman Scattering Microscopy

Minbiao Ji¹,*, Daniel A. Orringer²,*, Christian W. Freudiger¹, Shakti Ramkissoon³,⁴, Xiaohui Liu⁵, …, Sandra Camelo-Piragua⁸, Keith L. Ligon³,⁴,⁹, Oren Sagher² and X. Sunney Xie¹,†
Science Translational Medicine 04 Sep 2013: Vol. 5, Issue 201, pp. 201ra119 DOI: 10.1126/scitranslmed.3005954

Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.

Neural Stem Cell–Mediated Enzyme/Prodrug Therapy for Glioma: Preclinical Studies

Karen S. Aboody¹,²,*, Joseph Najbauer¹, Marianne Z. Metz¹, Massimo D’Apuzzo³,…., Christine E. Brown⁸, Seung U. Kim⁹, Behnam Badie² and Jana Portnow^{10

Science Translational Medicine 08 May 2013: Vol. 5, Issue 184, pp. 184ra59 DOI: 10.1126/scitranslmed.3005365}

High-grade gliomas are extremely difficult to treat because they are invasive and therefore not curable by surgical resection; the toxicity of current chemo- and radiation therapies limits the doses that can be used. Neural stem cells (NSCs) have inherent tumor-tropic properties that enable their use as delivery vehicles to target enzyme/prodrug therapy selectively to tumors. We used a cytosine deaminase (CD)–expressing clonal human NSC line, HB1.F3.CD, to home to gliomas in mice and locally convert the prodrug 5-fluorocytosine to the active chemotherapeutic 5-fluorouracil. In vitro studies confirmed that the NSCs have normal karyotype, tumor tropism, and CD expression, and are genetically and functionally stable. In vivo biodistribution studies demonstrated NSC retention of tumor tropism, even in mice pretreated with radiation or dexamethasone to mimic clinically relevant adjuvant therapies. We evaluated safety and toxicity after intracerebral administration of the NSCs in non–tumor-bearing and orthotopic glioma–bearing immunocompetent and immunodeficient mice. We detected no difference in toxicity associated with conversion of 5-fluorocytosine to 5-fluorouracil, no NSCs outside the brain, and no histological evidence of pathology or tumorigenesis attributable to the NSCs. The average tumor volume in mice that received HB1.F3.CD NSCs and 5-fluorocytosine was about one-third that of the average volume in control mice. On the basis of these results, we conclude that combination therapy with HB1.F3.CD NSCs and 5-fluorocytosine is safe, nontoxic, and effective in mice. These data have led to approval of a first-in-human study of an allogeneic NSC-mediated enzyme/prodrug-targeted cancer therapy in patients with recurrent high-grade glioma.

Magnetic Resonance Metabolic Imaging of Glioma

Philippe Metellus¹,²,^* and Dominique Figarella-Branger²,^{3

Science Translational Medicine 11 Jan 2012: Vol. 4, Issue 116, pp. 116ps1 DOI: 10.1126/scitranslmed.3003591}

2-Hydroxyglutarate (2-HG) is a potential oncometabolite involved in gliomagenesis that has been identified as an aberrant product of isocitrate dehydrogenase (IDH)–mutated glial tumors. Recent genomics studies have shown that heterozygous mutation of IDH genes 1 and 2, present in up to 86% of grade II gliomas, is associated with a favorable outcome. Two reports in this issue describe both ex vivo and in vivo methods that could noninvasively detect the presence of 2-HG in glioma patients. This approach could have valuable implications for diagnosis, prognosis, and stratification of brain tumors, as well as for monitoring of treatment in glioma patients.

Exploiting the Immunogenic Potential of Cancer Cells for Improved Dendritic Cell Vaccines

Lien Vandenberk,^1,^*Jochen Belmans,¹Matthias Van Woensel,^2,³Matteo Riva,^1,⁴ andStefaan W. Van Gool^1,^5,^6,^*

Front Immunol. 2015; 6: 663. doi: 10.3389/fimmu.2015.00663

Cancer immunotherapy is currently the hottest topic in the oncology field, owing predominantly to the discovery of immune checkpoint blockers. These promising antibodies and their attractive combinatorial features have initiated the revival of other effective immunotherapies, such as dendritic cell (DC) vaccinations. Although DC-based immunotherapy can induce objective clinical and immunological responses in several tumor types, the immunogenic potential of this monotherapy is still considered suboptimal. Hence, focus should be directed on potentiating its immunogenicity by making step-by-step protocol innovations to obtain next-generation Th1-driving DC vaccines. We review some of the latest developments in the DC vaccination field, with a special emphasis on strategies that are applied to obtain a highly immunogenic tumor cell cargo to load and to activate the DCs. To this end, we discuss the effects of three immunogenic treatment modalities (ultraviolet light, oxidizing treatments, and heat shock) and five potent inducers of immunogenic cell death [radiotherapy, shikonin, high-hydrostatic pressure, oncolytic viruses, and (hypericin-based) photodynamic therapy] on DC biology and their application in DC-based immunotherapy in preclinical as well as clinical settings.

Cancer immunotherapy has gained considerable momentum over the past 5 years, owing predominantly to the discovery of immune checkpoint inhibitors. These inhibitors are designed to release the brakes of the immune system that under physiological conditions prevent auto-immunity by negatively regulating cytotoxic T lymphocyte (CTL) function. Following the FDA approval of the anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) monoclonal antibody (mAb) ipilimumab (Yervoy) in 2011 for the treatment of metastatic melanoma patients (1), two mAbs targeting programed death (PD)-1 receptor signaling (nivolumab and pembrolizumab) have very recently joined the list of FDA-approved checkpoint blockers (respectively, for the treatment of metastatic squamous non-small cell lung cancer and relapsed/refractory melanoma patients) (2, 3).

However, the primary goal of cancer immunotherapy is to activate the immune system in cancer patients. This requires the induction of tumor-specific T-cell-mediated antitumor immunity. Checkpoint blockers are only able to abrogate the brakes of a functioning antitumoral immune response, implying that only patients who have pre-existing tumor-specific T cells will benefit most from checkpoint blockade. This is evidenced by the observation that ipilimumab may be more effective in patients who have pre-existing, albeit ineffective, antitumor immune responses (4). Hence, combining immune checkpoint blockade with immunotherapeutic strategies that prime tumor-specific T cell responses might be an attractive and even synergistic approach. This relatively new paradigm has lead to the revival of existing, and to date disappointing (as monotherapies), active immunotherapeutic treatment modalities. One promising strategy to induce priming of tumor-specific T cells is dendritic cell (DC)-based immunotherapy.

Dendritic cells are positioned at the crucial interface between the innate and adaptive immune system as powerful antigen-presenting cells capable of inducing antigen-specific T cell responses (5). Therefore, they are the most frequently used cellular adjuvant in clinical trials. Since the publication of the first DC vaccination trial in melanoma patients in 1995, the promise of DC immunotherapy is underlined by numerous clinical trials, frequently showing survival benefit in comparison to non-DC control groups (6–8). Despite the fact that most DC vaccination trials differ in several vaccine parameters (i.e., site and frequency of injection, nature of the DCs, choice of antigen), DC vaccination as a monotherapy is considered safe and rarely associates with immune-related toxicity. This is in sharp contrast with the use of mAbs or cytokine therapies. Ipilumumab has, for instance, been shown to induce immune-related serious adverse events in up to one-third of treated melanoma patients (1). The FDA approval of Sipuleucel-T (Provenge), an autologous DC-enriched vaccine for hormone-resistant metastatic prostate cancer, in 2010 is really considered as a milestone in the vaccination community (9). After 15 years of extensive clinical research, Sipileucel-T became the first cellular immunotherapy ever that received FDA approval, providing compelling evidence for the substantial socio-economic impact of DC-based immunotherapy. DC vaccinations have most often been applied in patients with melanoma, prostate cancer, high-grade glioma, and renal cell cancer. Although promising objective responses and tumor-specific T cell responses have been observed in all these cancer-types (providing proof-of-principle for DC-based immunotherapy), the clinical success of this treatment is still considered suboptimal (6). This poor clinical efficacy can in part be attributed to the severe tumor-induced immune suppression and the selection of patients with advanced disease status and poor survival prognostics (6, 10–12).

There is a consensus in the field that step-by-step optimization and standardization of the production process of DC vaccines, to obtain a Th1-driven immune response, might enhance their clinical efficacy (13). In this review, we address some recent DC vaccine adaptations that impact DC biology. Combining these novel insights might bring us closer to an ideal DC vaccine product that can trigger potent CTL- and Th1-driven antitumor immunity.

One factor requiring more attention in this production process is the immunogenicity of the dying or dead cancer cells used to load the DCs. It has been shown in multiple preclinical cancer models that the methodology used to prepare the tumor cell cargo can influence the in vivo immunogenic potential of loaded DC vaccines (14–19). Different treatment modalities have been described to enhance the immunogenicity of cancer cells in the context of DC vaccines. These treatments can potentiate antitumor immunity by inducing immune responses against tumor neo-antigens and/or by selectively increasing the exposure/release of particular damage-associated molecular patterns (DAMPs) that can trigger the innate immune system (14, 17–19). The emergence of the concept of immunogenic cell death (ICD) might even further improve the immunogenic potential of DC vaccines. Cancer cells undergoing ICD have been shown to exhibit excellent immunostimulatory capacity owing to the spatiotemporally defined emission of a series of critical DAMPs acting as potent danger signals (20, 21). Thus far, three DAMPs have been attributed a crucial role in the immunogenic potential of nearly all ICD inducers: the surface-exposed “eat me” signal calreticulin (ecto-CRT), the “find me” signal ATP and passively released high-mobility group box 1 (HMGB1) (21). Moreover, ICD-experiencing cancer cells have been shown in various mouse models to act as very potent Th1-driving anticancer vaccines, already in the absence of any adjuvants (21, 22). The ability to reject tumors in syngeneic mice after vaccination with cancer cells (of the same type) undergoing ICD is a crucial hallmark of ICD, in addition to the molecular DAMP signature (21).

Here, we review the effects of three frequently used immunogenic modalities and four potent ICD inducers on DC biology and their application in DC vaccines in preclinical as well as clinical settings (Tables (Tables11 and and2).2). Moreover, we discuss the rationale for combining different cell death-inducing regimens to enhance the immunogenic potential of DC vaccines and to ensure the clinical relevance of the vaccine product.

Table 1

A list of prominent enhancers of immunogenicity and ICD inducers applied in DC vaccine setups and their associations with DAMPs and DC biology.

Table 2

A list of preclinical tumor models and clinical studies for evaluation of the in vivo potency of DC vaccines loaded with immunogenically killed tumor cells.

The Impact of DC Biology on the Efficacy of DC Vaccines

Over the past years, different DC vaccine parameters have been shown to impact the clinical effectiveness of DC vaccinations. In the next section, we will elaborate on some promising adaptations of the DC preparation protocol.

Given the labor-intensive ex vivo culturing protocol of monocyte-derived DCs and inspired by the results of the Provenge study, several groups are currently exploiting the use of blood-isolated naturally circulating DCs (76–78). In this context, De Vries et al. evaluated the use of antigen-loaded purified plasmacytoid DCs for intranodal injection in melanoma patients (79). This strategy was feasible and induced only very mild side effects. In addition, the overall survival of vaccinated patients was greatly enhanced as compared to historical control patients. However, it still remains to be determined whether this strategy is more efficacious than monocyte-derived DC vaccine approaches (78). By contrast, experiments in the preclinical GL261 high-grade glioma model recently showed that vaccination with tumor antigen-loaded myeloid DCs resulted in more robust Th1 responses and a stronger survival benefit as compared to mice vaccinated with their plasmacytoid counterparts (80).

In view of their strong potential to stimulate cytotoxic T cell responses, several groups are currently exploring the use of Langerhans cell-like DCs as sources for DC vaccines (81–83). These so-called IL-15 DCs can be derived from CD14⁺monocytes by culturing them with IL-15 (instead of the standard IL-4). Recently, it has been shown that in comparison to IL-4 DCs, these cells have an increased capacity to stimulate antitumor natural killer (NK) cell cytotoxicity in a contact- and IL-15-dependent manner (84). NK cells are increasingly being recognized as crucial contributors to antitumor immunity, especially in DC vaccination setups (85, 86). Three clinical trials are currently evaluating these Langerhans cell-type DCs in melanoma patients (NCT00700167, NCT 01456104, and NCT01189383).

Targeting cancer stem cells is another promising development, particularly in the setting of glioma (87). Glioma stem cells can foster tumor growth, radio- and chemotherapy-resistance, and local immunosuppression in the tumor microenvironment (87, 88). Furthermore, glioma stem cells may express higher levels of tumor-associated antigens and MHC complex molecules as compared to non-stem cells (89, 90). A preclinical study in a rodent orthotopic glioblastoma model has shown that DC vaccines loaded with neuropsheres enriched in cancer stem cells could induce more immunoreactivity and survival benefit as compared to DCs loaded with GL261 cells grown under standard conditions (91). Currently there are four clinical trials ongoing in high-grade glioma patients evaluating this approach (NCT00890032, NCT00846456, NCT01171469, and NCT01567202).

With regard to the DC maturation status of the vaccine product, a phase I/II clinical trial in metastatic melanoma patients has confirmed the superiority of mature antigen-loaded DCs to elicit immunological responses as compared to their immature counterparts (92). This finding was further substantiated in patients diagnosed with prostate cancer and recurrent high-grade glioma (93, 94). Hence, DCs need to express potent costimulatory molecules and lymph node homing receptors in order to generate a strong T cell response. In view of this finding, the route of administration is another vaccine parameter that can influence the homing of the injected DCs to the lymph nodes. In the context of prostate cancer and renal cell carcinoma it has been shown that vaccination routes with access to the draining lymph nodes (intradermal/intranodal/intralymphatic/subcutaneous) resulted in better clinical response rates as compared to intravenous injection (93). In melanoma patients, a direct comparison between intradermal vaccination and intranodal vaccination concluded that, although more DCs reached the lymph nodes after intranodal vaccination, the melanoma-specific T cells induced by intradermal vaccination were more functional (95). Furthermore, the frequency of vaccination can also influence the vaccine’s immunogenicity. Our group has shown in a cohort-comparison trial involving relapsed high-grade glioma patients that shortening the interval between the four inducer DC vaccines improved the progression-free survival curves (58, 96).

Another variable that has been systematically studied is the cytokine cocktail that is applied to mature the DCs. The current gold standard cocktail for DC maturation contains TNF-α, IL-1β, IL-6, and PGE₂ (97, 98). Although this cocktail upregulates DC maturation markers and the lymph node homing receptor CCR7, IL-12 production by DCs could not be evoked (97, 98). Nevertheless, IL-12 is a critical Th1-driving cytokine and DC-derived IL-12 has been shown to associate with improved survival in DC vaccinated high-grade glioma and melanoma patients (99, 100). Recently, a novel cytokine cocktail, including TNF-α, IL-1β, poly-I:C, IFN-α, and IFN-γ, was introduced (101, 102). The type 1-polarized DCs obtained with this cocktail produced high levels of IL-12 and could induce strong tumor-antigen-specific CTL responses through enhanced induction of CXCL10 (99). In addition, CD40-ligand (CD40L) stimulation of DCs has been used to mature DCs in clinical trials (100, 103). Binding of CD40 on DCs to CD40L on CD4⁺ helper T cells licenses DCs and enables them to prime CD8⁺ effector T cells.

A final major determinant of the vaccine immunogenicity is the choice of antigen to load the DCs. Two main approaches can be applied: loading with selected tumor antigens (tumor-associated antigens or tumor-specific antigens) and loading with whole tumor cell preparations (13). The former strategy enables easier immune monitoring, has a lower risk of inducing auto-immunity, and can provide “off-the-shelf” availability of the antigenic cargo. Whole tumor cell-based DC vaccines, on the other hand, are not HLA-type dependent, have a reduced risk of inducing immune-escape variants, and can elicit immunity against multiple tumor antigens. Meta-analytical data provided by Neller et al. have demonstrated enhanced clinical efficacy in several tumor types of DCs loaded with whole tumor lysate as compared to DCs pulsed with defined tumor antigens (104). This finding was recently also substantiated in high-grade glioma patients, although this study was not set-up to compare survival parameters (105).

Toward a More Immunogenic Tumor Cell Cargo

The majority of clinical trials that apply autologous whole tumor lysate to load DC vaccines report the straightforward use of multiple freeze–thaw cycles to induce primary necrosis of cancer cells (8, 93). Freeze–thaw induced necrosis is, however, considered non-immunogenic and has even been shown to inhibit toll-like receptor (TLR)-induced maturation and function of DCs (16). To this end, many research groups have focused on tackling this roadblock by applying immunogenic modalities to induce cell death.

Immunogenic Treatment Modalities

Tables Tables11 and and22 list some frequently applied treatment methods to enhance the immunogenic potential of the tumor cell cargo that is used to load DC vaccines in an ICD-independent manner (i.e., these treatments do not meet the molecular and/or cellular determinants of ICD). Immunogenic treatment modalities can positively impact DC biology by inducing particular DAMPs in the dying cancer cells (Table (Table1).1). Table Table22 lists the preclinical and clinical studies that investigated their in vivo potential. Figure Figure11 schematically represents the application and the putative modes of action of these immunogenic enhancers in the setting of DC vaccines.

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A schematic representation of immunogenic DC vaccines. Cancer cells show enhanced immunogenicity upon treatment with UV irradiation, oxidizing treaments, and heat shock, characterized by the release of particular danger signals and the (increased) production of tumor (neo-)antigens. Upon loading onto DCs, DCs undergo enhanced phagocytosis and antigen uptake and show phenotypic and partial functional maturation. Upon in vivo immunization, these DC vaccines elicit Th1- and cytotoxic T lymphocyte (CTL)-driven tumor rejection.

Ultraviolet Irradiation ….

Oxidation-Inducing Modalities

In recent years, an increasing number of data were published concerning the ability of oxidative stress to induce oxidation-associate molecular patterns (OAMPs), such as reactive protein carbonyls and peroxidized phospholipids, which can act as DAMPs (28, 29) (Table (Table1).1). Protein carbonylation, a surrogate indicator of irreversible protein oxidation, has for instance been shown to improve cancer cell immunogenicity and to facilitate the formation of immunogenic neo-antigens (30, 31).

One prototypical enhancer of oxidation-based immunogenicity is radiotherapy (21,23). In certain tumor types, such as high-grade glioma and melanoma, clinical trials that apply autologous whole tumor lysate to load DC vaccines report the random use of freeze–thaw cycles (to induce necrosis of cancer cells) or a combination of freeze–thaw cycles and subsequent high-dose γ-irradiation (8, 18) (Table (Table2).2). However, from the available clinical evidence, it is unclear which of both methodologies has superior immunogenic potential. In light of the oxidation-based immunogenicity that is associated with radiotherapy, we recently demonstrated the superiority of DC vaccines loaded with irradiated freeze–thaw lysate (in comparison to freeze–thaw lysate) in terms of survival advantage in a preclinical high-grade glioma model (18) (Table (Table2).2). ….

Heat Shock Treatment

Heat shock is a term that is applied when a cell is subjected to a temperature that is higher than that of the ideal body temperature of the organisms of which the cell is derived. Heat shock can induce apoptosis (41–43°C) or necrosis (>43°C) depending on the temperature that is applied (110). The immunogenicity of heat shock treated cancer cells largely resides within their ability to produce HSPs, such as HSP60, HSP70, and HSP90 (17, 32) (Table (Table1).1). …

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Figure 2

A schematic representation of immunogenic cell death (ICD)-based DC vaccines. ICD causes cancer cells to emit a spatiotemporally defined pattern of danger signals. Upon loading of these ICD-undergoing cancer cells onto DCs, they induce extensive phagocytosis and antigen uptake. Loaded DCs show enhanced phenotypic and functional maturation and immunization with these ICD-based DC vaccines instigates Th1-, Th17-, and cytotoxic T lymphocyte (CTL)-driven antitumor immunity in vivo.

Inducers of Immunogenic Cell Death

Immunogenic cell death is a cell death regimen that is associated with the spatiotemporally defined emission of immunogenic DAMPs that can trigger the immune system (20, 21, 113). ICD has been found to depend on the concomitant induction of reactive oxygen species (ROS) and activation of endoplasmatic reticulum (ER) stress (111). Besides the three DAMPs that are most crucial for ICD (ecto-CRT, ATP, and HMGB1), other DAMPs such as surface-exposed or released HSPs (notably HSP70 and HSP90) have also been shown to contribute to the immunogenic capacity of ICD inducers (20, 21). The binding of these DAMPs to their respective immune receptors (CD91 for HSPs/CRT, P2RX7/P2RY2 for ATP, and TLR2/4 for HMGB1/HSP70) leads to the recruitment and/or activation of innate immune cells and facilitates the uptake of tumor antigens by antigen-presenting cells and their cross-presentation to T cells eventually leading to IL-1β-, IL-17-, and IFN-γ-dependent tumor eradiation (22). This in vivo tumor rejecting capacity induced by dying cancer cells in the absence of any adjuvant, is considered as a prerequisite for an agent to be termed an ICD inducer. …

Although the list of ICD inducers is constantly growing (113), only few of these immunogenic modalities have been tested in order to generate an immunogenic tumor cell cargo to load DC vaccines (Tables (Tables11 and and2).2). Figure Figure22 schematically represents the preparation of ICD-based DC vaccines and their putative modes of action.

Radiotherapy

Ionizing X-ray or γ-ray irradiation exerts its anticancer effect predominantly via its capacity to induce DNA double-strand breaks leading to intrinsic cancer cell apoptosis (114). The idea that radiotherapy could also impact the immune system was derived from the observation that radiotherapy could induce T-cell-mediated delay of tumor growth in a non-irradiated lesion (115). This abscopal (ab-scopus, away from the target) effect of radiotherapy was later explained by the ICD-inducing capacity (116). Together with anthracyclines, γ-irradiation was one of the first treatment modalities identified to induce ICD. …

Shikonin

The phytochemical shikonin, a major component of Chinese herbal medicine, is known to inhibit proteasome activity. It serves multiple biological roles and can be applied as an antibacterial, antiviral, anti-inflammatory, and anticancer treatment. …

High-hydrostatic pressure

High-hydrostatic pressure (HHP) is an established method to sterilize pharmaceuticals, human transplants, and food. HHP between 100 and 250 megapascal (MPa) has been shown to induce apoptosis of murine and human (cancer) cells (121–123). While DNA damage does not seem to be induced by HHP <1000 MPa, HHP can inhibit enzymatic functions and the synthesis of cellular proteins (122). Increased ROS production was detected in HHP-treated cancer cell lines and ER stress was evidenced by the rapid phosphorylation of eIF2α (42). …

Oncolytic Viruses

Oncolytic viruses are self-replicating, tumor selective virus strains that can directly lyse tumor cells. Over the past few years, a new oncolytic paradigm has risen; entailing that, rather than utilizing oncolytic viruses solely for direct tumor eradication, the cell death they induce should be accompanied by the elicitation of antitumor immune responses to maximize their therapeutic efficacy (128). One way in which these oncolytic viruses can fulfill this oncolytic paradigm is by inducing ICD (128).

Thus far, three oncolytic virus strains can meet the molecular requirements of ICD; coxsackievirus B3 (CVB3), oncolytic adenovirus and Newcastle disease virus (NDV) (Table (Table1)1) (113). Infection of tumor cells with these viruses causes the production of viral envelop proteins that induce ER stress by overloading the ER. Hence, all three virus strains can be considered type II ICD inducers (113). …

Photodynamic therapy

Photodynamic therapy (PDT) is an established, minimally invasive anticancer treatment modality. It has a two-step mode of action involving the selective uptake of a photosensitizer by the tumor tissue, followed by its activation by light of a specific wavelength. This activation results in the photochemical production of ROS in the presence of oxygen (129–131). One attractive feature of PDT is that the ROS-based oxidative stress originates in the particular subcellular location where the photosensitizer tends to accumulate, ultimately leading to the destruction of the tumor cell (132). …

Combinatorial Regimens

In DC vaccine settings, cancer cells are often not killed by a single treatment strategy but rather by a combination of treatments. In some cases, the underlying rationale lies within the additive or even synergistic value of combining several moderately immunogenic modalities. The combination of radiotherapy and heat shock has, for instance, been shown to induce higher levels of HSP70 in B16 melanoma cells than either therapy alone (16). In addition, a combination therapy consisting of heat shock, γ-irradiation, and UV irradiation has been shown to induce higher levels of ecto-CRT, ecto-HSP90, HMGB1, and ATP in comparison to either therapy alone or doxorubicin, a well-recognized inducer of ICD (57). ….

Triggering antitumor immune responses is an absolute requirement to tackle metastatic and diffusely infiltrating cancer cells that are resistant to standard-of-care therapeutic regimens. ICD-inducing modalities, such as PDT and radiotherapy, have been shown to be able to act as in situ vaccines capable of inducing immune responses that caused regression of distal untreated tumors. Exploiting these ICD inducers and other immunogenic modalities to obtain a highly immunogenic antigenic tumor cell cargo for loading DC vaccines is a highly promising application. In case of the two prominent ICD inducers, Hyp-PDT and HHP, preclinical studies evaluating this relatively new approach are underway and HHP-based DC vaccines are already undergoing clinical testing. In the preclinical testing phase, more attention should be paid to some clinically driven considerations. First, one should consider the requirement of 100% mortality of the tumor cells before in vivo application. A second consideration from clinical practice (especially in multi-center clinical trials) is the fact that most tumor specimens arrive in the lab in a frozen state. This implies that a significant number of cells have already undergone non-immunogenic necrosis before the experimental cell killing strategies are applied. ….

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Treatments for macular degenaration

Posted in Auditory and vision, Clinical & Translational, Diabetes Mellitus, Disease Biology, Neurological Diseases, Pharmaceutical Analytics, Pharmaceutical Drug Discovery, Population Health Management, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, tagged diabetic retinopathy, macular edema, VEGF on February 29, 2016| Leave a Comment »

Treatments for macular degenaration

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Eylea outperforms Avastin for diabetic macular edema with moderate or worse vision loss

NIH-funded clinical trial shows Eylea, Avastin, and Lucentis perform similarly when vision loss is mild.

http://www.nih.gov/news-events/news-releases/eylea-outperforms-avastin-diabetic-macular-edema-moderate-or-worse-vision-loss

image of a patient having an eye exam

A two-year clinical trial that compared three drugs for diabetic macular edema (DME) found that gains in vision were greater for participants receiving the drug Eylea (aflibercept) than for those receiving Avastin (bevacizumab), but only among participants starting treatment with 20/50 or worse vision. Gains after two years were about the same for Eylea and Lucentis (ranibizumab), contrary to year-one results from the study, which showed Eylea with a clear advantage. The three drugs yielded similar gains in vision for patients with 20/32 or 20/40 vision at the start of treatment. The clinical trial was conducted by the Diabetic Retinopathy Clinical Research Network (DRCR.net), which is funded by the National Eye Institute, part of the National Institutes of Health.

“This rigorous trial confirms that Eylea, Avastin, and Lucentis are all effective treatments for diabetic macular edema,” said NEI Director Paul A. Sieving, M.D., Ph.D. “Eye care providers and patients can have confidence in all three drugs.”

Eylea, Avastin, and Lucentis are all widely used to treat DME, a consequence of diabetes that can cause blurring of central vision due to the leakage of fluid from abnormal blood vessels in the retina. The macula is the area of the retina used when looking straight ahead. The drugs are injected into the eye and work by inhibiting vascular endothelial growth factor (VEGF), a substance that can promote abnormal blood vessel growth and leakage. Although the drugs have a similar mode of action, they differ significantly in cost. Based on Medicare allowable charges, the per-injection costs of each drug at the doses used in this study were about $1850 for Eylea, about $60 for Avastin, and about $1200 for Lucentis.

DRCR.net investigators enrolled 660 people with DME at 89 clinical trial sites across the United States. When the study began, participants on average were 61 years old with 17 years of type 1 or type 2 diabetes. Only people with a visual acuity of 20/32 or worse were eligible to participate (to see clearly, a person with 20/32 vision would have to be 20 feet away from an object that a person with normal vision could see clearly at 32 feet). At enrollment, about half the participants had 20/32 to 20/40 vision. The other half had 20/50 or worse vision. In many states, a corrected visual acuity of 20/40 or better in at least one eye is required for a driver’s license that allows both day- and nighttime driving.

Each participant was assigned randomly to receive Eylea (2.0 milligrams/0.05 milliliter), Avastin (1.25 mg/0.05 mL), or Lucentis (0.3 mg/0.05 mL). Participants were evaluated monthly during the first year and every 4-16 weeks during the second year. Most participants received monthly injections during the first six months. Thereafter, participants received additional injections of assigned study drug until DME resolved or stabilized with no further vision improvement. Subsequently, injections were resumed if DME worsened. Additionally, laser treatment was given if DME persisted without continual improvement after six months of injections. Laser treatment alone was the standard treatment for DME until widespread adoption of anti-VEGF drugs a few years ago.

Among participants with 20/40 or better vision at the trial’s start, all three drugs improved vision similarly on an eye chart. On average, participants’ vision improved from 20/40 vision to 20/25.

Among participants with 20/50 or worse vision at the trial’s start, visual acuity on average improved substantially in all three groups. At two years, Eylea participants were able to read about 3.5 additional lines on an eye chart; Lucentis participants were able to read about three additional lines, and Avastin participants improved about 2.5 lines, compared with visual acuity before treatment. Eylea outperformed Avastin at the one- and two-year time points. While Eylea outperformed Lucentis at the one-year time point, by the two-year time point gains in visual acuity were statistically no different. At the end of the trial, average visual acuity was 20/32 to 20/40 among participants in all three groups.

“The results of the DRCR Network’s comparison of Eylea, Avastin, and Lucentis will help doctors and their patients with diabetic macular edema choose the most appropriate therapy,” said John A. Wells, M.D., the lead author of the study and a retinal specialist at the Palmetto Retina Center, Columbia, South Carolina. “The study suggests there is little advantage of choosing Eylea or Lucentis over Avastin when a patient’s loss of visual acuity from macular edema is mild, meaning a visual acuity of 20/40 or better. However, patients with 20/50 or worse vision loss may benefit from Eylea, which over the course of the two-year study outperformed Lucentis and Avastin.”

The number of injections participants needed was about the same for all three treatment groups. Eylea, Avastin, and Lucentis participants on average required nine injections in the first year of the study and five in the second year.

The need for laser treatment varied among the three treatment groups. By two years, 41 percent of participants in the Eylea group received laser treatment to treat their macular edema, compared with 64 percent of participants in the Avastin group and 52 percent in the Lucentis group.

The risk of heart attack, stroke, or death from a cardiovascular condition or an unknown cause by end of the trial was higher among participants in the Lucentis group. Twelve percent of Lucentis participants had at least one event, compared with five percent of participants in the Eylea group and eight percent of participants in the Avastin group. This difference in cardiovascular rates has not been seen across all other studies, and therefore may be due to chance. Continued assessment of these serious cardiovascular events and their association with these drugs is important in future studies. Cardiovascular events such as heart attack and stroke are common complications of diabetes. The occurrence of eye complications, such as eye infections and inflammation, was similar for all three drugs.

Results of the study were published online today in Ophthalmology, the journal of the American Academy of Ophthalmology. Eylea and Lucentis were provided by drug manufacturers Regeneron and Genentech, respectively. Additional research funding for this study was provided by the National Institute of Diabetes and Digestive and Kidney Diseases, also a part of NIH.

“This important study would not have happened without funding from the National Institutes of Health and the cooperation of two competing companies,” said Adam R. Glassman, M.S., principal investigator of the DRCR.Net Coordinating Center at the Jaeb Center for Health Research.

The DRCR.net is dedicated to facilitating multicenter clinical research of diabetic eye disease. The Network formed in 2002 and comprises more than 350 physicians practicing at more than 140 clinical sites across the country. For more information, visit the DRCR.net website at http://drcrnet.jaeb.org/(link is external).

The study was funded by grants EY14231, EY14229, and EY18817.

The study is registered as NCT01627249 at ClinicalTrials.gov.

Macular edema can arise during any stage of diabetic retinopathy and is the most common cause of diabetes-related vision loss. About 7.7 million Americans have diabetic retinopathy. Of these, about 750,000 have DME. The NEI provides information about diabetic eye disease athttp://www.nei.nih.gov/health/diabetic. View an NEI video about how diabetic retinopathy can be detected through a comprehensive dilated eye exam at http://youtu.be/sQ-0RkPu35o(link is external).

NEI leads the federal government’s research on the visual system and eye diseases. NEI supports basic and clinical science programs that result in the development of sight-saving treatments. For more information, visit http://www.nei.nih.gov.

Vascular Endothelial Growth Factor (VEGF) and Its Role in Non-Endothelial Cells: Autocrine Signalling by VEGF

Angela M. Duffy, David J. Bouchier-Hayes, and Judith H. Harmey. http://www.ncbi.nlm.nih.gov/books/NBK6482/

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and was first described as an essential growth factor for vascular endothelial cells. VEGF is up-regulated in many tumors and its contribution to tumor angiogenesis is well defined. In addition to endothelial cells, VEGF and VEGF receptors are expressed on numerous non-endothelial cells including tumor cells. This review examines the relevance of VEGF signalling in non-endothelial cells and explores the probable mechanisms involved.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), was originally described as an endothelial cell-specific mitogen.¹ VEGF is produced by many cell types including tumor cells,²^,³ macrophages,⁴ platelets,⁵ keratinocytes,⁶ and renal mesangial cells.⁷ The activities of VEGF are not limited to the vascular system; VEGF plays a role in normal physiological functions such as bone formation,⁸ hematopoiesis,⁹wound healing,¹⁰ and development.¹¹

Anti-VEGF strategies to treat cancers were designed to target the pro-angiogenic function of VEGF and thereby inhibit neovascularization. However, anti-VEGF therapies may have a dual effect since evidence is accumulating to support the existence of both paracrine and autocrine VEGF loops within tumors. It has been suggested that direct stimulation of tumor cells by VEGF may protect the cells from apoptosis and increase their resistance to conventional chemotherapy and radiotherapy.¹² Chemotherapy and radiotherapy have been shown to increase VEGF within tumors,¹³ and this increased VEGF may in fact protect tumor cells from these interventions. Anti-VEGF therapies are therefore likely to target both the pro-angiogenic activity of VEGF and the anti-apoptotic/pro-survival functions of VEGF.

VEGF and the Central Nervous System (CNS)

In the central nervous system (CNS) both positive (pro-migratory) and negative (anti-migratory) regulatory factors are essential for axonal guidance.¹⁷ Following prolonged exposure, Sema3A, a member of the semaphorin family, acts as an inhibitor of neuronal migration and induces neuronal cell death¹⁸ through the neuropilin-1 receptor (NP-1).¹⁹However, in addition to Sema3A binding, NP-1 also acts as an additional receptor for VEGF₁₆₅ isoform.²⁰ The relationship between Sema3A and VEGF was explored in Dev cells,²¹ undifferentiated cells derived from a cerebellar medullablastoma that behave as pluripotential neural progenitor cells.²² NP-1 mRNA expression was detected in Dev cells by RT-PCR and in situ hybridization. Western blotting and immunohistochemical analysis confirmed that NP-1 was expressed on the cell surface. VEGF₁₆₅ or anti-NP-1 antibody blocked the effect of Sema3A on these cells, suggesting that VEGF₁₆₅ binds competitively to NP-1 to block Sema3A signalling.

Dev cells also expressed VEGFR-1 and blockade of VEGFR-1 reduced the inhibition of neuronal cell migration by Sema3A.²¹ It appears that both NP-1 and VEGFR-1 are required for Sema3A activity in these neuronal cells. NP-1 binds with high affinity to VEGFR-1.²⁴NP-1 has a very short intracellular domain and appears to require a coreceptor to transduce a signal²⁰ thus, VEGFR-1 may serve as a coreceptor for NP-1 in the modulation of Sema3A signalling. Both VEGF₁₂₁ and VEGF₁₆₅ inhibited Sema3A-induced apoptosis, and at higher concentrations reduced apoptosis below basal levels indicating an additional neuroprotective effect.

VEGF is induced in many CNS pathologies where it may have a neuroprotective role. VEGF has a neurotrophic effect and enhances survival of Schwann cells,²⁵ and protects hippocampal neurons from ischemic injury.²⁶ Impaired VEGF induction in the spinal cord results in motor neuron degeneration.²⁷ In addition, when cerebellar granule neurons (CGNs) were exposed to 5% hypoxia for 9 hours VEGF, VEGFR-1 and VEGFR-2 expression increased, and a neutralizing antibody to VEGF, DC 101, inhibited hypoxic preconditioning.²⁸ Thus, VEGF autocrine or paracrine mechanisms appear to play a role in CGN cell survival following hypoxic preconditioning. In CGNs Akt (also known as Protein Kinase B/ PKB) was phosphorylated in response to VEGF and other studies have shown that VEGF stimulation in neurons is linked to PI3-K (Phosphatidylinositol 3′-kinase) and Akt activation and neuronal protection.²⁹

VEGFA http://www.uniprot.org/uniprot/P15692

Growth factor active in angiogenesis, vasculogenesis and endothelial cell growth. Induces endothelial cell proliferation, promotes cell migration, inhibits apoptosis and induces permeabilization of blood vessels. Binds to the FLT1/VEGFR1 and KDR/VEGFR2 receptors, heparan sulfate and heparin. NRP1/Neuropilin-1 binds isoforms VEGF-165 and VEGF-145. IsoformVEGF165B binds to KDR but does not activate downstream signaling pathways, does not activate angiogenesis and inhibits tumor growth.

GO:1902336 positive regulation of retinal ganglion cell axon guidance

ID	GO:1902336
Name	positive regulation of retinal ganglion cell axon guidance
Ontology	Biological Process
Definition	Any process that activates or increases the frequency, rate or extent of retinal ganglion cell axon guidance. PMID:21658587
GONUTS	GO:1902336 Wiki Page
Acknowledgements	This term was created by the GO Consortium

Synonym

up-regulation of retinal ganglion pathfinding

cell axon pathfinding

up regulation of retinal ganglion cell axon pathfinding

activation of retinal ganglion cell axon pathfinding

activation of retinal ganglion cell axon guidance

upregulation of retinal ganglion cell axon pathfinding

up regulation of retinal ganglion cell axon guidance

up-regulation of retinal ganglion cell axon guidance

upregulation of retinal ganglion cell axon guidance

positive regulation of retinal ganglion cell axon pathfinding

What Is Age-Related Macular Degeneration? http://www.webmd.com/eye-health/macular-degeneration/age-related-macular-degeneration-overview
Macular degeneration is the leading cause of severe vision loss in people over age 60. It occurs when the small central portion of the retina, known as the macula, deteriorates. The retina is the light-sensing nerve tissue at the back of the eye. Because the disease develops as a person ages, it is often referred to as age-related macular degeneration (AMD). Although macular degeneration is almost never a totally blinding condition, it can be a source of significant visual disability.

There are two main types of age-related macular degeneration:

Dry form. The “dry” form of macular degeneration is characterized by the presence of yellow deposits, called drusen, in the macula. A few small drusen may not cause changes in vision; however, as they grow in size and increase in number, they may lead to a dimming or distortion of vision that people find most noticeable when they read. In more advanced stages of dry macular degeneration, there is also a thinning of the light-sensitive layer of cells in the macula leading to atrophy, or tissue death. In the atrophic form of dry macular degeneration, patients may have blind spots in the center of their vision. In the advanced stages, patients lose central vision.
Wet form. The “wet” form of macular degeneration is characterized by the growth of abnormal blood vessels from the choroid underneath the macula. This is called choroidal neovascularization. These blood vessels leak blood and fluid into the retina, causing distortion of vision that makes straight lines look wavy, as well as blind spots and loss of central vision. These abnormal blood vessels and their bleeding eventually form a scar, leading to permanent loss of central vision.
Most patients with macular degeneration have the dry form of the disease and can lose some form of central vision. However, the dry form of macular degeneration can lead to the wet form. Although only about 10% of people with macular degeneration develop the wet form, they make up the majority of those who experience serious vision loss from the disease.

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Inflammatory Disorders: Articles published @ pharmaceuticalintelligence.com

Posted in Alzheimer's Disease, Autoimmune Inflammatory DIseases, Autophagy, Bacterial Resistance, Cancer and Current Therapeutics, Crohn's disease, Cytokines, Diabetes Mellitus, Disease Biology, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Enzymes and isoenzymes, Etiology, Gastroenterology, Hematology, Human Immune System in Health and in Disease, Inflammasome, Lung and pulmonology, Lymphoma, Metabolism, Metabolomics, Microbiology, Micronutrients, Nephrology, Neurodegenerative Diseases, Neurological Diseases, Nitric Oxide in Health and Disease, Nutrition, Obesity, Protein-energy malnutrition, Proteolysis, Proteomics, Ulcerative Colitis, Viral diseases, Virology, tagged autoimmunity, systemic inflammatory response, T cells on February 28, 2016| Leave a Comment »

Inflammatory Disorders: Articles published @ pharmaceuticalintelligence.com

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

This is a compilation of articles on Inflammatory Disorders that were published

@ pharmaceuticalintelligence.com, since 4/2012 to date

There are published works that have not been included. However, there is a substantial amount of material in the following categories:

Proteomics, metabolomics and diabetes

http://pharmaceuticalintelligence.com/2015/11/16/reducing-obesity-related-inflammation/

http://pharmaceuticalintelligence.com/2015/10/25/the-relationship-of-stress-hypermetabolism-to-essential-protein-needs/

http://pharmaceuticalintelligence.com/2015/10/24/the-relationship-of-s-amino-acids-to-marasmic-and-kwashiorkor-pem/

http://pharmaceuticalintelligence.com/2015/10/24/the-significant-burden-of-childhood-malnutrition-and-stunting/

http://pharmaceuticalintelligence.com/2015/04/14/protein-binding-protein-protein-interactions-therapeutic-implications-7-3/

http://pharmaceuticalintelligence.com/2015/03/07/transthyretin-and-the-stressful-condition/

http://pharmaceuticalintelligence.com/2015/02/13/neural-activity-regulating-endocrine-response/

http://pharmaceuticalintelligence.com/2015/01/31/proteomics/

http://pharmaceuticalintelligence.com/2015/01/17/proteins-an-evolutionary-record-of-diversity-and-adaptation/

http://pharmaceuticalintelligence.com/2014/11/01/summary-of-signaling-and-signaling-pathways/

http://pharmaceuticalintelligence.com/2014/10/31/complex-models-of-signaling-therapeutic-implications/

http://pharmaceuticalintelligence.com/2014/10/24/diabetes-mellitus/

http://pharmaceuticalintelligence.com/2014/10/16/metabolomics-summary-and-perspective/

http://pharmaceuticalintelligence.com/2014/10/14/metabolic-reactions-need-just-enough/

http://pharmaceuticalintelligence.com/2014/11/03/introduction-to-protein-synthesis-and-degradation/

http://pharmaceuticalintelligence.com/2015/09/25/proceedings-of-the-nyas/

http://pharmaceuticalintelligence.com/2014/10/31/complex-models-of-signaling-therapeutic-implications/

http://pharmaceuticalintelligence.com/2014/03/21/what-is-the-key-method-to-harness-inflammation-to-close-the-doors-for-many-complex-diseases/

http://pharmaceuticalintelligence.com/2013/03/05/irf-1-deficiency-skews-the-differentiation-of-dendritic-cells/

http://pharmaceuticalintelligence.com/2012/11/26/new-insights-on-no-donors/

http://pharmaceuticalintelligence.com/2012/11/20/the-potential-for-nitric-oxide-donors-in-renal-function-disorders/

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Making Sense of Hypertensive Retinopathy

Posted in Cerebrovascular and Neurodegenerative Diseases, Clinical Diagnostics, Curation, Dialectic, Discovery process, Disease Biology, Experimental validation, Explanatory, Historical relevance, Neurological Diseases, Neuroscience, Pharmaceutical Drug Discovery, Pharmacodynamics and Pharmacokinetics, Pharmacotherapy of Cardiovascular Disease, Systemic Inflammatory Response Related Disorders, Translational Science, Vision on February 21, 2016| Leave a Comment »

High blood pressure can damage the retina’s blood vessels and limit the retina’s function. It can also put pressure on the optic nerve.

Sourced through Scoop.it from: www.healthline.com

See on Scoop.it – Cardiovascular Disease: PHARMACO-THERAPY

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Sheila Mathews: The right to refuse psychiatric services

Posted in Neurological Diseases on February 21, 2016| Leave a Comment »

Sheila Mathews: The right to refuse psychiatric services

Reporter: Aviva Lev-Ari, PhD, RN

Sheila Mathews, co-founder the organization AbleChild joins us to talk about the labeling and drugging of children and the Hatch Amendment meant to protect them from this kind of abuse. Sheila will join the show at 7:45 pm CST.

Sourced through Scoop.it from: www.blogtalkradio.com

See on Scoop.it – Cardiotoxicity

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Alzheimer’s Disease – tau art thou, or amyloid

Posted in Alzheimer's Disease, Behavior, Clinical & Translational, Clinical Diagnostics, Cognition, Etiology, Neurodegenerative Diseases, Neurological Diseases, Neuroscience, Pharmacotherapy and Cell Activity, tagged Alzheimer's pathology, Tau protein on February 15, 2016| Leave a Comment »

Alzheimer’s Disease – tau art thou, or amyloid

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Alzheimer’s Disease and Tau

http://www.nyas.org/Publications/Ebriefings/Detail.aspx

Pathogenic Mechanisms and Therapeutic Approaches

Organizers: Robert Martone (St. Jude Children’s Research Hospital) and Sonya Dougal (The New York Academy of Sciences)Presented by the Brain Dysfunction Discussion Group

Reported by Caitlin McOmish | Posted February 2, 2016

http://www.nyas.org/image.axd?id=7391dacc-ddda-4b9a-ad45-06a05953b756&t=635690168670270000

Microtubule-associated protein tau helps maintain the stability and flexibility of microtubules in neuronal axons. Alternative splicing of the tau gene, MAPT, produces 6 isoforms of tau in the brain and many more in the peripheral nervous system. Tau can be phosphorylated at over 30 sites, and it undergoes many posttranslational modifications to operate as a substrate for multiple enzymes. However, tau also mediates pathological functions including neuroinflammatory response, seizure, and amyloid-β (Aβ) toxicity, and tau pathology is a hallmark of conditions including frontotemporal dementia, traumatic brain injury (TBI), Down syndrome, focal cortical dysplasia, and Alzheimer’s disease (AD), as well as some tumors and infections. On September 18, 2015, speakers at the Brain Dysfunction Discussion Group’s Alzheimer’s Disease and Tau: Pathogenic Mechanisms and Therapeutic Approaches symposium discussed the mechanisms by which tau becomes pathological and how the pathology spreads. They also described emerging therapeutic strategies for AD focused on tau.

http://www.nyas.org/image.axd?id=4a8bd710-d75a-4655-8bee-ef48fac6a783&t=635888888349400000

Microtubule-associated protein tau has a complex biology, including multiple splice variants and phosphorylation sites. Tau is a key component of microtubules, which contribute to neuronal stability. In AD, tau changes, causing microtubules to collapse, and tau proteins clump together to form neurofibrillary tangles. (Image presented by Robert Martone courtesy of the National Institute on Aging)

Tau is ubiquitous in the brain, with widespread effects, but has historically been overlooked as a driving force in AD. In his introduction to the symposium, Robert Martone from St. Jude Children’s Research Hospital highlighted tau’s activity and emergence as a treatment target for this devastating disorder. Hyperphosphorylated tau (p-tau) has long been recognized as a principle component of neurofibrillary tangles in AD; tau monomers are misfolded into oligomers that form tau filaments. As Hartmuth Kolb from Johnson & Johnson explained, the development in 2012 of a tau-specific positron emission tomography (PET) tracer led to important insights into the presence and spread of tau pathology over the course of tauopathies, including AD, in humans. Notably, researchers demonstrated that tau pathology propagates through the brain in a predictable pattern, corresponding to the Braak stages of AD.

http://www.nyas.org/image.axd?id=16b77dfd-391b-4495-913f-95a2fba38458&t=635888888240470000

Tau pathology spreads through the brain in a predictable pattern. Abnormal tau protein is first observed in the transentorhinal region (stages I and II) and spreads to the limbic regions in stages III and IV, when early signs of AD begin to be observed. Pathology subsequently extends throughout the neocortex, driving fully developed AD. This staging was first described by Braak and Braak in 1991. (Image courtesy of Hartmuth Kolb)

It is likely that the symptoms of AD are produced by the combined effects of tau and Aβ pathologies. George Bloom from the University of Virginia described how Aβ and tau interact to cause mature neurons to reenter the cell cycle, leading to cell death. In a healthy brain, insulin acts as a gatekeeper that maintains adult neurons in the G0 phase after the cells permanently exit the cell cycle. In AD, amyloid oligomers sequester neuronal insulin receptors, causing insulin resistance. In parallel, tau phosphorylation at key sites—pY18 (fyn site), pS409 (PKA site), pS416 (CAM Kinase site), and pS262—drives mTOR signaling at the plasma membrane but not at the lysosome, resulting in cell cycle reentry. In a normal cell, activation of mTOR at the lysosome overrides the cell cycle reentry signal—creating an important regulatory mechanism for maintaining healthy neurons. However, lysosomal activation of mTOR is insulin dependent and thus affected by Aβ-induced insulin insensitivity. Amyloid oligomers, via insulin regulation, release the brakes on a cascade of events driven by p-tau that leads to cell cycle reentry and cell death.

http://www.nyas.org/image.axd?id=35ed7190-cb27-4bed-bc0d-1fa27091e733&t=635888888067800000

Hallmark dysfunction produced by Aβ is dependent on tau. Pathological Aβ drives the formation of p-tau in the brain, resulting in synaptic dysfunction, cell death, and broad neurocognitive symptoms. This process can be influenced by a range of factors including genetic predisposition, environmental risk factors, and biochemical signaling pathways. (Image courtesy of George Bloom)

Khalid Iqbal from the New York State Institute for Basic Research in Developmental Disabilities described research showing that p-tau spreads through the brain in a rodent model, well beyond the injection site, in a prion-like manner, and that the spread of pathology can be mitigated by the addition of PP2A—a phosphatase known to be decreased in gray and white matter in AD. PP2A regulation is affected in AD, stroke, and brain acidosis, providing a link between these disorders and tau pathology.

Discussion of the pathophysiology of AD commonly focuses on Aβ plaques and neurofibrillary tangles (NFTs) composed of misassembled hyperphosphorylated tau; it has generally been thought that these plaques and tangles are the primary causes of symptoms. However, recent evidence indicates that oligomeric variants of tau are actually far more toxic than the form of tau present in NFTs. Michael Hutton from Eli Lilly and Company studies the properties needed for tau to become pathological. He used animal models to show that the abnormal p-tau “seed,” from which a prion-like spread develops, must be of a high molecular weight (with at minimum three tau units) and highly phosphorylated to induce healthy tau to become pathological. These characteristics are necessary but not sufficient for effective seeding. There is also evidence that tau pathology propagates via an autocatalytic cycle of seeded aggregation and fragmentation.

Propagation, in addition to requiring a large number of p-tau units in aggregates, may be affected by the isomerization of those monomers. Kun Ping Lu from Harvard Medical School provided data suggesting that cis but not trans pT231-tau is a precursor of tauopathy, linking TBI to the later development of neurodegenerative diseases such as chronic traumatic encephalopathy and AD. He demonstrated a role for Pin1, a phosphorylation-specific prolyl isomerase, in this process using animal models of TBI and AD. Pin1, which is regulated in response to stress, prevents the accumulation of toxic cis p-tau by converting it to the trans isoform, but this process is inhibited in AD and TBI. Lu showed that cis p-tau’s ability to cause and spread neurodegeneration can be blocked by a cis p-tau monoclonal antibody in vitro and in animal models, pointing to the therapeutic potential of targetingcis p-tau for treatment of TBI and AD.

Culturing p-tau seeds in vitro produces a broad array of tau aggregate structures. Marc Diamond from the University of Texas Southwestern Medical Center discussed the diverse structures produced by different tau seeds, which his team has studied in a series of experiments using in vitro models, animal models, and human postmortem analyses. His lab showed that distinct conformations of aggregate seeds propagate stably, infecting normal cells and leading them to acquire abnormal tau aggregates with distinct, reproducible structures and different biochemical properties. In another study, the team showed that the morphology of the p-tau aggregates was related to diagnosis. Seeds sourced from postmortem human tissue produced reliable phenotypes in culture, which tracked with different diagnoses, retroactively predicting biological outcome. Thus, the characteristics of the p-tau seed have a large influence on the biological outcome, providing a new prospect for presymptomatic diagnosis.

http://www.nyas.org/image.axd?id=53f0c661-b6ca-4366-b289-a610fa12572f&t=635888888166270000

Tau seeds obtained from postmortem brain tissue from AD, argyrophilic grain disease (AGD), corticobasal degeneration (CBD), Pick’s disease (PiD), and progressive supranuclear palsy (PSP) produce unique aggregate pathologies in cell culture, including toxic, mosaic, ordered, disordered, and speckled. AD-derived seeds largely produce the speckled phenotype. (Images courtesy of Marc Diamond)

With the mechanisms by which p-tau forms, converts healthy tau, and seeds dysfunction established, the question of how p-tau exits the cell and moves through the brain arises. The pattern of spread and the speed with which the pathology progresses suggests that p-tau propagates trans-synaptically. Nicole Leclerc from the University of Montreal provided evidence to support this view. It is likely, her lab has shown, that tau is secreted and taken up by neurons in an active process, in response to neuronal activity. Tau secretion in vitro increases under conditions such as starvation and lysosomal dysfunction, phenomena found in the early stages of AD. Moreover, hyperphosphorylation appears to increase the targeting of tau to the secretory pathway, potentially accelerating the spread of p-tau. Intriguingly, however, the extracellular tau is hypophosphorylated, suggesting large-scale dephosphorylation during the secretory process. This hypo-tau may activate muscarinic acetylcholine receptors, increasing intracellular Ca²⁺ and promoting cell death.

These findings suggest that the synapse plays a critical role in the development of AD; the extrasynaptic environment is known to be exquisitely regulated by microglia. The focus of studies into neurodegenerative disorders is often neurons, but genetic studies have repeatedly identified changes in expression of microglial genes in AD, including in one of the leading AD candidate genes, TREM2, demonstrating a fundamental contribution of these cells to AD. Richard Ransohoff of Biogen discussed the importance of this cell type. Microglia enter the brain at around embryonic day (E) 9.5 in rodents and are crucially involved in maintaining brain health. During development the cells play a major role in large-scale synaptic pruning required for effective neural maturation. They are also highly responsive to the environment, and stress in adulthood can reengage microglial synaptic pruning—a process that is adaptive during development but maladaptive in adulthood. The process is regulated by complement system cascades. TGF-β expressed by astrocytes drives neurons to express C1q presynaptically, initiating complement elements to accumulate at the site, ultimately activating microglia to prune the synaptic connection. In AD, inappropriate activation of this cascade may lead to the removal of otherwise healthy connections. Ransohoff described a role for CXCR3, the fractalkine receptor, in regulating reactivity of microglia, and thus mitigating pruning of adult synapses. Regulation of microglia reactivity is driven by epigenetically induced changes in inflammatory response genes. Correspondingly, in the absence of CXCR3, tau pathology is aggravated in htau mice (which express human tau isoforms), suggesting a protective effect of the CXCR3 pathway. Ransohoff closed with the caveat that microglia are not intrinsically helpful or harmful; their properties are context dependent and must be unraveled by empirical observations in appropriate models.

Peter Davies from the Feinstein Institute for Medical Research discussed the need to better incorporate current knowledge into research model design, particularly to develop monoclonal antibodies for the treatment of AD. Monoclonal antibodies are a promising strategy, but translating preclinical findings into successful clinical outcomes will require careful consideration of the context of the early research. Most transgenic animal models for AD express p-tau in all neurons, but such extensive p-tau spread is not found in human AD brains. There are several hurdles to determine the drugs’ efficacy and safety in humans; it is difficult to assess specificity and find appropriate dosages. In a series of studies with a focus on external reproducibility, Davies presented evidence from animal models showing that immunotherapy can block the spread of p-tau but cannot undo pathology already present in the brain. In the htau mouse model several putative antibodies lacked efficacy and in some cases appeared to worsen pathology. These findings underscore the need for both better models and improved understanding of mechanisms of action before moving drugs to the clinic.

The New York Academy of Sciences. Alzheimer’s Disease and Tau: Pathogenic Mechanisms and Therapeutic Approaches. Academy eBriefings. 2015. Available at: www.nyas.org/Tau2015-eB

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Archive for the ‘Neurological Diseases’ Category

references:

Microglial phagocytosis study

References

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Box 1 Schizophrenia and Depression

Box 2 Epigenetic regulators and chromatin structure and function

MBD5

MBD6

MECP2

SETDB1

SETDB2

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Ultraviolet Irradiation ….

Oxidation-Inducing Modalities

Heat Shock Treatment

Radiotherapy

Shikonin

Oncolytic Viruses

Combinatorial Regimens

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Inflammatory Disorders: Articles published @ pharmaceuticalintelligence.com

This is a compilation of articles on Inflammatory Disorders that were published

@ pharmaceuticalintelligence.com, since 4/2012 to date

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Sheila Mathews: The right to refuse psychiatric services

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