Gilbert Bernier

BMI1 Sustains Human Glioblastoma Multiforme Stem Cell Renewal

Glioblastoma multiforme (GBM) is one of the most common and aggressive types of brain tumors. In GBM, a subpopulation of CD133-positive cancer initiating cells displays stem cell characteristics. The Polycomb group (PcG) and oncogene BMI1 is part of the Polycomb repressive complex 1 (PRC1) that regulates gene expression by modifying chromatin organization. Here we show that BMI1 is expressed in human GBM tumors and highly enriched in CD133-positive cells. Stable BMI1 knockdown using short hairpin RNA-expressing lentiviruses resulted in inhibition of clonogenic potential in vitro and of brain tumor formation in vivo. Cell biology studies support the notion that BMI1 prevents CD133-positive cell apoptosis and/or differentiation into neurons and astrocytes, depending on the cellular context. Gene expression analyses suggest that BMI1 represses alternate tumor suppressor pathways that attempt to compensate for INK4A/ARF/P53 deletion and PI(3)K/AKT hyperactivity. Inhibition of EZH2, the main component of the PRC2, also impaired GBM tumor growth. Our results reveal that PcG proteins are involved in GBM tumor growth and required to sustain cancer initiating stem cell renewal.

BMI1 Confers Radioresistance to Normal and Cancerous Neural Stem Cells through Recruitment of the DNA Damage Response Machinery

Glioblastoma multiforme (GBM) is an aggressive brain tumor that is resistant to all known therapies. Within these tumors, a CD133-positive cancer-initiating neural stem cell (NSC) population was shown to be resistant to gamma radiation through preferential activation of the DNA double-strand break (DSB) response machinery, including the ataxia-telangiectasia-mutated (ATM) kinase. The polycomb group protein BMI1 is enriched in CD133-positive GBM cells and required for their self-renewal in an INK4A/ARF-independent manner through transcriptional repression of alternate tumor suppressor pathways. We report here that BMI1 copurifies with DNA DSB response and nonhomologous end joining (NHEJ) repair proteins in GBM cells. BMI1 was enriched at the chromatin after irradiation and colocalized and copurified with ATM and the histone γH2AX. BMI1 also preferentially copurified with NHEJ proteins DNA-PK, PARP-1, hnRNP U, and histone H1 in CD133-positive GBM cells. BMI1 deficiency in GBM cells severely impaired DNA DSB response, resulting in increased sensitivity to radiation. In turn, BMI1 overexpression in normal NSCs enhanced ATM recruitment to the chromatin, the rate of γH2AX foci resolution, and resistance to radiation. BMI1 thus displays a previously uncharacterized function in controlling DNA DSB response and repair. Pharmacological inhibition of BMI1 combined with radiation therapy may provide an effective mean to target GBM stem cells.

The LIM homeobox transcription factor Lhx2 is required to specify the retina field and synergistically cooperates with Pax6 for Six6 trans-activation.

In mammals, a limited set of homeobox-containing transcription factors are expressed in the presumptive eye field and required to initiate eye development. How these factors interact together at the genetic and molecular level to coordinate this developmental process is poorly understood. We found that the Lhx2 and Pax6 transcription factors operate in a concerted manner during retinal development to promote transcriptional activation of the Six6 homeobox-gene in primitive and mature retinal progenitors. Lhx2 demarcates the presumptive retina field at the neural plate stage and Lhx2 inactivation delays initiation of Rx, Six3 and Pax6 expression in this domain. The later expressed Six6 is properly activated in the pituitary/hypothalamic axis of Lhx2(-/-) embryos, but expression fails to be initiated in the optic vesicle. Lhx2 and Pax6 associate with the chromatin at several regions of Six6 in vivo and cooperate for trans-activation of Six6 regulatory elements in vitro. In retinal progenitor/stem cells, both Lhx2 and Pax6 are genetically required for proper Six6 expression and forced co-expression of Lhx2 and Pax6 can synergistically trans-activate the Six6 locus. Our work reveals how two master regulators of eye development coordinate their action to sequentially promote tissue-specific transcriptional initiation and full activation of a retinal determinant gene.

Brain Cancer Stem Cells: Current Status on Glioblastoma Multiforme

Glioblastoma multiforme (GBM), an aggressive brain tumor of astrocytic/neural stem cell origin, represents one of the most incurable cancers. GBM tumors are highly heterogeneous. However, most tumors contain a subpopulation of cells that display neural stem cell characteristics in vitro and that can generate a new brain tumor upon transplantation in mice. Hence, previously identified molecular pathways regulating neural stem cell biology were found to represent the cornerstone of GBM stem cell self-renewal mechanism. GBM tumors are also notorious for their resistance to radiation therapy. Notably, GBM “cancer stem cells” were also found to be responsible for this radioresistance. Herein, we will analyze the data supporting or not the cancer stem cell model in GBM, overview the current knowledge regarding GBM stem cell self-renewal and radioresistance molecular mechanisms, and discuss the potential therapeutic application of these findings.

ASPP1/2 Regulate p53-Dependent Death of Retinal Ganglion Cells through PUMA and Fas/CD95 Activation In Vivo

The transcription factor p53 mediates neuronal death in a variety of stress-related and neurodegenerative conditions. The proapoptotic activity of p53 is tightly regulated by the apoptosis-stimulating proteins of p53 (ASPP) family members: ASPP1 and ASPP2. However, whether ASPP1/2 play a role in the regulation of p53-dependent neuronal death in the CNS is currently unknown. To address this, we asked whether ASPP1/2 contribute to the death of retinal ganglion cells (RGCs) using in vivo models of acute optic nerve damage in mice and rats. Here, we show that p53 is activated in RGCs soon after injury and that axotomy-induced RGC death is attenuated in p53 heterozygote and null mice. We demonstrate that ASPP1/2 proteins are abundantly expressed by injured RGCs, and that short interfering (si)RNA-based ASPP1 or ASPP2 knockdown promotes robust RGC survival. Comparative gene expression analysis revealed that siASPP-mediated downregulation of p53-upregulated-modulator-of-apoptosis (PUMA), Fas/CD95, and Noxa depends on p53 transcriptional activity. Furthermore, siRNA against PUMA or Fas/CD95 confers neuroprotection, demonstrating a functional role for these p53 targets in RGC death. Our study demonstrates a novel role for ASPP1 and ASPP2 in the death of RGCs and provides evidence that blockade of the ASPP–p53 pathway is beneficial for central neuron survival after axonal injury.

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.

p53 Pro-Oxidant Activity in the Central Nervous System: Implication in Aging and Neurodegenerative Diseases

Recent advances in delineating the biological functions of p53 had shed the light on its key role in the multifacets of cellular homeostasis. After its activation, via DNA damage, oxidative stress, or aberrant expression of oncogenes, p53 transduces its classical effect through several mechanisms comprising activation of the DNA repair machinery, cell cycle arrest, and initiation of apoptosis or senescence. In the mammalian brain, p53 plays critical functions in normal development, tumor suppression, neurodegenerative diseases, and aging. Herein, we focus on the constitutive pro-oxidant activity of p53 in neurons and discuss the potential implication of this finding in the context of neurodegenerative diseases and normal brain aging.

Bmi1 Distinguishes Immature Retinal Progenitor/Stem Cells from the Main Progenitor Cell Population and Is Required for Normal Retinal Development

The developing mammalian retina is generated by the proliferation and differentiation of multipotent retinal progenitor cells (RPCs) giving rise to neuronal and glial lineages. Whether an immature progenitor/stem cell subpopulation is present in the developing mammalian retina remains undefined. Deficiency in the polycomb group gene Bmi1 results in reduced proliferation and postnatal depletion of neural and hematopoietic stem cells. Here, we show that Bmi1 is required for the self‐renewal of most immature RPCs and for postnatal retinal development. In the embryo, Bmi1 is highly enriched in a rare stage‐specific embryonic antigen‐1‐positive RPC subpopulation expressing the stem cell markers Sox2, Lhx2, and Musashi. Gain‐of‐function experiments revealed that Bmi1 overexpression could convert RPCs having limited proliferation capacity into RPCs showing extensive proliferation and multiple differentiation capacities over time. At all developmental stages analyzed using the neurosphere assay, Bmi1 deficiency resulted in reduced proliferation and self‐renewal of most immature RPCs. Reduced RPCs proliferation was also observed in the peripheral retina of Bmi1−/− fetus and newborn mice. The biological impact of these developmental anomalies was revealed by the reduced retinal diameter of Bmi1‐deficient pups. P19Arf and p16Ink4a were upregulated in vivo and in vitro and coinactivation of p53, which lies downstream of p19Arf, partially restored Bmi1‐deficient RPCs self‐renewal phenotype. Bmi1 thus distinguishes immature RPCs from the main RPC population and is required for normal retinal development. STEM CELLS 2010;28:1412–1423

Loss of Bmi1 causes anomalies in retinal development and degeneration of cone photoreceptors.

ABSTRACT Retinal development occurs through the sequential but overlapping generation of six types of neuronal cells and one glial cell type. Of these, rod and cone photoreceptors represent the functional unit of light detection and phototransduction and are frequently affected in retinal degenerative diseases. During mouse development, the Polycomb group protein Bmi1 is expressed in immature retinal progenitors and differentiated retinal neurons, including cones. We show here that Bmi1 is required to prevent post natal degeneration of cone photoreceptors and bipolar neurons and that inactivation of Chk2 or p53 could improve but not overcome cone degeneration in Bmi1−/− mice. The retinal phenotype of Bmi1−/− mice was also characterized by loss of heterochromatin, activation of tandem repeats, oxidative stress and Rip3-associated necroptosis. In the human retina, BMI1 was preferentially expressed in cones at heterochromatic foci. BMI1 inactivation in human embryonic stem cells was compatible with retinal induction but impaired cone terminal differentiation. Despite this developmental arrest, BMI1-deficient cones recapitulated several anomalies observed in Bmi1−/− photoreceptors, such as loss of heterochromatin, activation of tandem repeats and induction of p53, revealing partly conserved biological functions between mouse and man. Summary: Bmi1 is required to prevent postnatal degeneration of cone photoreceptors and bipolar neurons, while BMI1 inactivation in human embryonic stem cells impairs cone terminal differentiation.

The type II transmembrane serine protease matriptase cleaves the amyloid precursor protein and reduces its processing to β amyloid

Recent studies have reported that many proteases, besides the canonical α-, β-, and γ-secretases, cleave the amyloid precursor protein (APP) and modulate β-amyloid (Aβ) peptide production. Moreover, specific APP isoforms contain Kunitz protease-inhibitory domains, which regulate the proteolytic activity of serine proteases. This prompted us to investigate the role of matriptase, a member of the type II transmembrane serine protease family, in APP processing. Using quantitative RT-PCR, we detected matriptase mRNA in several regions of the human brain with an enrichment in neurons. RNA sequencing data of human dorsolateral prefrontal cortex revealed relatively high levels of matriptase RNA in young individuals, whereas lower levels were detected in older individuals. We further demonstrate that matriptase and APP directly interact with each other and that matriptase cleaves APP at a specific arginine residue (Arg-102) both in vitro and in cells. Site-directed (Arg-to-Ala) mutagenesis of this cleavage site abolished matriptase-mediated APP processing. Moreover, we observed that a soluble, shed matriptase form cleaves endogenous APP in SH-SY5Y cells and that this cleavage significantly reduces APP processing to Aβ40. In summary, this study identifies matriptase as an APP-cleaving enzyme, an activity that could have important consequences for the abundance of Aβ and in Alzheimers disease pathology.