Добірка наукової літератури з теми "CHCHD10"

CHCHD2 mutations have been identified in various neurological diseases such as Parkinson’s disease (PD), frontotemporal dementia (FTD), and Alzheimer’s disease (AD). It is also the first mitochondrial gene whose mutations lead to PD. CHCHD10 is a homolog of CHCHD2; similar to CHCHD2, various mutations of CHCHD10 have been identified in a broad spectrum of neurological disorders, including FTD and AD, with a high frequency of CHCHD10 mutations found in motor neuron diseases. Functionally, CHCHD2 and CHCHD10 have been demonstrated to interact with each other in mitochondria. Recent studies link the biological functions of CHCHD2 to the MICOS complex (mitochondrial inner membrane organizing system). Multiple experimental models suggest that CHCHD2 maintains mitochondrial cristae and disease-associated CHCHD2 mutations function in a loss-of-function manner. However, both CHCHD2 and CHCHD10 knockout mouse models appear phenotypically normal, with no obvious mitochondrial defects. Strategies to maintain or enhance mitochondria cristae could provide opportunities to correct the associated cellular defects in disease state and unravel potential novel targets for CHCHD2-linked neurological conditions.

Coiled-coil-helix-coiled-coil-helix domain-containing 10 (CHCHD10) is a nuclear-encoded mitochondrial protein which is primarily mutated in the spectrum of familial and sporadic amyotrophic lateral sclerosis (ALS)–frontotemporal dementia (FTD). Endogenous CHCHD10 levels decline in the brains of ALS–FTD patients, and the CHCHD10S59L mutation in Drosophila induces dominant toxicity together with PTEN-induced kinase 1 (PINK1), a protein critical for the induction of mitophagy. However, whether and how CHCHD10 variants regulate mitophagy flux in the mammalian brain is unknown. Here, we demonstrate through in vivo and in vitro models, as well as human FTD brain tissue, that ALS/FTD-linked CHCHD10 mutations (R15L and S59L) impair mitophagy flux and mitochondrial Parkin recruitment, whereas wild-type CHCHD10 (CHCHD10WT) normally enhances these measures. Specifically, we show that CHCHD10R15L and CHCHD10S59L mutations reduce PINK1 levels by increasing PARL activity, whereas CHCHD10WT produces the opposite results through its stronger interaction with PARL, suppressing its activity. Importantly, we also demonstrate that FTD brains with TAR DNA-binding protein-43 (TDP-43) pathology demonstrate disruption of the PARL–PINK1 pathway and that experimentally impairing mitophagy promotes TDP-43 aggregation. Thus, we provide herein new insights into the regulation of mitophagy and TDP-43 aggregation in the mammalian brain through the CHCHD10–PARL–PINK1 pathway.

Mutations of coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) and 10 (CHCHD10) have been found to be linked to Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and/or frontotemporal lobe dementia (FTD). CHCHD2 and CHCHD10 proteins, which are homologous proteins with 54% identity in amino acid sequence, belong to the mitochondrial coiled-coil-helix-coiled-coil-helix (CHCH) domain protein family. A series of studies reveals that these twin proteins form a multimodal complex, producing a variety of pathophysiology by the disease-causing variants of these proteins. In this review, we summarize the present knowledge about the physiological and pathological roles of twin proteins, CHCHD2 and CHCHD10, in neurodegenerative diseases.

OBJECTIVES/GOALS: Understanding the mechanism of transcriptional adaptation may contribute to an explanation for variation in clinical manifestations of Amyotrophic lateral sclerosis patient phenotypes. METHODS/STUDY POPULATION: To examine transcriptional adaptation, we utilized gene editing tools in HT1080 cells and patient samples with known CHCHD10 mutations causative for Amyotrophic lateral sclerosis. Frameshift mutations were performed via CRISPR-Cas9. Ribonucleoprotein electroporation was used to transfect cells and DNA sequencing was conducted to validate gene editing. To validate transcriptional adaption, changes in levels of protein and gene expression will be measured via immunoblot and quantification of CHCHD10 and CHCHCD2 from whole cells lysates of the edited cells. RESULTS/ANTICIPATED RESULTS: We anticipate that CHCHD2 transcriptional adaptation can functionally compensate for the locus loss of function of CHCHD10. This mechanism of transcriptional adaptation may contribute to an explanation for variation in clinical manifestations of patient phenotypes. DISCUSSION/SIGNIFICANCE: Our approach would advance discovery science towards by exploring CHCHD10/2 transcriptional adaptation mechanism that can lead to novel therapies for rare Amyotrophic lateral sclerosis, such as CHCHD10-R15L.

OBJECTIVES/GOALS: Rare mutations in CHCHD10 gene are found in 1% of patients with familial Amyotrophic lateral sclerosis (ALS). The overall goal of this study is to utilize induced pluripotent stem cells (iPSCs) as an in vitro model organism for rare ALS variants to evaluate the mechanism of transcription adaptation of CHCHD10/2 as a potential therapeutic. METHODS/STUDY POPULATION: Point mutations on normal iPSCs was performed via Donorguide CRISPR/Cas9. The single stranded RNA/DNA donors contain genetic alterations of CHCHD10: Pro12Ser, Arg15Leu, Pro23Leu, Pro34Ser, Ser59Leu, Gly66Val, Pro80Leu, Tyr92Cys and Gln102His. Ribonucleoprotein electroporation was used to transfect iPSCs and DNA sequencing was used to validate gene editing. To validate transcriptional adaption, changes in levels of protein and gene expression were measured via immunoblot and quantification of CHCHD10 and CHCHCD2 was performed from whole cells lysates of the edited iPSCs. RESULTS/ANTICIPATED RESULTS: We anticipate that CHCHD2 transcriptional adaptation can functionally compensate for the locus loss of function of CHCHD10. This mechanism of transcriptional adaptation may contribute to an explanation for variation in clinical manifestations of patient phenotypes. DISCUSSION/SIGNIFICANCE: This study supplies further evidence for genetic modification as a treatment option for diseases with point mutation causal or enabling mechanisms, including some variants of ALS. Future work will explore the gene-correction from an ALS patient with a known CHCHD10-R15L variant.

OBJECTIVES/GOALS: Transcriptional adaptation is a phenomenon in which a mutation in one gene leads to the genetic compensation of another homogenous gene. Understanding the mechanism of transcriptional adaptation may contribute to an explanation for variation in clinical manifestations of rare Amyotrophic lateral sclerosis patient phenotypes. METHODS/STUDY POPULATION: The presence of a premature termination codon triggers transcriptional activation. Therefore, we utilized CRISPR-Cas9 tool to generate a premature termination codon in CHCHD10 gene in multiple types of cells, including induced pluripotent stem cells derived from patient samples with known CHCHD10 mutations causative for Amyotrophic lateral sclerosis. CRISPR-Cas9 tool was delivered via ribonucleoprotein electroporation and transfect cell’s DNA was sequenced to validate gene editing. To confirm transcriptional adaption, changes in levels of protein and gene expression will be measured via immunoblot and quantification of CHCHD10 and CHCHCD2 from whole cells lysates of the edited cells. RESULTS/ANTICIPATED RESULTS: We anticipate that CHCHD2 transcriptional adaptation can functionally compensate for the locus loss of function of CHCHD10. This mechanism of transcriptional adaptation may contribute to an explanation for variation in clinical manifestations of patient phenotypes. DISCUSSION/SIGNIFICANCE: Our approach would advance discovery science towards by exploring transcriptional adaptation mechanism in humans, which can lead to novel therapies for rare Amyotrophic lateral sclerosis, such as CHCHD10.

ObjectiveTo present the postmortem neuropathologic report of a patient with a CHCHD10 mutation exhibiting an amyotrophic lateral sclerosis (ALS) clinical phenotype.MethodsA 54-year-old man without significant medical history or family history presented with arm weakness, slowly progressed over 19 years to meet the El Escorial criteria for clinically probable ALS with bulbar and respiratory involvement, and was found to have a CHCHD10 p.R15L mutation. Postmortem neuropathologic examination took place including immunohistochemical staining with CHCHD10, and double immunofluorescence combining CHCHD10 with TDP43 and neurofilament was performed and the results were compared with normal controls and sporadic ALS cases.ResultsPostmortem examination of the CHCHD10 mutation carrier showed severe loss of hypoglossal and anterior horn motor neurons, mild corticospinal tract degeneration, and a relative lack of TDP43 immunopathology. CHCHD10 immunohistochemistry for the 3 controls and the 5 sporadic ALS cases showed strong neuronal cytoplasmic and axonal labeling, with the CHCHD10 mutation carrier also having numerous CHCHD10 aggregates within their anterior horns. These aggregates may be related to the CHCHD10 aggregates recently described to cause mitochondrial degeneration and disease in a tissue-selective toxic gain-of-function fashion in a CHCHD10 knock-in mouse model. The CHCHD10 aggregates did not colocalize with TDP43 and were predominantly extracellular on double immunofluorescence labeling with neurofilament.ConclusionsThe neuropathology of CHCHD10 mutated ALS includes predominantly lower motor neuron degeneration, absent TDP43 immunopathology, and aggregates of predominantly extracellular CHCHD10, which do not contain TDP43.

ObjectiveSince the first report of CHCHD10 gene mutations in amyotrophiclateral sclerosis (ALS)/frontotemporaldementia (FTD) patients, genetic variation in CHCHD10 has been inconsistently linked to disease. A pathological assessment of the CHCHD10 protein in patient neuronal tissue also remains to be reported. We sought to characterise the genetic and pathological contribution of CHCHD10 to ALS/FTD in Australia.MethodsWhole-exome and whole-genome sequencing data from 81 familial and 635 sporadic ALS, and 108 sporadic FTD cases, were assessed for genetic variation in CHCHD10. CHCHD10 protein expression was characterised by immunohistochemistry, immunofluorescence and western blotting in control, ALS and/or FTD postmortem tissues and further in a transgenic mouse model of TAR DNA-binding protein 43 (TDP-43) pathology.ResultsNo causal, novel or disease-associated variants in CHCHD10 were identified in Australian ALS and/or FTD patients. In human brain and spinal cord tissues, CHCHD10 was specifically expressed in neurons. A significant decrease in CHCHD10 protein level was observed in ALS patient spinal cord and FTD patient frontal cortex. In a TDP-43 mouse model with a regulatable nuclear localisation signal (rNLS TDP-43 mouse), CHCHD10 protein levels were unaltered at disease onset and early in disease, but were significantly decreased in cortex in mid-stage disease.ConclusionsGenetic variation in CHCHD10 is not a common cause of ALS/FTD in Australia. However, we showed that in humans, CHCHD10 may play a neuron-specific role and a loss of CHCHD10 function may be linked to ALS and/or FTD. Our data from the rNLS TDP-43 transgenic mice suggest that a decrease in CHCHD10 levels is a late event in aberrant TDP-43-induced ALS/FTD pathogenesis.

Abstract The neuromuscular junction (NMJ) is a synapse between motoneurons and skeletal muscles to control motor behavior. Acetylcholine receptors (AChRs) are restricted at the synaptic region for proper neurotransmission. Mutations in the mitochondrial CHCHD10 protein have been identified in multiple neuromuscular disorders; however, the physiological roles of CHCHD10 at NMJs remain elusive. Here, we report that CHCHD10 is highly expressed at the postsynapse of NMJs in skeletal muscles. Muscle conditional knockout CHCHD10 mice showed motor defects, abnormal neuromuscular transmission and NMJ structure. Mechanistically, we found that mitochondrial CHCHD10 is required for ATP production, which facilitates AChR expression and promotes agrin-induced AChR clustering. Importantly, ATP could effectively rescue the reduction of AChR clusters in the CHCHD10-ablated muscles. Our study elucidates a novel physiological role of CHCHD10 at the peripheral synapse. It suggests that mitochondria dysfunction contributes to neuromuscular pathogenesis.

The central role of energy metabolism in cellular activities is becoming widely recognized. However, there are many gaps in our knowledge of the mechanisms by which mitochondria evaluate their status and call upon the nucleus to make adjustments. Recently, a protein family consisting of twin CX9C proteins has been shown to play a role in human pathophysiology. We focus here on two family members, the isoforms CHCHD2 (renamed MNRR1) and CHCHD10. The better studied isoform, MNRR1, has the unusual property of functioning in both the mitochondria and the nucleus and of having a different function in each. In the mitochondria, it functions by binding to cytochromecoxidase (COX), which stimulates respiration. Its binding to COX is promoted by tyrosine-99 phosphorylation, carried out by ABL2 kinase (ARG). In the nucleus, MNRR1 binds to a novel promoter element inCOX4I2and itself, increasing transcription at 4% oxygen. We discuss mutations in both MNRR1 and CHCHD10 found in a number of chronic, mostly neurodegenerative, diseases. Finally, we propose a model of a graded response to hypoxic and oxidative stresses, mediated under different oxygen tensions by CHCHD10, MNRR1, and HIF1, which operate at intermediate and very low oxygen concentrations, respectively.

Les maladies mitochondriales sont des maladies métaboliques caractérisées par une atteinte de la chaîne respiratoire, c’est-à-dire un déficit de production d’énergie sous la forme d’ATP. En 2014, l’équipe du Pr. Paquis-Flucklinger a analysé plusieurs patients d’une même famille atteints de myopathie mitochondriale couplée à une atteinte du motoneurone de type Sclérose Latérale Amyotrophique/Démence Fronto Temporale. L’équipe a découvert par séquençage d’exome la mutation p.Ser59Leu dans le gène CHCHD10. Ce gène code pour la protéine mitochondriale CHCHD10, dont la fonction était alors inconnue. Notre équipe a pu montrer que la mutation p.Ser59Leu provoque la désorganisation du complexe MICOS, un complexe multi protéique dont le rôle est de maintenir l’intégrité des crêtes mitochondriales, provoquant la myopathie mitochondriale et l’atteinte du motoneurone. Il s’agissait de la première évidence d’un lien de causalité entre une atteinte mitochondriale et la mort des motoneurones. La Sclérose Latérale Amyotrophique est une maladie neurodégénérative terrible qui provoque la mort des motoneurones des patients. Ceci provoque une paralysie progressive des muscles du corps, entrainant la mort du patient entre deux et trois ans après le début des symptômes, lorsque la paralysie atteint le système respiratoire. Aucun traitement curatif n’existe aujourd’hui. C’est ce qui nous a amené à commencer ce projet de thèse : essayer de trouver une molécule pouvant améliorer les symptômes dus à la mutation p.Ser59Leu et qui pourrait devenir un médicament contre cette maladie.Pour cela nous avons réalisé un crible de deux banques de molécules repositionnables, la banques Prestwick et la banque Selleckchem. Nous avons opté pour des molécules repositionnables dans le but d’écourter au maximum le temps de mise sur le marché d’une potentielle molécule efficace. Dans un premier temps, nous avons réalisé un crible haut débit sur un mutant de levure S.cerevisiae reproduisant la désorganisation du complexe MICOS. Ce premier crible nous a permis de tester plus de 1500 molécules et de trouver deux molécules efficaces sur ce modèle. Après les premiers tests toxicologiques effectués sur des cellules humaines, nous nous sommes concentrés sur l’une d’entre elle, le Nifuroxazide. Nous avons pu montrer que le Nifuroxazide permet d’améliorer les phénotypes délétères liés à la mutation p.Ser59Leu dans des fibroblastes de patients mais également dans des motoneurones issus d’IPSCs porteurs de la mutation p.Ser59Leu. Le Nifuroxazide permet notamment de retrouver des crêtes mitochondriales normales et d’augmenter la longueur du réseau mitochondrial. Nous avons ensuite essayé de comprendre le mécanisme d’action du Nifuroxazide. Nous avons pu montrer que l’action du Nifuroxazide ne dépend pas de STAT3 mais qu’il permet d’augmenter l’interaction entre Mic60, le composant majeur du complexe MICOS et OPA1, un autre acteur majeur du maintien des crêtes mitochondriales. Les investigations sont toujours en cours sur cet aspect. La prochaine étape est de réaliser un test préclinique chez la souris. Pour cela nous allons utiliser le modèle murin CHCHD10S59L/+ que nous avons caractérisé et qui reproduit les phénotypes observés chez les patients porteurs de la mutation p.Ser59Leu. Nous avons également travaillé à mieux comprendre la cascade moléculaire qui provoque la désorganisation des crêtes mitochondriales. Nous avons montré que les protéines CHCHD10 et SLP2 contribuent à la stabilité du complexe PHB1/PHB2. Nous avons également démontré, en conditions p.Ser59Leu, la formation de complexes SLP2/PHB et la déstabilisation du complexe PHB1/PHB2, ce qui active la protéase OMA1 qui clive alors OPA1 et entraine la désorganisation du complexe MICOS et la perte des crêtes mitochondriales.Cette thèse à permit de mieux comprendre les mécanismes liés à CHCHD10 et de placer les bases pour ce qui, nous espérons, deviendra dans le futur, un traitement contre la SLA/DFT
Mitochondrial diseases are metabolic diseases characterized by a deficit of the respiratory chain, implicating a deficit in the production of energy under the form of ATP. In 2014, Pr. Paquis-Flucklinger’s team analysed several families suffering from mitochondrial myopathy coupled to a motor neuron disorder resembling Amyotrophic Lateral Sclerosis/FrontoTemporal Dementia. By exome sequencing, the team discovered the point mutation p.Ser59Leu in the gene CHCHD10. This gene codes for the mitochondrial protein CHCHD10, which function was unknown. Our team showed that the p.Ser59Leu mutation induces the disorganisation of the MICOS complex, a multiproteic complex which major role is to maintain mitochondrial cristae structure, leading to the mitochondrial myopathy and the motor neuron disease. It was the first evidence of a causal link between a mitochondrial disorder and a motor neuron loss. Amyotrophic Lateral Sclerosis is a terrible disease that provokes the death of motor neurons, inducing a progressive paralysis and patient death, two to three years after onset when the paralysis affects the respiratory system. Today, no curative treatment exists. This led us to start this PhD project: To try to find a molecule able to counter the symptoms due to the p.Ser59Leu mutation in CHCHD10 that could become a drug against the disease. For that, we screened two libraries of molecules containing repurposable drugs, the Prestwick library and the Selleckchem library. We chose this approach to reduce as much as possible the time between a potential discovery and the placing on the market of the new drug. First, we carried out a high throughput screening on an S.cerevisiae mutant that mimics the MICOS complex disassembly. This first screen allowed us to test more than 1500 molecules and to find two efficient molecules in this model. After the first toxicological tests performed on human cells, we decided to focus on one drug: the Nifuroxazide. We have been able to show that Nifuroxazide improves the deleterious phenotypes linked to the p.Ser59Leu mutation in patient fibroblasts but also in motor neurons derived from IPSCs carrying the p.Ser59Leu mutation. Notably, treatment with Nifuroxazide increases the number of normal mitochondrial cristae and elongates the mitochondrial network. We tried to understand the mechanism of action of the Nifuroxazide. We showed that the mechanism of action of the Nifuroxazide does not depend on STAT3 but it increases the interaction between two major actors of the maintaining of mitochondrial cristae structure: Mic60, the main component of the MICOS complex and OPA1. Investigation are still ongoing. The next step will be the pre-clinical analysis in mouse. For that, we will use the mutant murine model CHCHD10S59L/+ that we characterized and that recapitulates the phenotypes observed in patients carrying the p.Ser59Leu mutation. We also worked on the understanding of the molecular cascade that leads to the disorganisation of mitochondrial cristae. We showed that CHCHD10 interacts with SLP2 and that they stabilize the PHB1/PHB2 complex. We demonstrated that in mutant conditions, SLP2/PHB aggregates and the destabilisation of the PHB1/PHB2 complex trigger the activation the protease OMA1 that will cleave OPA1 and provoke the disorganisation of the MICOS complex and the loss of mitochondrial cristae.The work done during this PhD project allowed us to better understand the mechanisms linked to CHCHD10 and to set the bases of what we hope will become, in the future, a new treatment for ALS/FTD linked to the p.Ser59Leu mutation in CHCHD10

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative dévastatrice causée par la dégénérescence progressive des motoneurones (MNs) supérieurs et inférieurs menant à une faiblesse et une atrophie musculaire qui progresse jusqu’à la paralysie. Deux gènes majeurs identifiés chez les patients SLA sont le gène FUS (FUSed in sarcoma), impliqué dans le métabolisme de l’ARN, et CHCHD10, qui joue un rôle dans la stabilité des mitochondries. Ces deux gènes ont été étudiés à travers différents modèles, de petits modèles invertébrés aux biopsies de patients. Cependant, les différents traits phénotypiques observés sont complexes et parfois controversés. L’objectif de cette thèse est de fournir de nouvelles informations sur l’implication de ces deux gènes dans la SLA à travers l’utilisation de nouveaux modèles. Pour étudier les mécanismes pathologiques induits par FUS et CHCHD10, nous avons généré et caractérisé deux nouveaux modèles de poisson-zèbres présentant une mutation non-sens des orthologues de ces gènes, et nous avons mis en évidence différents traits phénotypiques propres à la SLA. Nous avons montré, pour FUS, une espérance de vie réduite, une locomotion altérée, des axones moteurs aberrants, une jonction neuromusculaire (JNM) désorganisée, une altération des muscles et mitochondries, ainsi que des changements moléculaires. Ces résultats montrent que la perte de fonction de fus est responsable de l’apparition de signes pathologiques distaux au niveau de la JNM, indiquant une neuronopathie en « dying-back », dans laquelle les traits pathologiques de la SLA commencent au niveau de la JNM et progressent vers les corps cellulaires des MNs
Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder caused by progressive degeneration of upper and lower motor neurons (MNs), with a very rapid clinical course. It leads to muscle weakness and atrophy progressing to paralysis, with respiratory failure being the major cause of death within years following clinical diagnosis. Two major genes mutated in ALS patients are the RNA-binding protein FUS (FUSed in sarcoma), implicated in RNA metabolism, and coiled-coil-helix-coiled-coil-helix domain 10 (CHCHD10), which plays a role in mitochondria stability. Both these genes have been investigated through different model systems, from small invertebrate models to patient biopsies. However, the major phenotypic features obtained in these models are complex and often controversial. The objective of this work is to provide new insights on the implication of these genes in ALS through the use of new models.To investigate the pathogenic mechanisms induced by FUS and CHCHD10, we generated and characterized two novel stable non-sense mutant zebrafish models for the orthologues of these genes and highlighted several ALS phenotypic features. We demonstrated, for the FUS model but not for CHCHD10, reduced lifespan, locomotor disabilities, aberrant motor axons, disorganized neuromuscular junction (NMJ), muscle and mitochondrial alteration, as well as molecular changes. These findings indicate that loss of fus expression is responsible for the occurrence of distal pathological signs at the NMJ, thus supporting a “dying-back” neuronopathy, in which early disease hallmarks start at the level of the NMJ and progress towards MN cell bodies

La Sclérose Latérale Amyotrophique (SLA) est une maladie neurodégénérative fatale caractérisée par la dégénérescence des motoneurones centraux et périphériques. Elle est le plus souvent sporadique (SALS, 90% des cas), tandis que les formes familiales (FALS) représentent 10% des patients. Une vingtaine de gènes liés à la SLA ont été identifiés et sont responsables de 70% des FALS et 10% des SALS. Le but de ce projet était d’étudier la contribution de 6 gènes rares dans une large cohorte de patients français atteints de SLA et d’étudier les conséquences fonctionnelles de certains variants identifiés. La première partie de ce projet a consisté à réaliser l’analyse génétique des gènes MATR3, CHCHD10, SS18L1, SQSTM1, UBQLN2 et PFN1. Aucun variant causal ne fut identifié pour les 2 premiers gènes alors que 2 nouveaux variants possiblement pathogènes ont été identifiés dans le gène SS18L1, 4 mutations pour SQSTM1, 5 dans UBQLN2 et 2 mutations déjà répertoriées dans le gène PFN1. Cette analyse génétique a permis de souligner un chevauchement génétique entre SLA et maladie de Paget pour SQSTM1 et entre SLA et Paraplégie Spastique pour UBQLN2. La deuxième partie de ce projet a consisté en l’étude de la pathogénicité de certains variants que nous avons identifiés dans les gènes SQSTM1, UBQLN2 et PFN1, par l’analyse (i) des inclusions dans les tissus post mortem de patients, (ii) de l’expression et de la dégradation protéique dans des lymphoblastes issus de patients SLA et/ou (iii) des conséquences cellulaires après surexpression in vitro et in vivo. Ces analyses ont montré, concernant SQSTM1, qui est impliquée dans la formation des autophagosomes, que les mutations perturbaient l’agrégation protéique, que les mutations dans le gène UBQLN2 altéraient la dégradation lysosomale et la liaison de la protéine avec HSP70 et que celles dans PFN1 dérégulaient la voie de l’autophagie alternative et la mitophagie. Notre travail a permis (i) d’évaluer la contribution chez les patients français atteints de SLA de plusieurs gènes incriminés dans la SLA, (i) d’élargir le spectre génétique commun à la SLA et à d’autres pathologies et (iii) de mettre en avant la pertinence de l’implication des voies de dégradation protéique, notamment l’autophagie, dans la pathogénèse de la maladie
The fatal Amyotrophic Lateral Sclerosis (ALS) motor neuron disease is characterized by the degeneration of upper and lower motor neurons. Most ALS cases are sporadic (SALS) whereas ~10% are familial (FALS). A growing number of genes has been identified in ALS and represent 70% of FALS and 10% of SALS. The aims of this project were to analyze the contribution of 6 rare genes in a large population of French ALS patients and to study the pathogenic impact of some identified variants.The first part of this work was dedicated to the genetic analysis of MATR3, CHCHD10, SS18L1, SQSTM1, UBQLN2 and PFN1 genes. No causing variants were identified for MATR3 and CHCHD10 while 2 new variants, probably pathogenic, were identified for SS18L1, as well as 4 mutations for SQSTM1, 5 for UBQLN2 and 2 already reported mutations for PFN1. These analyses also highlighted a genetic overlap between ALS and other diseases: the Paget disease of bone for SQSTM1 and spastic paraplegia for UBQLN2. The second part of this work was to study the pathogenicity of some of the mutations identified in SQSTM1, UBQLN2 and PFN1 genes using analyses of (i) inclusions in ALS patient post-mortem tissue, (ii) protein expression and degradation pathways in patient lymphoblasts and/or (iii) cellular consequences after in vitro and in vivo overexpression. Our results showed prominent aggregation of mutant SQSTM1 (involved in autophagosomes formation), impaired lysosomal degradation and disrupted protein binding to HSP70 for mutant UBQLN2 and deregulated alternative autophagy and mitophagy pathways for mutant PFN1. Our results (i) precised the contribution of several genes in French ALS patients, (i) documented the genetic overlap between ALS and other diseases and (iii) highlighted the role of protein degradation pathways, especially autophagy, in the pathogenesis of ALS

Beside its apoptotic function, the Apoptosis Inducing Factor (AIF), a highly conserved mitochondrial flavoprotein, plays a pro-vital role in eukaryotic cells through its interaction with CHCHD4, a mitochondrial protein that contributes to oxidative folding of respiratory complexes’ subunits. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer complex (CT complex) upon reaction with NAD(H), which leads to protein dimerization modulating the affinity for its ligands. To date, nine point mutations of the human AIF gene were found to cause rare and severe neurodegenerative mitochondriopathies. To define the molecular bases of the pathogenicity of AIF variants, we selected a set of AIF mutations (G337E, D236G, G261S and F133L) and investigated their effects on both AIF molecular properties and its interaction with CHCHD4. To this aim, a combination of biophysical techniques, Microscale Thermophoresis (MST) and structural biology was used. AIF variants CT complex stability was evaluated studying its reoxidation by O2. Interestingly, CT complex of G337E and G261S forms displayed a faster oxidation compared to wild type AIF, indicating a lower stability. Moreover, the 3D structures of the CT complex of AIF-D236G and of AIF-F133L both in oxidized and CT complex state were obtained, showing, however, no significant structural rearrangements with respect to the wild type protein. Hence, we investigated possible alterations of the interaction with CHCHD4 induced by AIF pathogenic mutations. Through a MST approach, for the first time we quantitatively studied redox-dependent effects of AIF amino acid replacements on its affinity for CHCHD4, revealing a possible involvement of the G337 residue in protein-protein complex formation. We characterized the AIF-CHCHD4 complex also from a structural point of view using the Small-Angle X-ray Scattering (SAXS) technique, through which we identified a putative interaction region between the two proteins. In addition, all SAXS models obtained revealed that the solvent-exposed G337 residue is localized in the neighborhood of the identified region, in agreement with a possible role of this residue in complex formation. Our results, not only shed new light on AIF-CHCHD4 relationship, but also provide a possible explanation of the pathogenicity of the AIF G337E allelic variant.

Dans les conditions physiologiques, les mitochondries jouent un rôle fondamental dans la survie, la différentiation et l'état d'activation des cellules, en participant au métabolisme bioénergétique mais aussi à la synthèse des macromolécules, la régulation des voies de signalisation ou le contrôle de l'épigénome. Cet organite est bifonctionnel car son implication est également établie dans la réponse cellulaire au stress ou à des signaux apoptotiques. L'activité mitochondriale est étroitement liée à sa morphologie, qui est contrôlée par un ensemble de protéines impliquées dans le remodelage de son ultrastructure et de sa dynamique fusion/fission. Ces protéines sont cruciales pour l'adaptation de l'activité mitochondriale aux besoins bioénergétique de la cellule. Elles sont également des acteurs clés dans la régulation des processus cellulaires et des voies de signalisation qui nécessitent l'interaction des mitochondries avec d'autres compartiments cellulaires tels que le réticulum endoplasmique.Récemment, une nouvelle classe de protéines façonnant les mitochondries (TRIAP1, CHCHD2, CHCHD3, CHCHD6 et CHCHD10) a été décrite. Ces protéines contiennent un domaine coiled-coil-helix (CHCHD) et sont importées dans l'espace intermembranaire de l'organite grâce à l'activité de la machinerie d'import redox-dépendante Mia40/CHCHD4. Elles représentent des cibles thérapeutiques potentielles car leur expression anormale ou leur activité déficiente est associée à divers types de pathologies humaines telle que les maladies neurodégénératives et le cancer. Au cours de ma thèse je me suis particulièrement intéressée à la protéine TRIAP1 qui est surexprimée dans de nombreux types de cancers. Les expériences d'ARN interférence ou de surexpression de la protéine recombinante, dans un modèle de cancer colorectal, ont montré que l'expression de TRIAP1 favorise la prolifération des cellules et la croissance tumorale. Nos résultats montrent que la déplétion de TRIAP1, altère l'ultrastructure mitochondriale, impacte le profil métabolomique et lipidomique des cellules et engendre une signalisation rétrograde vers le noyau qui modifie le programme d'expression génique. Par ailleurs, nos résultats montrent que la perte de TRIAP1 modifie la réponse des cellules cancéreuses à des conditions de stress métabolique. Dans l'ensemble, nos résultats soulignent l'importance de la protéine TRIAP1 dans la plasticité métabolique des cellules cancéreuses. Une meilleure compréhension des bases moléculaires de l'activité mitochondriale de TRIAP1 dans les cellules cancéreuses permettrait de mieux appréhender l'avantage sélectif qu'apporte sa surexpression aux cellules tumorales
Under physiological conditions, mitochondria play a fundamental role in cell survival, differentiation and activation by participating in bioenergetic metabolism, synthesis of macromolecules, regulation of signaling pathways or control of the epigenome. This organelle is bifunctional as its involvement is also well established in the cellular response to stress or apoptotic signals. The regulation of the mitochondrial activity is closely linked to its morphology, which is controlled by a set of proteins involved in the remodeling of its ultrastructure and fusion/fission dynamics. These proteins are crucial for the adaptation of mitochondrial biogenesis and activity to the bioenergetic needs of the cell. They are also key players in the regulation of cellular processes and signaling pathways that require the interaction of mitochondria with other cellular compartments such as the endoplasmic reticulum.Recently, a new class of mitochondria shaping proteins (TRIAP1, CHCHD2, CHCHD3, CHCHD6 and CHCHD10) was described. These proteins contain a coiled-coil-helix (CHCHD) domain and are imported into the intermembrane space of the organelle through the activity of the redox-dependent Mia40/CHCHD4 import machinery. They represent potential therapeutic targets as their abnormal expression or deficient activity has been associated with various human pathologies such as neurodegenerative diseases and cancer. During my thesis I studied the TRIAP1 protein which is overexpressed in many types of cancers. RNA interference or recombinant protein overexpression experiments , in a colorectal cancer model, showed that TRIAP1 expression promotes cell proliferation and tumor growth. Our results show that TRIAP1 depletion alters mitochondrial ultrastructure, impacts the metabolomic and lipidomic profile of the cells and induces a retrograde signaling to the nucleus that modifies the gene expression program. Furthermore, our results show that loss of TRIAP1 alters the response of cancer cells to metabolic stress conditions. Overall, our results highlight the relevance of TRIAP1 in the metabolic plasticity of cancer cells. A better understanding of the molecular basis of the mitochondrial activity of TRIAP1 in cancer cells should provide a better understanding of the selective advantage that its overexpression provides to tumor cells

Dans la majorité des cas, les mitochondries sont nécessaires à la tumorigenèse et à la réponse des cellules cancéreuses aux signaux générés par les facteurs micro-environnementaux (exemples : privation de nutriments, hypoxie) ou par les traitements anticancéreux (exemples : chimiothérapie, radiothérapie). Presque toutes les protéines mitochondriales sont codées par le génome nucléaire et importées dans l'organelle. Des machineries d'import ont donc évolué afin de répondre aux besoins d'import protéique. Dans ce contexte, la machinerie régulée par CHCHD4/Mia40 fonctionne dans l’espace intermembranaire et contrôle l’import d’un groupe de protéines (substrats) qui joue des rôles importants dans la survie et la réponse au stress. Les substrats de CHCHD4/Mia40 sont impliqués dans un vaste panel d’activités mitochondriales qui inclut la biogenèse des complexes de la chaîne respiratoire, l’homéostasie lipidique, le stockage du calcium, ainsi que l'ultrastructure et la dynamique mitochondriale. Ce programme de thèse a été dédié à l’étude de la voie d’import CHCHD4/Mia40 dans les cellules cancéreuses et a porté un intérêt tout particulier à l'un des substrats CHCHD4/Mia40 qui façonne l'ultrastructure mitochondriale. En utilisant des techniques d’ARN interférence et de sur-expression de protéines recombinantes, dans un modèle de cancer du côlon, nous avons montré que l’expression du substrat étudié a un effet crucial sur la prolifération et la croissance tumorale. Nos données ont également impliqué cette protéine dans la réponse aux traitements anticancéreux. Dans l'ensemble, ces travaux ouvrent un nouveau champ d'investigations qui non seulement permettra de mieux comprendre la plasticité métabolique des cellules cancéreuses, mais aidera également à identifier de nouveaux biomarqueurs métaboliques
In the vast majority of cases, mitochondria are required for tumorigenesis and also for the tumoral response to signals generated by the microenvironmental factors (e.g. nutrient deprivation, hypoxia) or to the effects of anti-cancer treatments (e.g. chemotherapy, radiotherapy). As almost all mitochondrial proteins are nuclear-encoded and imported into the organelle, specialized import machineries have evolved in order to meet the need for protein import. Among these machineries, the one that operates in the intermembrane space and is controlled by CHCHD4/Mia40, regulates the import of a group of proteins (substrates) that play important roles in survival and stress response. Substrates of CHCHD4/Mia40 are involved in a broad panel of mitochondrial activities that includes the biogenesis of respiratory chain complexes, lipid homeostasis, calcium storage, as well as ultrastructure and mitochondrial dynamics. This thesis program was dedicated to the study of the CHCHD4/Mia40 import pathway in cancer cells, with a particular interest for one of the CHCHD4/Mia40 substrates that shapes mitochondrial ultrastructure. Using RNA interference approach and recombinant protein overexpression technique, in a colon cancer model, we showed that the expression of this substrate had a crucial effect on proliferation and tumor growth. Our data also involved this protein in the response to anti-cancer treatments. All together, this work opens a new field of investigations that will not only shed new lights on the metabolic plasticity of cancer cells but also help to identify new metabolic biomarkers

Introduction: Parkinson’s disease - PD is the second most common agerelated neurodegenerative disorder. Characterized by a variety of motor and non-motor symptoms that relate to the loss of dopaminergic neurons in the midbrain black substance. Although most cases of PD are sporadic, 5–10% of patients have monogenetic mutations with a description of more than 20 genes for the familial form. Mitochondrial mutation in CHCHD10 has also been reported to be associated with a wide spectrum of neurodegenerative disorders, including PD. Objectives: Description of a rare recently described genetic cause of autosomal dominant parkinsonism. Methodology: Describe the case of a Brazilian woman with atypical parkinsonism due to CHCHD10 pathogenic variant that was followed up in our service. Result: Female, 64 years old. “. He started episodes of imbalance about 5 years ago, with falls, in addition to limb stiffness, worse on the left. 4 years ago, he started myalgia to great efforts with low subsequent tolerance to light effort. 1 year ago with urinary incontinence and choking past of poor performance in physical activities without pre-motor symptoms FAMILY: mother with clinical picture of possible dementia syndrome at age 60, history in the maternal family of myalgia, intolerance to physical exercise and hearing loss in adulthood. EXOMA: presence of variant c.146C > T (p.Ala49Val) in simple heterozygosity without CHCHD10 gene. MRI with thigh muscle hypotrophy in anterior and posterior thigh compartments; slight muscle edema in the legs. Conclusion: Pathogenic variants in the CHCHD10 gene should be considered in cases of atypical parkinsonism, especially in cases of positive familial history of mitochondrial myopathy or dementia.