Étude moléculaire de la fonction du gène Bmi1 dans le processus de sénescence du système nerveux

Des études présentées dans cette thèse ont permis de démontrer que le gène du groupe Polycomb (PcG) Bmi1 est essentiel à l’auto-renouvellement des progéniteurs rétiniens immatures et pour le développement rétinien après la naissance. Ce travail illustre chez l’embryon que Bmi1 est hautement enrichie dans une sous-population de progéniteurs rétiniens exprimant le marqueur de surface SSEA-1 et différents marqueurs de cellules souches. À tous les stades de développement analysés, l’absence de Bmi1 résulte en une diminution de la prolifération et de l’auto-renouvellement des progéniteurs immatures. Pour mieux comprendre la cascade moléculaire en absence de Bmi1, nous avons inactivé p53 dans les colonies Bmi1-/-. Cette inactivation a permis une restauration partielle du potentiel d’auto-renouvellement. De plus, en absence de Bmi1, la prolifération et la maintenance de la population de progéniteurs rétiniens immatures localisés dans le corps ciliaire sont aussi affectées après la naissance. Bmi1 permet donc de distinguer les progéniteurs immatures de la population principale de progéniteurs, et est requis pour le développement normal de la rétine. Nous avons également démontré que l’oncogène Bmi1 est requis dans les neurones pour empêcher l’apoptose et l’induction d’un programme de vieillissement prématuré, causé par une baisse des défenses anti-oxydantes. Nous avons observé dans les neurones Bmi1-/- une augmentation des niveaux de p53, de la concentration des ROS et de la sensibilité aux agents neurotoxiques. Nous avons démontré ainsi que Bmi1 contrôle les défenses anti-oxydantes dans les neurones en réprimant l’activité pro-oxydante de p53. Dans les neurones Bmi1-/-, p53 provoque la répression des gènes anti-oxydants, induisant une augmentation des niveaux de ROS. Ces résultats démontrent pour la première fois que Bmi1 joue un rôle critique dans la survie et le processus de vieillissement neuronal.The studies presented in this thesis establish that the Polycomb Group (PcG) gene Bmi1 is required for the self-renewal of immature retinal progenitor cells (RPCs) and for postnatal retinal development. Work performed in mouse embryos reveals that Bmi1 is highly enriched in a RPC subpopulation expressing the cell surface antigen SSEA-1 and different stem cell markers. Furthermore, at all developmental stages analysed, Bmi1 deficiency resulted in reduced proliferation and self-renewal of immature RPCs. To better understand the molecular cascade leading to this phenotype, we inactivated p53 in Bmi1-deficient colonies. p53 inactivation partially restored RPCs self-renewal potential. Moreover, the proliferation and the postnatal maintenance of an immature RPC population located in the ciliary body was also impaired in absence of Bmi1. Thus, Bmi1 distinguishes immature RPCs from the main RPC population and is required for normal retinal development. We have also shown that the oncogene Bmi1 is required in neurons to prevent apoptosis and the induction of a premature aging-like program characterized by reduced antioxidant defenses. We observed in Bmi1-deficient neurons an increased p53 and ROS levels, and a hypersensitivity to neurotoxic agents. We demonstrated that Bmi1 regulate antioxidant defenses in neurons by suppressing p53 pro-oxidant activity. In Bmi1-/- neurons, p53 induces antioxidant genes repression, resulting in increased ROS levels. These findings reveal for the first time the major role of Bmi1 on neuronal survival and aging

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

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

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 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

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