Regulation of the Permeability Transition Pore in Skeletal Muscle Mitochondria MODULATION BY ELECTRON FLOW THROUGH THE RESPIRATORY CHAIN COMPLEX I

We have investigated the regulation of the permeability transition pore (PTP), a cyclosporin A-sensitive channel, in rat skeletal muscle mitochondria. As is the case with mitochondria isolated from a variety of sources, skeletal muscle mitochondria can undergo a permeability transition following Ca2+uptake in the presence of Pi. We find that the PTP opening is dramatically affected by the substrates used for energization, in that much lower Ca2+ loads are required when electrons are provided to complex I rather than to complex II or IV. This increased sensitivity of PTP opening does not depend on differences in membrane potential, matrix pH, Ca2+ uptake, oxidation-reduction status of pyridine nucleotides, or production of H2O2, but is directly related to the rate of electron flow through complex I. Indeed, and with complex I substrates only, pore opening can be observed when depolarization is induced with uncoupler (increased electron flow) but not with cyanide (decreased electron flow). Consistent with pore regulation by electron flow, we find that PTP opening is inhibited by ubiquinone 0 at concentrations that partially inhibit respiration and do not depolarize the inner membrane. These data allow identification of a novel site of regulation of the PTP, suggest that complex I may be part of the pore complex, and open new perspectives for its pharmacological modulation in living cells

Spatial Relational Memory Requires Hippocampal Adult Neurogenesis

The dentate gyrus of the hippocampus is one of the few regions of the mammalian brain where new neurons are generated throughout adulthood. This adult neurogenesis has been proposed as a novel mechanism that mediates spatial memory. However, data showing a causal relationship between neurogenesis and spatial memory are controversial. Here, we developed an inducible transgenic strategy allowing specific ablation of adult-born hippocampal neurons. This resulted in an impairment of spatial relational memory, which supports a capacity for flexible, inferential memory expression. In contrast, less complex forms of spatial knowledge were unaltered. These findings demonstrate that adult-born neurons are necessary for complex forms of hippocampus-mediated learning

Non-Antioxidant Properties of α-Tocopherol Reduce the Anticancer Activity of Several Protein Kinase Inhibitors In Vitro

The antioxidant properties of α-tocopherol have been proposed to play a beneficial chemopreventive role against cancer. However, emerging data also indicate that it may exert contrasting effects on the efficacy of chemotherapeutic treatments when given as dietary supplement, being in that case harmful for patients. This dual role of α-tocopherol and, in particular, its effects on the efficacy of anticancer drugs remains poorly documented. For this purpose, we studied here, using high throughput flow cytometry, the direct impact of α-tocopherol on apoptosis and cell cycle arrest induced by different cytotoxic agents on various models of cancer cell lines in vitro. Our results indicate that physiologically relevant concentrations of α-tocopherol strongly compromise the cytotoxic and cytostatic action of various protein kinase inhibitors (KI), while other classes of chemotherapeutic agents or apoptosis inducers are unaffected by this vitamin. Interestingly, these anti-chemotherapeutic effects of α-tocopherol appear to be unrelated to its antioxidant properties since a variety of other antioxidants were completely neutral toward KI-induced cell cycle arrest and cell death. In conclusion, our data suggest that dietary α-tocopherol could limit KI effects on tumour cells, and, by extent, that this could result in a reduction of the clinical efficacy of anti-cancer treatments based on KI molecules

Mécanismes d'activation de la proteine pro-apoptotique Bax

L'apoptose est un processus de mort cellulaire génétiquement programmé, dont tout dysfonctionnement par défaut peut générer la formation de tumeurs cancéreuses. La protéine pro-apoptique Bax agit au coeur de la signalisation apoptotique impliquant la mitochondrie, et son activation, qui précède l'étape de non retour de l'apoptose passe par des changements conformationnels aboutissant à l'exposition de l'hélice a9 C-terminale, supposée être une ancre transmembranaire et du domaine de multimérisation BH3. Cependant, la coordination des événements de multimérisation, relocalisation mitochondriale, ancrage transmembranaire reste confuse ; de même que l'implication des différents domaines structuraux et acides aminés de la protéine. Les travaux présentés se proposent de participer à l'analyse détaillée du mode d'activation et des relations structure/fonctions de Bax. Ces données ont permis dans une optique de thérapie anti-cancéreuse d'envisager la synthèse organique de drogues ciblant l'activation de Bax et donc la mort cellulaire.Apoptosis is a highly ordered and controlled cell death program, that, when dysregulated, can contribute to several diseases including cancer. The pro-apoptotic protein Bax is largely implicated in the mitochondrial apoptotic pathway. Its activation, that precedes the point of no return of apoptosis requires some conformational changes resulting in the exposure of C-terminus a9 helix, supposed to be a transmembrane anchor and of the BH3 multimerization domain. However, the coordination of proteic multimerization, mitochondrial translocation, transmembrane anchoring events remains elusive ; as well as the involvement of the different structural domains and amino acids of the protein. So, this work essentially focalise its interest on Bax's activation dynamic and structure / function relatioship. In a perspective of anti-cancerous therapy, these data also allowed us to envisage the organic synthesis of drugs developped to target Bax's activation and consequently cellular death.BORDEAUX2-BU Santé (330632101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Mise au point d'une nouvelle génération de capteurs recombinants fluorescents pour la mesure de l'activité des caspases au sein de cellules vivantes au cours de l'apoptose

L'apoptose est un processus de mort cellulaire génétiquement programmé, dont tout dysfonctionnement peut générer la formation de tumeurs cancéreuses. Un événement clef de l'apoptose est représenté par l'activation des caspases, principaux effecteurs. Les travaux proposés sont basés sur le développement de nouveaux outils pour l'étude des événements moléculaires de l'apoptose. La mise au point d'une nouvelle génération de biocapteurs "positionnels" permettant de mesurer l'activité de la caspase 3, dans des cellules vivantes au cours de l'apoptose. Ainsi le transfert industriel de ces capteurs pour leur exploitation sur une station robotisée de criblage en cytométrie en flux à haut débit. Ils ont été exploités pour disséquer les voies de signalisation de l'apoptose à travers une approche bi-oaramétrique d'analyse par imagerie confocale, des cinétiques intracellulaires des événements clés de la phase effectrice de l'apoptose. Ainsi le capteur à caspase 3 a été utilisé lors de la caractérisation d'un modèle particulier de mort cellulaire induite par stress oxydatif.Apoptosis is a genetically programmed process of cell death, which, if non-functional, can lead to the formation of cancerous tumors. A key element of apoptosis in the activation of its principal effector, the caspases. The proposed work is based on the development of novel tools for the study of molecular mechanisms of apoptosis. The perfection of a new generation of "positioned" biosensors, allowing to measure the activity of caspase 3 in living cells undergoing apopotosis, as well as the industrial transfer of these sensors for their exploitation on a robotic station for High Content Screening. They were used for dissecting the apoptotic signalling pathways through a bi-parametrical approach of analysis by confocal imaging of intracellular kinetics of the effector part of apoptosis. Furthermore, the sensor of caspase 3 was employed for the characterization of a specific model of cell death, induced by oxidative stress.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

α-Synuclein Fibrils as Penrose Machines: A Chameleon in the Gear

In 1957, Lionel Penrose built the first man-made self-replicating mechanical device and illustrated its function in a series of machine prototypes, prefiguring our current view of the genesis and the proliferation of amyloid fibrils. He invented and demonstrated, with the help of his son Roger, the concepts that decades later, would become the fundamentals of prion and prion-like neurobiology: nucleation, seeding and conformational templating of monomers, linear polymer elongation, fragmentation, and spread. He published his premonitory discovery in a movie he publicly presented at only two conferences in 1958, a movie we thus reproduce here. By making a 30-year-jump in the early 90’s, we evoke the studies performed by Peter Lansbury and his group in which α-Synuclein (α-Syn) was for the first time (i) compared to a prion; (ii) shown to contain a fibrillization-prone domain capable of seeding its own assembly into fibrils; (iii) identified as an intrinsically disordered protein (IDP), and which, in the early 2000s, (iv) was described by one of us as a protein chameleon. We use these temporally distant breakthroughs to propose that the combination of the chameleon nature of α-Syn with the rigid gear of the Penrose machine is sufficient to account for a phenomenon that is of current interest: the emergence and the spread of a variety of α-Syn fibril strains in α-Synucleinopathies

α-Synuclein Fibrils as Penrose Machines: A Chameleon in the Gear

In 1957, Lionel Penrose built the first man-made self-replicating mechanical device and illustrated its function in a series of machine prototypes, prefiguring our current view of the genesis and the proliferation of amyloid fibrils. He invented and demonstrated, with the help of his son Roger, the concepts that decades later, would become the fundamentals of prion and prion-like neurobiology: nucleation, seeding and conformational templating of monomers, linear polymer elongation, fragmentation, and spread. He published his premonitory discovery in a movie he publicly presented at only two conferences in 1958, a movie we thus reproduce here. By making a 30-year-jump in the early 90’s, we evoke the studies performed by Peter Lansbury and his group in which α-Synuclein (α-Syn) was for the first time (i) compared to a prion; (ii) shown to contain a fibrillization-prone domain capable of seeding its own assembly into fibrils; (iii) identified as an intrinsically disordered protein (IDP), and which, in the early 2000s, (iv) was described by one of us as a protein chameleon. We use these temporally distant breakthroughs to propose that the combination of the chameleon nature of α-Syn with the rigid gear of the Penrose machine is sufficient to account for a phenomenon that is of current interest: the emergence and the spread of a variety of α-Syn fibril strains in α-Synucleinopathies

Amplification of Fas-Mediated Apoptosis in Type II Cells via Microdomain Recruitment

Fas triggers apoptosis via the caspase cascade when bound to its ligand FasL. In type I cells, Fas is concentrated into the plasma membrane lipid rafts, and these domains are required for the apoptotic signal to occur. In contrast, Fas is excluded from the microdomains in type II cells. We report that the coligation with Fas of the membrane receptor CD28 strongly increases Fas-induced apoptosis in type II T lymphocytes, whereas it has no effect in a type I cell line. The effect of CD28 is independent of its intracellular region and requires the recruitment of the microdomains. Indeed, upon CD28 costimulation, Fas is redistributed in the lipid rafts, and their disruption with a cholesterol chelator abrogates the effect of CD28. The microdomain-mediated cell death amplification does not alter death-induced signaling complex formation and is mediated by the enhancement of the mitochondrial apoptotic pathway. These findings indicate that the sensitivity to Fas-induced apoptosis of type II cells can be amplified in vivo by the recruitment of lipid rafts following interactions between nonapoptotic ligand/receptor pairs during cell-to-cell contacts