A study of the polycomb group complexes in the maintenance of heterochromatic genome stability and Alzheimer's disease

La démence d'Alzheimer est une maladie neurodégénérative caractérisée par une perte progressive et irreversible des fonctions cognitives et des compétences intellectuelles. La maladie d’Alzheimer se présente sous deux formes: la forme familiale ou précoce (EOAD) qui représente 5% des cas et elle est liée à des mutations génétiques affectant le métabolisme des peptides amyloïde; et la forme tardive ou sporadique (LOAD) qui représente 95% des cas mais son étiologie est encore mal définie. Cependant, le vieillissement reste le principal facteur de risque pour développer LOAD. Les changements épigénétiques impliquant des modifications des histones jouent un rôle crucial dans les maladies neurodégénératives et le vieillissement lié à l'âge. Des données récentes ont décrit LOAD comme un désordre de l'épigénome et ont associé ce trouble à l'instabilité génomique. Les protéines Polycomb sont des modificateurs épigénétiques qui induisent le remodelage de la chromatine et la répression des gènes à l'hétérochromatine facultative. Nous rapportons que les souris hétérozygotes pour une protéine Polycomb développent avec l'âge un trouble neurologique ressemblant à LOAD caractérisé par l’altération des fonctions cognitives, la phosphorylation de la protéine tau, l'accumulation des peptides amyloïde, et le dysfonctionnement synaptique. Ce phénotype pathologique est précédé par la décondensation de l’hétérochromatine neuronale et l'activation de la réponse aux dommages à l'ADN. Parallèlement, une réduction d’expression de polycomb, malformations de l'hétérochromatine neuronale, et l'accumulation de dommages à l'ADN étaient également présents dans les cerveaux de patients LOAD. Remarquablement, les dommages de l'ADN ne sont pas distribués de façon aléatoire sur le génome mais sont enrichis au niveau des séquences répétitives. Les conclusions présentées dans cette thèse ont identifié des modifications épigénétiques spécifiques qui conduisent à une instabilité génomique aberrante menant à la formation de LOAD. Ces résultats vont aider au développement de nouveaux traitements qui peuvent potentiellement ralentir la neurodégénérescence.Alzheimer’s disease (AD) is the most common neurodegenerative disorder characterized by progressive and irreversible decline in cognitive functions and thinking skills. There are two types of AD: early-onset or familial AD (EOAD) that accounts for 5% of cases and is linked to mutations affecting the amyloid metabolism and late-onset or sporadic AD (LOAD), which accounts for 95% of cases, however the etiology of this type remains poorly delineated with ageing presenting the main risk factor. Epigenetic changes involving histone modifications play a critical role in ageing and age- related neurodegenerative diseases. Recent evidence describing LOAD as an epigenetic disorder has accrued, associating this disorder to global genomic instability. Polycomb group proteins are epigenetic modifiers initiating chromatin remodeling and gene repression at facultative heterochromatin. We report that mice heterozygous for a polycomb protein develop with advancing age a neurological disorder resembling LOAD characterized by impaired memory behaviour, tau phosphorylation, amyloid accumulation, and synaptic dysfunction. Interestingly, this phenotype was preceded by neuronal heterochromatin decondensation and activation of DNA damage response. Concomitantly, polycomb deficiency, de-compaction of neuronal heterochromatin, and accumulation of DNA damage machinery were also characteristic of LOAD brains. Remarkably, DNA damage was not randomly distributed on the genome but enriched at heterochromatin. The findings presented in this thesis identified specific epigenetic modifications that lead to aberrant genomic instability in LOAD and will aid in the development of novel therapeutics, which may potentially slow neurodegeneration

Bmi1 Is Down-Regulated in the Aging Brain and Displays Antioxidant and Protective Activities in Neurons

Aging increases the risk to develop several neurodegenerative diseases, although the underlying mechanisms are poorly understood. Inactivation of the Polycomb group gene Bmi1 in mice results in growth retardation, cerebellar degeneration, and development of a premature aging-like phenotype. This progeroid phenotype is characterized by formation of lens cataracts, apoptosis of cortical neurons, and increase of reactive oxygen species (ROS) concentrations, owing to p53-mediated repression of antioxidant response (AOR) genes. Herein we report that Bmi1 expression progressively declines in the neurons of aging mouse and human brains. In old brains, p53 accumulates at the promoter of AOR genes, correlating with a repressed chromatin state, down-regulation of AOR genes, and increased oxidative damages to lipids and DNA. Comparative gene expression analysis further revealed that aging brains display an up-regulation of the senescence-associated genes IL-6, p19Arf and p16Ink4a, along with the pro-apoptotic gene Noxa, as seen in Bmi1-null mice. Increasing Bmi1 expression in cortical neurons conferred robust protection against DNA damage-induced cell death or mitochondrial poisoning, and resulted in suppression of ROS through activation of AOR genes. These observations unveil that Bmi1 genetic deficiency recapitulates aspects of physiological brain aging and that Bmi1 over-expression is a potential therapeutic modality against neurodegeneration

Modeling Late-Onset Sporadic Alzheimer’s Disease through BMI1 Deficiency

Late-onset sporadic Alzheimer’s disease (AD) is the most prevalent form of dementia, but its origin remains poorly understood. The Bmi1/Ring1 protein complex maintains transcriptional repression of developmental genes through histone H2A mono-ubiquitination, and Bmi1 deficiency in mice results in growth retardation, progeria, and neurodegeneration. Here, we demonstrate that BMI1 is silenced in AD brains, but not in those with early-onset familial AD, frontotemporal dementia, or Lewy body dementia. BMI1 expression was also reduced in cortical neurons from AD patient-derived induced pluripotent stem cells but not in neurons overexpressing mutant APP and PSEN1. BMI1 knockout in human post-mitotic neurons resulted in amyloid beta peptide secretion and deposition, p-Tau accumulation, and neurodegeneration. Mechanistically, BMI1 was required to repress microtubule associated protein tau (MAPT) transcription and prevent GSK3beta and p53 stabilization, which otherwise resulted in neurodegeneration. Restoration of BMI1 activity through genetic or pharmaceutical approaches could represent a therapeutic strategy against AD

BMI1 is down-regulated in the aging human brain and retina.

<p>(A) Immunohistochemistry on human brain (hippocampus) sections using anti-Bmi1 (brown) and anti-GFAP (pink) antibodies. BMI1 is expressed in neurons, but not in GFAP+ astrocytes, and expression is highly reduced in old brain neurons. Note the virtual absence of BMI1 labeling in some neurons (red arrowheads). Scale bars; 20 µm. (B) Immunofluorescence analysis of BMI1 expression in the human retina (23 years old, frozen sections). BMI1 is highly expressed in human photoreceptors (white arrowheads), which cell body lies in the outer nuclear layer (ONL), while its expression is weaker in neurons of the inner nuclear (INL) and ganglion cell (GCL) layers (red arrowheads). Scale bars; 20 µm. (C) Human retina samples were analyzed by Western blot for BMI1 expression and protein content was normalized using <i>β</i>-actin. BMI1 protein levels are reduced in old retinas (65–75 years). Results are Mean ± s.d. (n = 2–5 retinas per group; *<i>P</i><0.05). (D)Immunofluorescence analysis of GFAP and P16^INK4A expression in young and old human retinas. Note increased GFAP and P16^INK4A immunoreactivity in the old retinas. Scale bars; 20 µm.</p

BMI1 is highly neuroprotective against topoisomerase I inhibition and mitochondrial poisoning.

<p>(A) Empty plasmid vectors (CMV-GFP) or human BMI1-carrying plasmid (CMV-GFP: BMI1) were transfected in 293FT cells and lysates were analyzed 72 hours later for Bmi1 expression by Western blot. β-actin was used as internal control for normalization of protein loading. Non-transfected cells were used as control (Ctl) for endogenous Bmi1 expression. (B) Experimental scheme showing the procedure used to electroporate plasmid vectors in primary neuronal cultures from e18.5 WT mouse embryo cortices. (C) After 7 days i<i>n vitro</i> (DIV), electroporated neurons were exposed to CA, 3-NP or their respective vehicles. 16 hours later, cultures were stained for apoptosis induction (caspase-3 in brown) and expression of GFP (in pink), in order to distinguish neurons carrying or not the transgene. (D) Cell viability was assessed in cultures photographed in (C) as the percentage of GFP⁺/Caspase-3⁻ cells <i>versus</i> total GFP⁺ cells. Results are Mean ± s.d. (n = 3; *<i>P</i><0.05; **<i>P</i><0.001). (E) After 7 DIV, electroporated WT and <i>p53^−/−</i> neurons were exposed to CA or vehicle (DMSO) and analyzed after 16 hours as described in (C). Results are Mean ± s.d. (n = 3; **<i>P</i><0.001).</p

Bmi1 deficiency during aging influences neurons resistance to genotoxic stresses and mitochondrial dysfunctions.

<p>Proposed model of Bmi1 function in neurons: (A) When over-expressed, Bmi1 represses p53 activity by an unknown mechanism, leading to complete inhibition of p53 pro-apoptotic and pro-oxidant activities and supra-activation of the antioxidant defense system. (B) In young neurons, where Bmi1 expression is robust, Bmi1 partially represses p53 activity, thus allowing modulation of p53-mediated apoptosis and repression of antioxidant response elements (ARE). These elements are present in antioxidant-coding genes activated by the Nrf2 transcription factor. (C) In aging neurons, where Bmi1 expression becomes deficient, p53 is activated (1), leading to induction of apoptosis and inflammation, and in transcriptional repression of antioxidant-coding genes (2). Elevated mitochondrial reactive oxygen species (mROS) concentrations ultimately induce damages to lipids and DNA, which further activate p53 (3), resulting in the formation of a vicious circle. This situation renders old neurons particularly more vulnerable to genotoxic stresses (gs) and mitochondrial dysfunctions. This model is based on data from the present work, and those published previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031870#pone.0031870-Chatoo1" target="_blank">[20]</a>.</p

Antioxidant defenses are reduced in the aging mouse brain.

<p>(A) The relative expression of senescence-associated genes in cortices from young and old brains was analyzed by Q-PCR. Results are Mean ± s.d. (n = 3; *<i>P</i><0.05; **<i>P</i><0.01). The dashed line represents the basal gene expression level measured in young mice. (B) The relative expression of antioxidant genes in cortices from young and old brains, and from P25 <i>Bmi1^−/−</i> and WT mice was analyzed by Q-PCR. The dashed line represents the basal gene expression level measured in young compared to old and to WT compared to <i>Bmi1^−/−</i> mice. Results are Mean ± s.d. (n = 3; *<i>P</i><0.05; **<i>P</i><0.01). (C) Coronal sections from the cerebral cortex of young and old mice, and of P25 WT and <i>Bmi1^−/−</i> mice were labeled with antibodies against 8-oxo-guanine (8-OG; brown) and GFAP (pink). Note the increase in 8-oxo-guanine labeling in neurons from old and <i>Bmi1^−/−</i> mice compared to respective controls. Scale bars; 50 µm. (D) ChIP analysis of young and old brains revealing accumulation of p53 and heterochromatin marks (histone H3 K27^me2 or H3 k9^me2) at the <i>xCT</i>, <i>Sod1</i> and <i>Sod2</i> promoters in old brains. Antibodies against acetylated histone H4 and IgG were used as control. The <i>β-major</i> promoter region of <i>globin</i> was use as negative control. Results are Mean ± s.d. (n = 3; *<i>P</i><0.05).</p

Peripheral and local predictive immune signatures identified in a phase II trial of ipilimumab with carboplatin/paclitaxel in unresectable stage III or stage IV melanoma

BACKGROUND: Checkpoint blockade with ipilimumab provides long-term survival to a significant proportion of patients with metastatic melanoma. New approaches to increase survival and to predict which patients will benefit from treatment are needed. This phase II trial combined ipilimumab with carboplatin/paclitaxel (CP) to assess its safety, efficacy, and to search for peripheral and tumor-based predictive biomarkers. METHODS: Thirty patients with untreated unresectable/metastatic melanoma were treated with ipilimumab and CP. Adverse events (AEs) were monitored and response to treatment was evaluated. Tumor tissue and peripheral blood were collected at specified time points to characterize tumor immune markers by immunohistochemistry and systemic immune activity by multiplex assays and flow cytometry. RESULTS: Eighty three percent of patients received all 5 cycles of CP and 93% completed ipilimumab induction. Serious AEs occurred in 13% of patients, and no treatment-related deaths were observed. Best Overall Response Rate (BORR) and Disease Control Rate (DCR) were 27 and 57%, respectively. Median overall survival was 16.2 months. Response to treatment was positively correlated with a higher tumor CD3+ infiltrate (immune score) at baseline. NRAS and BRAF mutations were less frequent in patients who experienced clinical benefit. Assessment of peripheral blood revealed that non-responders had elevated baseline levels of CXCL8 and CCL4, and a higher proportion of circulating late differentiated B cells. Pre-existing high levels of chemokines (CCL3, CCL4 and CXCL8) and advanced B cell differentiation were strongly associated with worse patient overall survival. Elevated proportions of circulating CD8+/PD-1+ T cells during treatment were associated with worse survival. CONCLUSIONS: The combination of ipilimumab and CP was well tolerated and revealed novel characteristics associated with patients likely to benefit from treatment. A pre-existing systemic inflammatory state characterized by elevation of selected chemokines and advanced B cell differentiation, was strongly associated with poor patient outcomes, revealing potential predictive circulating biomarkers. TRIAL REGISTRATION: Clinicaltrials.gov , NCT01676649 , registered on August 29, 2012