Étude des voies de mort cellulaire induites par les rayons UVB dans les fibroblastes de derme humain

Les rayons ultraviolets (UVR) font partie du spectre solaire et sont classés selon leur longueur d'onde en UVA (315 nm-400 nm), UVB (280 nm-315 nm) et UVC (100 nm-280 nm). Les UVA et une portion des UVB traversent la couche d'ozone et atteignent la surface terrestre, affectant la peau et les yeux. La peau se compose de l'épiderme, du derme et de l'hypoderme. Les UVR peuvent causer des dommages cellulaires et des dommages directs à l'ADN sous forme de dimères de pyrimidines (CPD et 6-4PP). Les dommages à l'ADN peuvent mener à la formation de mutations impliquées dans l'initiation et la progression des cancers de peau. La réparation des dommages à l'ADN couplée à l'arrêt du cycle cellulaire, ainsi que la mort cellulaire programmée sont des mécanismes de protection contre la transformation tumorale. En effet, la mort cellulaire régulée (RCD), permet de supprimer les cellules endommagées. Il existe de nombreuses voies de RCD, parmi lesquelles se trouve l'apoptose, la nécroptose, la ferroptose et la parthanatos. L'apoptose est connue pour être activée par les UVR, tandis que peu d'études examinent les voies non-apoptotiques initiées par les UVR. Cette thèse s'intéresse à l'étude des voies de RCD activées par les UVB dans les fibroblastes de derme humain (NHDF). En premier lieu, l'activation potentielle des voies non-apoptotiques de nécroptose, ferroptose et parthanatos par les UVB, a été étudiée, en plus de l'apoptose. L'investigation de ces voies a été réalisée à l'aide d'inhibiteurs pharmacologiques de la nécroptose, ferroptose, parthanatose et de l'apoptose, suite à une irradiation létale d'UVB. Nos résultats démontrent que seule l'apoptose est mesurable dans les NHDF exposés aux UVB, tandis que la nécroptose, ferroptose et parthanatos ne sont pas activées par les UVB, et ce, même suite à l'inhibition de l'apoptose. Ainsi, l'apoptose est la réponse principale des NHDF à une exposition létale d'UVB. En parallèle de la mort cellulaire, une déplétion du pool de NAD+ est observée dans les NHDF, dû en partie à l'activation de la protéine de parylation PARP1. Par la suite, la participation des protéines RIPK3 et MLKL dans la mort UV-induite a été étudiée. En effet, une précédente étude a mis en évidence une augmentation de la transcription de RIPK3 à la suite d'irradiations chroniques d'UVB, faisant de RIPK3 une protéine d'intérêt. RIPK3, sous forme phosphorylée, est connue pour être impliquée dans la nécroptose au côté de la protéine MLKL. Dans cette partie, la contribution de RIPK3 et de MLKL dans la mort UVB-induite des NHDF a été mise en évidence par déplétion partielle des protéines cibles. L'utilisation d'inhibiteurs de la nécroptose et l'étude des niveaux de phosphorylation de RIPK3 et MLKL démontre que cette implication est indépendante de leur activité nécroptotique. RIPK3 protège les NHDF de la mort UV-induite par un mécanisme indirect à l'apoptose, tandis que MLKL sensibilise les cellules à l'apoptose UV-induite. Enfin, le régime d'irradiation, soit l'exposition des cellules à une irradiation unique ou à des irradiations chroniques d'UVB, peut influencer la réponse cellulaire. En effet, la réparation des dommages à l'ADN des NHDF a été étudiée au sein du laboratoire et est modifiée selon le régime d'irradiation. Le rôle du régime d'irradiation, unique et chronique, sur la mort cellulaire induite par les rayons UVB dans les NHDF a donc aussi été étudiée. Pour ce faire, les cellules ont été prétraitées ou non par de faibles irradiations chroniques d'UVB (CLUV) puis ont été exposées à une dose létale d'UVB. Les résultats montrent que le régime d'irradiation chronique ne modifie pas la voie de mort activée par les UVB, qui reste apoptotique, et n'entraine pas de changement dans la proportion de cellules en voie de mort. Ainsi, la réponse de mort cellulaire des NHDF n'est pas modifiée par le régime d'irradiation UVB, contrairement à la réparation de l'ADN. Cette thèse établie que l'exposition des NHDF à une dose létale d'UVB induit l'activation de l'apoptose, sans activation des voies non-apoptotiques (nécroptose, ferroptose, parthanatos), et ce même suite à un régime d'irradiation CLUV. De plus, cette étude révèle deux nouveaux acteurs de la mort UV-induite des NHDF : RIPK3 et MLKL. Cette thèse présente des éléments nouveaux qui s'ajoutent à l'ensemble des connaissances et permettent une compréhension plus complète de la réponse cellulaire au stress génotoxique UVB.Ultraviolet radiation (UVR) is part of the solar spectra. UVR are composed of UVA (315 nm-400 nm), UVB (280 nm-315 nm) and UVC (100 nm-280 nm). UVA and part of UVB reach the earth and affect our skin and eyes. Skin comprises epidermis, dermis and hypodermis layers. UVA and UVB lead to both cellular damage and direct DNA damage, mainly dipyrimidine dimers (CPD and 6-4PP). UV-induced DNA damage can be converted into skin cancer-driver mutations. DNA damage repair mechanisms and regulated cell death (RCD) are two mechanisms protecting cells from tumoral transformation. RCD, such as apoptosis, necroptosis, ferroptosis and parthanatos, remove damaged cells. UVR can induce apoptosis in skin cells, while little is known about non-apoptotic cell death following UVR. This thesis focuses on RCD pathways induced by UVB in normal human dermal fibroblasts (NHDF). First, possible activation of necroptosis, ferroptosis and parthanatos by UVB was investigated, as well as apoptosis, by use of pharmacologic inhibitors. Our results show that only apoptosis can be measured after UVB irradiation in NHDF, while no necroptosis, ferroptosis or parthanatos could be measured, with or without apoptosis inhibition. Apoptosis is thus a key response in NHDF exposed to a lethal UVB dose. Simultaneously with cell death, a drastic decrease in NAD+ was observed in irradiated NHDF, partly due to the activation of PARP1 polymerase. Secondly, the involvement of RIPK3 and MLKL proteins in UV-induced death was investigated. A previous study demonstrated an increase in RIPK3 transcription following chronic UVB irradiations, showing a potential involvement of RIPK3 in UV-induced cell response. Phosphorylated RIPK3 is known to be involved in necroptosis alongside phosphorylated MLKL. Using RIPK3 and MLKL knockdown, our results demonstrate RIPK3 and MLKL involvement in UVB-induced cell death. Use of necroptosis inhibitors plus the study of RIPK3 and MLKL phosphorylation confirmed that their roles are independent of their necroptotic activities. In fact, RIPK3 protects NHDF from UVB-induced cell death by a non-apoptotic mechanism, while MLKL sensitizes cells to UVB-induced apoptosis. Finally, irradiation regime, i.e. chronic irradiations versus unique irradiation, can influence the cellular response. Indeed, DNA damage repair has been studied in the laboratory and is modified by the irradiation regime in NHDF. Thus, we investigated the effect of chronic irradiations on UVB-induced cell death in NHDF. Cells were pre-treated or not with low chronic UVB doses (CLUV) and then exposed to a lethal UVB dose. The results show that with or without CLUV pre-treatment, UVB induced apoptosis with the same proportion of dying cells. Thus, NHDF cell death response is not altered by chronic expositions to UVB, unlike DNA repair. This thesis establishes that NHDF exposure to a lethal UVB dose induces the activation of apoptosis, without activation of non-apoptotic cell death pathways (necroptosis, ferroptosis, parthanatos), even after chronic irradiations (CLUV). In addition, two new actors of UVB-induced cell death, namely RIPK3 and MLKL, are presented here. This thesis presents new elements that help better understand the global cellular response to UVB genotoxic stress

Expression screening using a Medaka cDNA library identifies evolutionarily conserved regulators of the p53/Mdm2 pathway

Background: The p53 tumor suppressor protein is mainly regulated by alterations in the half-life of the protein, resulting in significant differences in p53 protein levels in cells. The major regulator of this process is Mdm2, which ubiquitinates p53 and targets it for proteasomal degradation. This process can be enhanced or reduced by proteins that associate with p53 or Mdm2 and several proteins have been identified with such an activity. Furthermore, additional ubiquitin ligases for p53 have been identified in recent years. Nevertheless, our understanding of how p53 abundance and Mdm2 activity are regulated remains incomplete. Here we describe a cell culture based overexpression screen to identify evolutionarily conserved regulators of the p53/Mdm2 circuit. The results from this large-scale screening method will contribute to a better understanding of the regulation of these important proteins. Methods: Expression screening was based on co-transfection of H1299 cells with pools of cDNA’s from a Medaka library together with p53, Mdm2 and, as internal control, Ror2. After cell lysis, SDS-PAGE/WB analysis was used to detect alterations in these proteins. Results: More than one hundred hits that altered the abundance of either p53, Mdm2, or both were identified in the primary screen. Subscreening of the library pools that were identified in the primary screen identified several potential novel regulators of p53 and/or Mdm2. We also tested whether the human orthologues of the Medaka genes regulate p53 and/or Mdm2 abundance. All human orthologues regulated p53 and/or Mdm2 abundance in the same manner as the proteins from Medaka, which underscores the suitability of this screening methodology for the identification of new modifiers of p53 and Mdm2. Conclusions: Despite enormous efforts in the last two decades, many unknown regulators for p53 and Mdm2 abundance are predicted to exist. This cross-species approach to identify evolutionarily conserved regulators demonstrates that our Medaka unigene cDNA library represents a powerful tool to screen for these novel regulators of the p53/Mdm2 pathway

Analysis of HIV Protease Killing Through Caspase 8 Reveals a Novel Interaction Between Caspase 8 and Mitochondria

Human Immunodeficiency Virus (HIV) protease initiates apoptosis of HIV-infected cells by proteolytic cleavage of procaspase 8, creating a novel peptide termed casp8p41. Expression of casp8p41 alone is sufficient to initiate caspase-dependent cell death associated with mitochondrial depolarization. Since casp8p41 does not contain the catalytic cysteine at position 360, the mechanism by which casp8p41 initiates apoptosis is unclear. We demonstrate that casp8p41 directly causes mitochondrial depolarization and release of cytochrome c with downstream caspase 9 activation. Moreover, death induced by casp8p41 requires the presence of mitochondria, and in intact cells, casp8p41 colocalizes with mitochondria. These results illuminate a novel mechanism of cell death induced by a caspase 8 cleavage fragment whereby mitochondrial interaction leads to depolarization and cytochrome c release

Encephalopathy induced by Alzheimer brain inoculation in a non-human primate.

Alzheimer's disease is characterized by cognitive alterations, cerebral atrophy and neuropathological lesions including neuronal loss, accumulation of misfolded and aggregated β-amyloid peptides (Aβ) and tau proteins. Iatrogenic induction of Aβ is suspected in patients exposed to pituitary-derived hormones, dural grafts, or surgical instruments, presumably contaminated with Aβ. Induction of Aβ and tau lesions has been demonstrated in transgenic mice after contamination with Alzheimer's disease brain homogenates, with very limited functional consequences. Unlike rodents, primates naturally express Aβ or tau under normal conditions and attempts to transmit Alzheimer pathology to primates have been made for decades. However, none of earlier studies performed any detailed functional assessments. For the first time we demonstrate long term memory and learning impairments in a non-human primate (Microcebus murinus) following intracerebral injections with Alzheimer human brain extracts. Animals inoculated with Alzheimer brain homogenates displayed progressive cognitive impairments (clinical tests assessing cognitive and motor functions), modifications of neuronal activity (detected by electroencephalography), widespread and progressive cerebral atrophy (in vivo MRI assessing cerebral volume loss using automated voxel-based analysis), neuronal loss in the hippocampus and entorhinal cortex (post mortem stereology). They displayed parenchymal and vascular Aβ depositions and tau lesions for some of them, in regions close to the inoculation sites. Although these lesions were sparse, they were never detected in control animals. Tau-positive animals had the lowest performances in a memory task and displayed the greatest neuronal loss. Our study is timely and important as it is the first one to highlight neuronal and clinical dysfunction following inoculation of Alzheimer's disease brain homogenates in a primate. Clinical signs in a chronic disease such as Alzheimer take a long time to be detectable. Documentation of clinical deterioration and/or dysfunction following intracerebral inoculations with Alzheimer human brain extracts could lead to important new insights about Alzheimer initiation processes

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Abstracts from the NIHR INVOLVE Conference 2017

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Fc Effector Function Contributes to the Activity of Human Anti-CTLA-4 Antibodies.

With the use of a mouse model expressing human Fc-gamma receptors (FcγRs), we demonstrated that antibodies with isotypes equivalent to ipilimumab and tremelimumab mediate intra-tumoral regulatory T (Treg) cell depletion in vivo, increasing the CD8+ to Treg cell ratio and promoting tumor rejection. Antibodies with improved FcγR binding profiles drove superior anti-tumor responses and survival. In patients with advanced melanoma, response to ipilimumab was associated with the CD16a-V158F high affinity polymorphism. Such activity only appeared relevant in the context of inflamed tumors, explaining the modest response rates observed in the clinical setting. Our data suggest that the activity of anti-CTLA-4 in inflamed tumors may be improved through enhancement of FcγR binding, whereas poorly infiltrated tumors will likely require combination approaches