Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes

Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6-(Hsp90)2-Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes

Glutamate Induces Mitochondrial Dynamic Imbalance and Autophagy Activation: Preventive Effects of Selenium

Glutamate-induced cytotoxicity is partially mediated by enhanced oxidative stress. The objectives of the present study are to determine the effects of glutamate on mitochondrial membrane potential, oxygen consumption, mitochondrial dynamics and autophagy regulating factors and to explore the protective effects of selenium against glutamate cytotoxicity in murine neuronal HT22 cells. Our results demonstrated that glutamate resulted in cell death in a dose-dependent manner and supplementation of 100 nM sodium selenite prevented the detrimental effects of glutamate on cell survival. The glutamate induced cytotoxicity was associated with mitochondrial hyperpolarization, increased ROS production and enhanced oxygen consumption. Selenium reversed these alterations. Furthermore, glutamate increased the levels of mitochondrial fission protein markers pDrp1 and Fis1 and caused increase in mitochondrial fragmentation. Selenium corrected the glutamate-caused mitochondrial dynamic imbalance and reduced the number of cells with fragmented mitochondria. Finally, glutamate activated autophagy markers Beclin 1 and LC3-II, while selenium prevented the activation. These results suggest that glutamate targets the mitochondria and selenium supplementation within physiological concentration is capable of preventing the detrimental effects of glutamate on the mitochondria. Therefore, adequate selenium supplementation may be an efficient strategy to prevent the detrimental glutamate toxicity and further studies are warranted to define the therapeutic potentials of selenium in animal disease models and in human

Hsp40 Couples with the CSPα Chaperone Complex upon Induction of the Heat Shock Response

In response to a conditioning stress, the expression of a set of molecular chaperones called heat shock proteins is increased. In neurons, stress-induced and constitutively expressed molecular chaperones protect against damage induced by ischemia and neurodegenerative diseases, however the molecular basis of this protection is not known. Here we have investigated the crosstalk between stress-induced chaperones and cysteine string protein (CSPα). CSPα is a constitutively expressed synaptic vesicle protein bearing a J domain and a cysteine rich “string” region that has been implicated in the long term functional integrity of synaptic transmission and the defense against neurodegeneration. We have shown previously that the CSPα chaperone complex increases isoproterenol-mediated signaling by stimulating GDP/GTP exchange of Gαs. In this report we demonstrate that in response to heat shock or treatment with the Hsp90 inhibitor geldanamycin, the J protein Hsp40 becomes a major component of the CSPα complex. Association of Hsp40 with CSPα decreases CSPα-CSPα dimerization and enhances the CSPα-induced increase in steady state GTP hydrolysis of Gαs. This newly identified CSPα-Hsp40 association reveals a previously undescribed coupling of J proteins. In view of the crucial importance of stress-induced chaperones in the protection against cell death, our data attribute a role for Hsp40 crosstalk with CSPα in neuroprotection

Genetic analysis of alpha-latrotoxin receptors reveals functional interdependence of CIRL/latrophilin 1 and neurexin 1 alpha

alpha-Latrotoxin triggers massive neurotransmitter release from nerve terminals by binding to at least two distinct presynaptic receptors, neurexin la and CIRL1/ latrophilinl (CL1). We have now generated knockout (KO) mice that lack CL1 and analyzed them alone or in combination with neurexin la KO mice. Mice lacking only CL1, or both CL1 and neurexin 1alpha, were viable and fertile. Ca2+-independent binding of alpha-latrotoxin to brain membranes was impaired similarly in CL1 single and in CL1/neurexin 1alpha double KO mice (similar to75% decrease) but not in neurexin 1alpha single KO mice. In contrast, Ca2+ dependent binding (similar to2 times above Ca2+-independent binding) was altered in both CL1 (similar to50% decrease) and neurexin 1alpha single KO mice (similar to25% decrease) and was decreased further in double KO mice (similar to75% decrease). Synaptosomes lacking CL1 exhibited the same decrease in alpha- latrotoxin-stimulated glutamate release in the presence and absence of Ca2+ (similar to75%). In contrast, synaptosomes lacking neurexin 1alpha exhibited only a small decrease in alpha-latrotoxin-triggered release in the absence of Ca2+ (similar to20%) but a major decrease in the presence of Ca2+ (similar to75%). Surprisingly, synaptosomes lacking both CL1 and neurexin 1alpha displayed a relatively smaller decrease in alpha-latrotoxin-stimulated glutamate release than synaptosomes lacking only CL1 in the absence of Ca2+ (similar to50 versus similar to75%), but the same decrease in the presence of Ca2+ (similar to75%). Our data suggest the following two major conclusions. 1) CL1 and neurexin 1alpha together account for the majority (75%) of alpha-latrotoxin receptors in brain, with the remaining receptor activity possibly due to other CL and neurexin isoforms, and 2) the two receptors act additively in binding alpha-latrotoxin but not in triggering release. Together these data suggest that the two receptors act autonomously in binding of alpha-latrotoxin but cooperatively in transducing the stimulation of neurotransmitter release by alpha-latrotoxin

Intelligence collective et complexité : L appropriation d une technologie d intelligence collective (Approches systémiques et affordancielles)

Comment comprendre, dans une approche communicationnelle, l appropriation d outils d intelligence collective ? En matière d intranet, on considère aisément que les outils de partage des connaissances ou de communication seront adoptés rapidement par des utilisateurs empressés et soucieux de bien faire. L appropriation collective de tels outils semble aller de soi pour bien des commanditaires comme pour bien des développeurs techniques. On est pourtant surpris par les fortes résistances qu opposent parfois les utilisateurs finaux. Ils ont le sentiment d être pris dans des jeux qui les désavantagent, ou bien que l on a peu considéré leurs véritables enjeux, leurs pratiques, leurs logiques ou en d autres termes, leur situation et la façon dont ils la définissent. Ils ne réussissent pas à développer les usages à partir des outils qu on leur propose. Dans ce travail de recherche, mené en compréhension, nous analyserons un processus d appropriation collective au travers de trois approches communicationnelles : une approche systémique sociotechnique par la modélisation des relations, une approche affordancielle par la modélisation de la situation d un acteur, une approche affordancielle par la modélisation d une situation de consultation. Nous mobilisons pour cela le cas d une communauté de chercheurs en toxicologie nucléaire : Clairatome. Chacune de ces analyses nous permet d appréhender le sens du site en situation, l affordance globale du site pour un acteur, et les usages possibles du site pour un acteur désigné. L appropriation collective est un questionnement s apparentant à la redéfinition de la situation par les acteurs et menant à des conduites intégrées d usages. Comment favoriser l engagement de chacun des acteurs dans une telle redéfinition ?How may we understand the appropriation of the collective intelligence technologies ? We easily believe, about intranets, that sharing and communicating will be quickly adopted and shared practises of enthusiastic users. For who places the orders and who develops these technologies, the collective appropriation poses initially no question. Nevertheless, strong resistances are noticed among the addressees They give as a pretext they are disfavoured. They argue their practises, their aims and goals, their strategies are not considered. In a word, their situation and how they define it are a far cry from what planned. Uses don t rise. In our comprehensive research, we will analyse the process of a collective appropriation thanks to three qualitative approaches. One is a systemic approach by modelling the relationship between the actors. The second is an affordancial approach by modelling the actors situation. The third is an affordancial approach by modelling the actors website s consultation. Our research will be enhanced by a case : a community of researchers in nuclear toxicology, Clairatome. Our analyses are aimed to understand the global sense of the website in the situation, its meaning for the researchers of the community, and the adequate uses for part of the actors. The collective appropriation is a long process of redefining the situation that leads to integrated and shared uses. How then can we coach the involvement of each actors ?MONTPELLIER-BU Lettres (341722103) / SudocSudocFranceF

Bcl-xL induces Drp1-dependent synapse formation in cultured hippocampal neurons

Maturation of neuronal synapses is thought to involve mitochondria. Bcl-xL protein inhibits mitochondria-mediated apoptosis but may have other functions in healthy adult neurons in which Bcl-xL is abundant. Here, we report that overexpression of Bcl-xL postsynaptically increases frequency and amplitude of spontaneous miniature synaptic currents in rat hippocampal neurons in culture. Bcl-xL, overexpressed either pre or postsynaptically, increases synapse number, the number and size of synaptic vesicle clusters, and mitochondrial localization to vesicle clusters and synapses, likely accounting for the changes in miniature synaptic currents. Conversely, knockdown of Bcl-xL or inhibiting it with ABT-737 decreases these morphological parameters. The mitochondrial fission protein, dynamin-related protein 1 (Drp1), is a GTPase known to localize to synapses and affect synaptic function and structure. The effects of Bcl-xL appear mediated through Drp1 because overexpression of Drp1 increases synaptic markers, and overexpression of the dominant-negative dnDrp1-K38A decreases them. Furthermore, Bcl-xL coimmunoprecipitates with Drp1 in tissue lysates, and in a recombinant system, Bcl-xL protein stimulates GTPase activity of Drp1. These findings suggest that Bcl-xL positively regulates Drp1 to alter mitochondrial function in a manner that stimulates synapse formation

Mitochondrial small conductance SK2 channels prevent glutamate-induced oxytosis and mitochondrial dysfunction.

Small conductance calcium-activated potassium (SK2/K(Ca)2.2) channels are known to be located in the neuronal plasma membrane where they provide feedback control of NMDA receptor activity. Here, we provide evidence that SK2 channels are also located in the inner mitochondrial membrane of neuronal mitochondria. Patch clamp recordings in isolated mitoplasts suggest insertion into the inner mitochondrial membrane with the C and N termini facing the intermembrane space. Activation of SK channels increased mitochondrial K+ currents, whereas channel inhibition attenuated these currents. In a model of glutamate toxicity, activation of SK2 channels attenuated the loss of the mitochondrial transmembrane potential, blocked mitochondrial fission, prevented the release of proapoptotic mitochondrial proteins, and reduced cell death. Neuroprotection was blocked by specific SK2 inhibitory peptides and siRNA targeting SK2 channels. Activation of mitochondrial SK2 channels may therefore represent promising targets for neuroprotective strategies in conditions of mitochondrial dysfunction