In Vivo Modulation of Mitochondrial Activity Determines HSC Engraftment and Post-Transplant Survival in Mice

Cellular metabolism is emerging as a potential fate determinant in cancer and stem cell biology, constituting a crucial regulator of the hematopoietic stem cell (HSC) pool [1-4]. The extremely low oxygen tension in the HSC microenvironment of the adult bone marrow forces HSCs into a low metabolic profile that is thought to enable their maintenance by protecting them from reactive oxygen species (ROS). Although HSC quiescence has for long been associated with low mitochondrial activity, as testified by the low rhodamine stain that marks primitive HSCs, we hypothesized that mitochondrial activation could be an HSC fate determinant in its own right. We thus set to investigate the implications of pharmacologically modulating mitochondrial activity during bone marrow transplantation, and have found that forcing mitochondrial activation in the post-transplant period dramatically increases survival. Specifically, we examined the mitochondrial content and activation profile of each murine hematopoietic stem and progenitor compartment. Long-term-HSCs (LT-HSC, Lin-cKit+Sca1+ (LKS) CD150+CD34-), short-term-HSCs (ST-HSC, LKS+150+34+), multipotent progenitors (MPPs, LKS+150-) and committed progenitors (PROG, Lin-cKit+Sca1-) display distinct mitochondrial profiles, with both mitochondrial content and activity increasing with differentiation. Indeed, we found that overall function of the hematopoietic progenitor and stem cell compartment can be resolved by mitochondrial activity alone, as illustrated by the fact that low mitochondrial activity LKS cells (TMRM low) can provide efficient long-term engraftment, while high mitochondrial activity LKS cells (TMRM high) cannot engraft in lethally irradiated mice. Moreover, low mitochondrial activity can equally predict efficiency of engraftment within the LT-HSC and ST-HSC compartments, opening the field to a novel method of discriminating a population of transitioning ST-HSCs that retain long-term engraftment capacity. Based on previous experience that a high-fat bone marrow microenvironment depletes short-term hematopoietic progenitors while conserving their long-term counterparts [5], we set to measure HSC mitochondrial activation in high-fat diet fed mice, known to decrease metabolic rate on a per cell basis through excess insulin/IGF-1 production. Congruently, we found lower mitochondrial activation as assessed by flow cytometry and RT-PCR analysis as well as a depletion of the short-term progenitor compartment in high fat versus control chow diet fed mice. We then tested the effects of a mitochondrial activator known to counteract the negative effects of high fat diet. We first analyzed the in vitro effect on HSC cell cycle kinetics, where no significant change in proliferation or division time was found. However, HSCs responded to the mitochondrial activator by increasing asynchrony, a behavior that is thought to directly correlate with asymmetric division [6]. As opposed to high-fat diet fed mice, mice fed with the mitochondrial activator showed an increase in ST-HSCs, while all the other hematopoietic compartments were comparable to mice fed on control diet. Given the dependency on short-term progenitors to rapidly reconstitute hematopoiesis following bone marrow transplantation, we tested the effect of pharmacological mitochondrial activation on the recovery of mice transplanted with a limiting HSC dose. Survival 3 weeks post-transplant was 80% in the treated group compared to 0% in the control group, as predicted by faster recovery of platelet and neutrophil counts. In conclusion, we have found that mitochondrial activation regulates the long-term to short-term HSC transition, unraveling mitochondrial modulation as a valuable drug target for post-transplant therapy. Identification of molecular pathways accountable for the metabolically mediated fate switch is currently ongoing.

The significance of pre-existing minority Y181C mutations on virological failure of NNRTI-based, first-line antiretroviral therapy

Background: Reduced re'nal function has been reported with tenofovir disoproxil fumarate (TDF). It is not clear whether TDF co-administered with a boosted protease inhibitor (PI) leads to a greater decline in renal function than TDF co-administered with a non-nucleoside reverse transcriptase inhibitor (NNRTI).Methods: We selected ail antiretroviral therapy-naive patients in the Swiss HIV Cohort Study (SHCS) with calibrated or corrected serum creatinine measurements starting antiretroviral therapy with TDF and either efavirenz (EFV) or the ritonavir-boosted PIs, lopinavir (LPV/r) or atazanavir (ATV/r). As a measure of renal function, we used the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation to estimate the glomerular filtration rate (eGFR). We calculated the difference in eGFR over time between two therapies using a marginal model for repeated measures. In weighted analyses, observations were weighted by the product of their point of treatment and censoring weights to adjust for differences both in the sort of patients starting each therapy and in the sort of patients remaining on each therapy over time.Results: By March 2011, 940 patients with at least one creatinine measurement on a first therapy with either TDF and EFV (n=484), TDF and LPVlr (n=269) or TDF and ATV/r (n=187) had been followed for a median of 1. 7, 1.2 and 1.3 years, respectively. Table 1 shows the difference in average estimated GFR (eGFR) over time since starting cART for two marginal models. The first model was not adjusted for potential confounders; the second mode! used weights to adjust for confounders. The results suggest a greater decline in renal function during the first 6 months if TDF is used with a PI rather than with an NNRTI, but no further difference between these therapies after the first 6 months. TDF and ATV/r may lead to a greater decline in the first 6 months than TDF and LPVlr.Conclusions: TDF co-administered with a boosted PI leads to a greater de cline in renal function over the first 6 months of therapy than TDF co-administered with an NNRTI; this decline may be worse with ATV/r than with LPV/r.

Mice Transgenic For Human Dominant Mutations Of The GUCY2D Gene

Purpose: Animal models are essential to study pathological mechanisms and to test new therapeutic strategies. Many mouse models mimic human rod loss but only a limited number simulate cone dystrophies. The importance of cone function for human vision highlights the need to engineer a model for cone degeneration. An approach of lentiviral-directed transgenesis was tested in mice to express a dominant mutant gene described in a human cone dystrophy.Methods: Lentiviral vectors (LV) encoding either hrGFPII or the human double mutant GUCY2DE837D/R838S cDNA under the control of a region of the pig arrestin-3 promoter (Arr3) were produced and used for lentiviral-derived transgenesis. PCR-genotyping determined the transgenic mouse ratio. The expression of GFP was then analyzed both in vivo and by immunohistochemistry in Arr3-GFPII mice. Functional analysis was performed by ERG at 5, 9, 16 and 24 weeks for Arr3-GUCY2DE837D/R838S mice. Mice were sacrificed at 10 months of age for both histological analysis and RNA extraction.Results: While all the newborns from the transgenesis using the LV-Arr3-GFPII were transgenic, one third of the newborns from the LV-Arr3-GUCY2DE837D/R838S transgenesis were positive. Expression of GFPII was demonstrated by in vivo imaging, while expression of the mutant GUCY2D transcript was detetected using RT-PCR. No severe alteration of the functional response was observed up to 24 weeks of age in the transgenic mice. No obvious modification of the retinal morphology was identified either.Conclusions: Lentiviral-directed transgenesis is a rapid and straightforward method to engineer transgenic mice. Protein expression can be specifically targeted to the retina and thus could help to study the effect of expression of dominant mutant proteins. In our case, Arr3-GUCY2DE837D/R838S mice have a less severe phenotype than that described for human patients. Further analyses are required to understand this difference but several modifications of the expression cassette might also help to increase the expression of the mutant protein and reinforce the phenotype. Interestingly, the same construct is less effective in mouse versus pig retina (see Arsenijevic et al. ARVO 2011 abstract).

HIV-1 reverse transcriptase connection domain mutations: dynamics of emergence and implications for success of combination antiretroviral therapy.

BACKGROUND: Factors promoting the emergence of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) connection domain mutations and their effect on antiretroviral therapy (ART) are still largely undetermined. We investigated this matter by analyzing genotypic resistance tests covering 400 amino acid positions in the RT of HIV-1 subtype B viruses and corresponding treatment histories and laboratory measurements. METHODS: The emergence of connection domain mutations was studied in 334 patients receiving monotherapy or dual therapy with thymidine analogues at the time of the genotypic resistance test. Response to subsequent combination ART (cART) was analyzed using Cox regression for 291 patients receiving unboosted protease inhibitors. Response was defined by ever reaching an HIV RNA level <50 copies/mL during the first cART. RESULTS: The connection domain mutations N348I, R356K, R358K, A360V, and A371V were more frequently observed in ART-exposed than ART-naive patients, of which only N348I and A360V were nonpolymorphic (with a prevalence of <1.5% in untreated patients). N348I correlated with M184V and predominantly occurred in patients receiving lamivudine and zidovudine concomitantly. A360V was not associated with specific drug combinations and was found to emerge later than M184V or thymidine analogue mutations. Nonpolymorphic connection domain mutations were rarely detected in the absence of established drug resistance mutations in ART-exposed individuals (prevalence, <1%). None of the 5 connection domain mutations associated with treatment showed a statistically significant effect on response to cART. CONCLUSIONS: Despite their frequent emergence, connection domain mutations did not show large detrimental effects on response to cART. Currently, routine implementation of connection domain sequencing seems unnecessary for developed health care settings.

Dangerous Liaisons: Mitochondrial DNA Meets the NLRP3 Inflammasome.

Danger signals released by damaged organelles can promote inflammation. In this issue of Immunity, Shimada et al. (2012) report that oxidized DNA, released by mitochondria, directly binds and activates the NLRP3 inflammasome.

Protective role of amantadine in mitochondrial dysfunction and oxidative stress mediated by hepatitis C virus protein expression.

Amantadine is an antiviral and antiparkinsonian drug that has been evaluated in combination therapies against hepatitis C virus (HCV) infection. Controversial results have been reported concerning its efficacy, and its mechanism of action remains unclear. Data obtained in vitro suggested a role of amantadine in inhibiting HCV p7-mediated cation conductance. In keeping with the fact that mitochondria are responsible to ionic fluxes and that HCV infection impairs mitochondrial function, we investigated a potential role of amantadine in modulating mitochondrial function. Using a well-characterized inducible cell line expressing the full-length HCV polyprotein, we found that amantadine not only prevented but also rescued HCV protein-mediated mitochondrial dysfunction. Specifically, amantadine corrected (i) overload of mitochondrial Ca(2+); (ii) inhibition of respiratory chain activity and oxidative phosphorylation; (iii) reduction of membrane potential; and (iv) overproduction of reactive oxygen species. The effects of amantadine were observed within 15 min following drug administration and confirmed in Huh-7.5 cells transfected with an infectious HCV genome. These effects were also observed in cells expressing subgenomic HCV constructs, indicating that they are not mediated or only in part mediated by p7. Single organelle analyzes carried out on isolated mouse liver mitochondria demonstrated that amantadine induces hyperpolarization of the membrane potential. Moreover, amantadine treatment increased the calcium threshold required to trigger mitochondrial permeability transition opening. In conclusion, these results support a role of amantadine in preserving cellular bioenergetics and redox homeostasis in HCV-infected cells and unveil an effect of the drug which might be exploited for a broader therapeutic utilization.

Role of ALK gene and mutations in initiation and progression of neuroblastoma

Introduction Le neuroblastome (NB) est la tumeur maligne solide extra-crânienne la plus fréquente chez l'enfant. Sa présentation clinique est très hétérogène, allant d'une tumeur localisée à une atteinte métastatique sévère. Malgré des traitements agressifs, environ 55% des NB de hauts risques sont actuellement résistants aux thérapies. L'espoir réside dans le développement de traitements ciblant les mécanismes moléculaires responsables du développement et de la progression du NB. Le gène Anaplastic Lymphoma Kinase (ALK) codant pour un récepteur tyrosine kinase a été particulièrement étudié ces dernières années car il est muté, amplifié ou surexprimé dans une majorité des NBs. Le but de ce projet était d'investiguer le rôle de ALK-wt, ainsi que de ces deux plus fréquentes mutations, ALK- F1174L et ALK-R1245Q, dans l'oncogenèse du NB. Le NB étant originaire des cellules de la crête neurale, nous avons analysé le potentiel oncogénique de ces différentes formes de ALK dans des cellules progénitrices de la crête neurale (NCPC). Méthode Des NCPC de souris (JoMal), possédant un c-MycER inductible pour leur maintien en culture in vitro, ont été transduites par un rétrovirus permettant l'expression stable de ALK-wt, ALK-F1174L et ALK-R1245Q. Des tests in vitro ont d'abord été effectués pour tester le système c-MycER, la stabilité de nos cellules transduites, leur phénotype, leur capacité de croissance et leur tumorigénicité. Les cellules transduites ont ensuite été injectées dans des souris immunosupprimées en sous-cutané, puis en orthotopique, c'est-à-dire dans leur glande surrénale, afin de mesurer leur tumorigénicité in vivo. Résultats La transduction et l'expression stable de ALK n'ont pas modifié le phénotype indifférencié des JoMal, ni de manière significative la capacité de croissance des cellules in vitro en absence d'activation de c-MycER. Par contre, lorsque c-MycER est actif, les cellules porteuses des mutations Fl 174L et R1245Q ont montré une meilleure capacité de prolifération et de formation de colonies, par rapport aux JoMal-ALK-wt et aux cellules contrôles en culture 3D dans de la méthylcellulose et dans un test de formation de neurosphères. In vivo, les souris injectées avec les cellules JoMal-ALK- F1174L en sous-cutané ou dans la glande surrénale ont rapidement développé des tumeurs, suivies par le groupe JoMal-ALK-R1245Q et le groupe JoMal-ALK-wt, alors que les groupes de souris contrôles n'ont présenté aucune tumeur. En orthotopique, nous avons obtenu 5/6 tumeurs ALK-F1174L, 7/7 tumeurs ALK-R1245Q et 6/7 tumeurs ALK-wt. Les tumeurs sous-cutanées ne présentaient pas de différences morphologiques et histologiques entre les différents groupes et montraient une histologie compatible avec un NB. Les tumeurs orthotopiques restent encore à analyser. Conclusion Cette étude a permis de démontrer que les mutations activatrices Fl 174L et R1245Q ont des propriétés tumorigéniques in vitro dans des NCPC et in vivo tandis que la forme sauvage de ALK montre une capacité oncogénique uniquement in vivo. Bien que la caractérisation des tumeurs orthotopiques n'a pas encore été effectuée, l'analyse des tumeurs sous-cutanées nous suggère que l'expression de ALK- wt ou muté est suffisante pour induire la formation de NB à partir des cellules progénitrices de la crête neurale. Le gène ALK semble donc jouer un rôle important dans l'oncogénèse du NB, aussi bien par la présence de mutations activatrices que par sa fréquente surexpression.

Mutations in FKBP10 cause recessive osteogenesis imperfecta and Bruck syndrome.

Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue characterized by bone fragility and alteration in synthesis and posttranslational modification of type I collagen. Autosomal dominant OI is caused by mutations in the genes (COL1A1 or COL1A2) encoding the chains of type I collagen. Bruck syndrome is a recessive disorder featuring congenital contractures in addition to bone fragility; Bruck syndrome type 2 is caused by mutations in PLOD2 encoding collagen lysyl hydroxylase, whereas Bruck syndrome type 1 has been mapped to chromosome 17, with evidence suggesting region 17p12, but the gene has remained elusive so far. Recently, the molecular spectrum of OI has been expanded with the description of the basis of a unique posttranslational modification of type I procollagen, that is, 3-prolyl-hydroxylation. Three proteins, cartilage-associated protein (CRTAP), prolyl-3-hydroxylase-1 (P3H1, encoded by the LEPRE1 gene), and the prolyl cis-trans isomerase cyclophilin-B (PPIB), form a complex that is required for fibrillar collagen 3-prolyl-hydroxylation, and mutations in each gene have been shown to cause recessive forms of OI. Since then, an additional putative collagen chaperone complex, composed of FKBP10 (also known as FKBP65) and SERPINH1 (also known as HSP47), also has been shown to be mutated in recessive OI. Here we describe five families with OI-like bone fragility in association with congenital contractures who all had FKBP10 mutations. Therefore, we conclude that FKBP10 mutations are a cause of recessive osteogenesis imperfecta and Bruck syndrome, possibly Bruck syndrome Type 1 since the location on chromosome 17 has not been definitely localized.

Correction: Frequent MAGE Mutations in Human Melanoma.

[This corrects the article on p. e12773 in vol. 5.].

New players driving inflammation in monogenic autoinflammatory diseases.

Systemic autoinflammatory diseases are caused by abnormal activation of the cells that mediate innate immunity. In the past two decades, single-gene defects in different pathways, driving clinically distinct autoinflammatory syndromes, have been identified. Studies of these aberrant pathways have substantially advanced understanding of the cellular mechanisms that contribute to mounting effective and balanced innate immune responses. For example, mutations affecting the function of cytosolic immune sensors known as inflammasomes and the IL-1 signalling pathway can trigger excessive inflammation. A surge in discovery of new genes associated with autoinflammation has pointed to other mechanisms of disease linking innate immune responses to a number of basic cellular pathways, such as maintenance of protein homeostasis (proteostasis), protein misfolding and clearance, endoplasmic reticulum stress and mitochondrial stress, metabolic stress, autophagy and abnormalities in differentiation and development of myeloid cells. Although the spectrum of autoinflammatory diseases has been steadily expanding, a substantial number of patients remain undiagnosed. Next-generation sequencing technologies will be instrumental in finding disease-causing mutations in as yet uncharacterized diseases. As more patients are reported to have clinical features of autoinflammation and immunodeficiency or autoimmunity, the complex interactions between the innate and adaptive immune systems are unveiled.

Phenotype of two Consanguineous Autosomic Recessive Retinitis Pigmentosa Families Caused by PDE6A and PDE6B Mutations

Purpose: To assess the clinical phenotype in two consanguineous Tunisian families with non syndromic autosomic recessive retinitis Pigmentosa (RP) caused by a PDE6A and PDE6B mutations.Methods: All accessible familiy members were included. Affected members from each family underwent full ophthalmic examination with best corrected Snellen visual acuity, fundus photography, optical coherence tomography and full field electroretinography. Haplotype analyses were used to test linkage in the family to 20 arRP loci, including ABCA4, LRAT, USH2A, RP29, CERKL, CNGA1, CNGB1, CRB1, EYS, RP28, MERTK, NR2E3, PDE6A, PDE6B, RGR, RHO, RLBP1, TULP1. All exons and intron-exon junctions of candidate genes not excluded by haplotype analysis were PCR amplified and directly sequenced.Results: Two family members were clinically affected with arRP in each pedigree. Age range at baseline was 43 to 54 years (mean age at baseline was 48 years). For all affected members, night blindness appeared since early childhood (at 4-5 years old) without nystagmus but with a severe progression and mild to severe loss of central vision at the second decade. Visual acuity at baseline ranged from 20/500 to 20/63. Kinetic visual field was severely constricted for one patient and unrealizable for the others. Funduscopic examination revealed bone spicule-shaped pigment deposits in the mid periphery along with atrophy of the retina, narrowing of the vessels and waxy optic discs. Tomograms showed macular atrophy in both cases of family A, and macular edema in the patients of family B. ERG showed a loss of both rod and cone responses. Haplotype analysis revealed homozygosity for microsatellites markers flanking PDE6A and PDE6B in family A and B, respectively. Sequencing of PDE6A in family A showed a homozygous R102S mutation. In family B, sequencing identified a D600N homozygous mutation. Both mutations cosegregated within each respective pedigree.Conclusions: For these families, affected members developed a severe form of non syndromic arRP. The two reported mutations have already been described. Our data further contribute to our understanding of genotype-phenotype correlations.

In-depth analysis of hyaline fibromatosis syndrome frameshift mutations at the same site reveal the necessity of personalized therapy.

Hyaline fibromatosis syndrome is an autosomal recessive disease caused by mutations in ANTXR2, a gene involved in extracellular matrix homeostasis. Sixty percent of patients carry frameshift mutations at a mutational hotspot in exon 13. We show in patient cells that these mutations lead to low ANTXR2 mRNA and undetectable protein levels. Ectopic expression of the proteins encoded by the mutated genes reveals that a two base insertion leads to the synthesis of a protein that is rapidly targeted to the ER-associated degradation pathway due to the modified structure of the cytosolic tail, which instead of being hydrophilic and highly disordered as in wild type ANTXR2, is folded and exposes hydrophobic patches. In contrast, one base insertion leads to a truncated protein that properly localizes to the plasma membrane and retains partial function. We next show that targeting the nonsense mediated mRNA decay pathway in patient cells leads to a rescue of ANTXR2 protein in patients carrying one base insertion but not in those carrying two base insertions. This study highlights the importance of in-depth analysis of the molecular consequences of specific patient mutations, which even when they occur at the same site can have drastically different consequences.

Regulation of the cardiac voltage-gated sodium channel Nav1.5 : regulation of Nav1.5 by ubiquitin-protein ligases of the Nedd4/Nedd4-like family and characterisation of two novel mutations in the gene encoding Nav1.5 (SCN5A) implicated in the Brugada syndrome triggered by fever

Abstract: The genesis of the cardiac action potential, which accounts for the cardiac contraction, is due to the sodium current INa mediated by the voltage-gated sodium channel Nav1.5. Several cardiac arrhythmias such as the Brugada syndrome are known te be caused by mutations in SCN5A, the gene encoding Nav1.5. Studies of these mutations allowed a better understanding of biophysical and functional properties of Nav1.5. However, only few investigations have been performed in order to understand the regulation of Nav1.5. During my thesis, I investigated different mechanisms of regulation of Nav1.5 using a heterologous expression system, HEK293 cells, coupled with a technique of sodium current recording: the patch clamp in whole cell configuration. In previous studies it has been shown that an enzyme of the Nedd4 family (Nedd4-2) regulates an epithelial sodium channel via the interaction with PY-motifs present in the latter. Interestingly, Nav1.5 contains a similar PY-motif, which motivated us to study the role of Nedd4-2 expressed in heart for the regulation of Nav1.5. In a second study, we investigated the implication of two Nav1.5 mutants, which were either less functional or net functional (Nav1.5 R535X and Nav1.5 L325R respectively) implied in the genesis of the Brugada syndrome by fever. Our results established two mechanisms implied in Nav1.5 regulation. The first one implies that following the interaction between the PY-motif of Nav1.5 and Nedd4- 2 Nav1.5 is ubiquitinated by Nedd4-2. This ubiquitination leads to the internalization of Nav1 .5. The second mechanism is a phenomenon called the "dominant negative" effect of Nav1.5 L325R on Nay1.5 where the decrease of 'Na is potentially due to the retention of Nav1.5 by Nav1.5 L325R in an undefined intracellular compartment. These studies defined two mechanisms of Nav1.5 regulation, which could play an important role for the genesis of cardiac arrhythmias where molecular processes are still poorly understood. Résumé La genèse du potentiel d'action cardiaque, permettant la contraction cardiaque, est due au courant sodique INa issu des canaux sodiques cardiaques dépendants du voltage Nav1.5. Nombreuses arythmies cardiaques telles que le syndrome de Brugada sont connues pour être liées à des mutations du gène SCN5A, codant pour Nav1.5. L'étude de ces mutations a permis une meilleure compréhension des propriétés structurelles et fonctionnelles de Nav1.5 et leurs implications dans la genèse de ces pathologies. Néanmoins peu d'études ont été menées afin de comprendre les mécanismes de régulation de Nav1.5. Mon travail de thèse a consisté à étudier des mécanismes de régulation de Nav1.5 en utilisant un système d'expression hétérologue, les cellules HEK293, couplé à une technique d'enregistrement des courants sodiques, le "patch clamp" en configuration cellule entière. La présence sur Nav1.5 d'un motif-PY similaire à ceux nécessaires pour la régulation d'un canal épithélial sodique par une enzyme de la famille de Nedd4, nous a amenée à étudier le rôle de ces ubiquitine-ligases, en particulier Nedd4-2, dans la régulation de Nav1.5. La seconde étude s'est intéressée aux conséquences de deux mutations de SCN5A codant pour deux mutants peu ou pas fonctionnels (Nav1.5 L325R et Nav1.5 R535X respectivement) retrouvées chez des patients présentant un syndrome de Brugada exacerbé par un état fébrile. Nos résultats ont permis d'établir deux mécanismes de régulation de Nav1.5 L'un par Nedd4-2 qui implique rubiquitination de Nav1.5 par cette ligase suite à l'interaction entre le motif-PY de Nav1.5 et Nedd4-2. Cette modification déclenche l'internalisation du canal impliquée dans la diminution d'INa. Le second mécanisme quant à lui est un effet "dominant négatif" de Nav1.5 L325R sur Nav1.5 aboutissant à une diminution d'INa suite à la séquestration intracellulaire potentielle de Nav1.5 par Nav1.5 L325R. Ces études ont mis en évidence deux mécanismes de régulation de Nav1.5 pouvant jouer un rôle majeur dans la genèse et/ou l'accentuation des arythmies cardiaques dont les processus moléculaires au sein des cardiomyocytes, impliquant des modifications du courant sodiques, sont encore mal compris. Résumé destiné à un large public La dépolarisation électrique de la membrane des cellules cardiaques permet la contraction du coeur. La génèse de cette activité électrique est due au courant sodique issu d'un type de canal à sodium situé dans la membrane des cellules cardiaques. De nombreuses pathologies provoquant des troubles du rythme cardiaque sont issues de mutations du gène qui code pour ce canal à sodium. Ces canaux mutants, entrainant diverses pathologies cardiaques telles que le syndrome de Brugada, ont été largement étudiées. Néanmoins, peu de travaux ont été réalisés sur les mécanismes de régulation de ce canal à sodium non muté. Mon travail de thèse a consisté à étudier certains des mécanismes de régulation de ce canal à sodium en utilisant une technique permettant l'enregistrement des courants sodiques issus de l'expression de ces canaux à sodium à la membrane de cellules mammifères. La présence sur ce canal à sodium d'une structure spécifique, similaire à celle nécessaire pour la régulation d'un canal épithélial à sodium par une enzyme appelée Nedd4-2, nous a amenée à étudier le rôle de cette enzyme dans la régulation de ce canal à sodium. La seconde étude s'est intéressée aux rôles de deux mutations du gène codant pour ce canal à sodium retrouvées chez des patients présentant un syndrome de Brugada exacerbé par la fièvre. Nos résultats nous ont permis d'établir deux mécanismes de régulation de ce canal à sodium diminuant le courant sodique l'un par l'action de l'enzyme Nedd4-2, suite à son interaction avec ce canal, qui modifie ce canal à sodium (ubiquitination) diminuant de ce fait la densité membranaire du canal. L'autre par un mécanisme suggérant un effet négatif de l'un des canaux mutants sur l'expression à la membrane du canal à sodium non muté. Ces études ont mis en évidence deux mécanismes de régulation de ce canal à sodium pouvant jouer un rôle majeur dans la genèse et/ou l'accentuation des troubles du rythme cardiaques dont les mécanismes cellulaires sont encore incompris.

Productive HIV-1 infection of primary CD4+ T cells induces mitochondrial membrane permeabilization leading to a caspase-independent cell death.

We have explored in vitro the mechanism by which human immunodeficiency virus, type 1 (HIV-1) induces cell death of primary CD4+ T cells in conditions of productive infection. Although HIV-1 infection primed phytohemagglutinin-activated CD4+ T cells for death induced by anti-CD95 antibody, T cell death was not prevented by a CD95-Fc decoy receptor, nor by decoy receptors of other members of the TNFR family (TNFR1/R2, TRAILR1/R2/OPG, TRAMP) or by various blocking antibodies, suggesting that triggering of death receptors by their cognate ligands is not involved in HIV-induced CD4 T cell death. HIV-1 induced CD4 T cell shrinkage, cell surface exposure of phosphatidylserine, loss of mitochondrial membrane potential (Deltapsim), and mitochondrial release of cytochrome c and apoptosis-inducing factor. A typical apoptotic phenotype (nuclear chromatin condensation and fragmentation) only occurred in around half of the dying cells. Treatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a broad spectrum caspase inhibitor, prevented nuclear chromatin condensation and fragmentation in HIV-infected CD4+ T cells and in a cell-free system (in which nuclei were incubated with cytoplasmic extracts from the HIV-infected CD4+ T cells). Nevertheless, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone did not prevent mitochondrial membrane potential loss and cell death, suggesting that caspases are dispensable for HIV-mediated cell death. Our findings suggest a major role of the mitochondria in the process of CD4 T cell death induced by HIV, in which targeting of Bax to the mitochondria may be involved.