Caractérisation de nouveaux acteurs de la mort des neurones granulaires de cervelet

Les acteurs moléculaires des modes de mort cellulaire alternatives à l'apoptose sont très mal connus en particulier pour les neurones. La mort des neurones granulaires de cervelet (NGC) induite par déplétion potassique présente toutes les caractéristiques de l'apoptose. L'induction de la voie de signalisation JNK constitue une étape primordiale dans l'activation des caspases. Cependant, la cascade de mort des NGC ne se limite pas à cette voie mais nécessite également une voie indépendante des caspases. Contrairement à d'autres modèles neuronaux, l'autophagie ne participe pas au déroulement de la mort des NGC. Nous avons également identifié la protéine Alix, qui joue un rôle crucial dans la formation des corps multivésiculaires, comme un acteur essentiel de la mort des NGC. Pour assurer sa fonction pro-apoptotique, Alix recrute les protéines Alg-2 et CIN85 suggérant qu'Alix pourrait connecter le trafic vésiculaire à la mort neuronale.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Survival response-linked Pyk2 activation during potassium depletion-induced apoptosis of cerebellar granule neurons.

International audienceNumerous extracellular stimuli trigger trans-autophosphorylation at Tyr402 of Pyk2, inducing its activation. Pyk2 is a key mediator of several signaling pathways and has been implicated in apoptosis induced by specific stress signals. We investigated whether Pyk2 participates in cerebellar granule neuron (CGN) apoptosis induced by the suppression of membrane depolarization. We demonstrate that shifting CGN cultures from 25 mM to 5 mM KCl-containing medium induces an early, transient 70% increase in phosphorylated Tyr402 and Tyr580 Pyk2 levels that is triggered by Ca(2+) released from intracellular stores and mediated by calmodulin (CaM). Overexpression of Pyk2 increases CGN survival after 24 h by 70% compared to the control, thus suggesting that Pyk2 is involved in an anti-apoptotic response to K+ lowering. Furthermore, we show that CGN grown in K25 medium exhibit detectable CaM-dependent Pyk2 activity. When silencing Pyk2 activity by expressing a dominant-negative form, only 40% of the transfected neurons were alive 24 h after transfection when compared to the control. Overall, the present findings demonstrate for the first time that Pyk2 is a critical mediator of CGN survival

Alix, a protein regulating endosomal trafficking, is involved in neuronal death.

International audienceAlix/AIP1 is a cytoplasmic protein, which was first characterized as an interactor of ALG-2, a calcium-binding protein necessary for cell death. Alix has also recently been defined as a regulator of the endo-lysosomal system. Here we have used post-mitotic cerebellar neurons to test Alix function in caspase-dependent and -independent cell death. Indeed, these neurons survived when cultured in 25 mm potassium-containing medium but underwent apoptosis soon after the extracellular potassium was lowered to 5 mm. In agreement with other studies, we show that caspases are activated after K+ deprivation, but that inhibition of these proteases, using the pancaspase inhibitor boc-aspartyl(OMe)-fluoromethylketone, has no effect on cell survival. Transfection experiments demonstrated that Alix overexpression is sufficient to induce caspase activation, whereas overexpression of its C-terminal half, Alix-CT, blocks caspase activation and cell death after K+ deprivation. We also define a 12-amino acid PXY repeat of the C-terminal proline-rich domain necessary for binding ALG-2. Deletion of this domain in Alix or in Alix-CT abolished the effects of the overexpressed proteins on neuronal survival, demonstrating that the ALG-2-binding region is crucial for the death-modulating function of Alix. Overall, these findings define the Alix/ALG-2 complex as a regulator of cell death controlling both caspase-dependent and -independent pathways. They also suggest a molecular link between the endo-lysosomal system and the effectors of the cell death machinery

Involvement of Mitochondrial Complex II Defects in Neuronal Death Produced by N-Terminus Fragment of Mutated Huntingtin

Alterations of mitochondrial function may play a central role in neuronal death in Huntington's disease (HD). However, the molecular mechanisms underlying such functional deficits of mitochondria are not elucidated yet. We herein showed that the expression of two important constituents of mitochondrial complex II, the 30-kDa iron-sulfur (Ip) subunit and the 70-kDa FAD (Fp) subunit, was preferentially decreased in the striatum of HD patients compared with controls. We also examined several mitochondrial proteins in striatal neurons that were infected with lentiviral vectors coding for the N-terminus part of huntingtin (Htt) with either a pathological (Htt171-82Q) or physiological (Htt171-19Q) polyglutamine tract. Compared with Htt171-19Q, expression of Htt171-82Q preferentially decreased the levels of Ip and Fp subunits and affected the dehydrogenase activity of the complex. The Htt171-82Q–induced preferential loss of complex II was not associated with a decrease in mRNA levels, suggesting the involvement of a posttranscriptional mechanism. Importantly, the overexpression of either Ip or Fp subunit restored complex II levels and blocked mitochondrial dysfunction and striatal cell death induced by Htt171-82Q in striatal neurons. The present results strongly suggest that complex II defects in HD may be instrumental in striatal cell death

D2 receptor inhibition reduces the DA-induced loss of mCII subunit expression and activity

Neurons were treated with 100 µ DA for 24 h in the presence or absence of the D2 receptor antagonists spiperone (SPI) and haloperidol (HAL), and the D1 receptor antagonist SCH23390 (SCH). () Quantification of the catalytic activity of mCII (succinate dehydrogenase). () Western blot analysis of Fp and Ip corresponding to the culture wells analyzed for mCII activity above. In both cases, the blockade of D2 receptors abolishes the loss of mCII triggered by DA, while D1 receptor blockade has no effect. * < 0.05; ANOVA and Fisher's PLSD test.<p><b>Copyright information:</b></p><p>Taken from "Dopamine determines the vulnerability of striatal neurons to the N-terminal fragment of mutant huntingtin through the regulation of mitochondrial complex II"</p><p></p><p>Human Molecular Genetics 2008;17(10):1446-1456.</p><p>Published online 11 Feb 2008</p><p>PMCID:PMC2367694.</p><p>© 2008 The Author(s)</p

Synergistic effects of mitochondrial complex II deficits and dopamine on striatal neuron degeneration

Cell viability assessed by the MTT assay after treatment for 24 h with 3-NP (75 µ), an irreversible inhibitor of mCII, and DA (100 µ). Note that when applied alone, 3-NP is non-toxic and DA is very mildly toxic. Treatment with both agents synergistically induces cell death. * < 0.0001; ANOVA and Fisher's PLSD test.<p><b>Copyright information:</b></p><p>Taken from "Dopamine determines the vulnerability of striatal neurons to the N-terminal fragment of mutant huntingtin through the regulation of mitochondrial complex II"</p><p></p><p>Human Molecular Genetics 2008;17(10):1446-1456.</p><p>Published online 11 Feb 2008</p><p>PMCID:PMC2367694.</p><p>© 2008 The Author(s)</p

Synergistic effects of mutated huntingtin and dopamine on striatal neuron degeneration

() Dose-dependent toxicity of DA (24 h treatment) assessed in striatal neurons (21 DIV) using the LDH and MTT cell death/viability assays. Note the clear-cut toxicity of DA above 250 µ. () Effect of 100 µ DA on the survival of striatal neurons 4 weeks after simultaneous transduction with a lentiviral vector encoding Htt171-82Q (mutated) or Htt171-19Q (wild-type), and a lentiviral vector encoding the reporter GFP. Neither DA alone nor Htt171-82Q alone is toxic to GFP-labeled neurons at this time point, but a combination of the two results in a 3-fold reduction in the number of neurons compared with Htt171-82Q controls. () Cell death assessed using the LDH assay in an experiment similar to that in (B), in the absence of lentiviral GFP. In the presence of DA, LDH release is significantly increased in cultures expressing Htt171-82Q. No LDH activity was observed without DA (data not shown). * < 0.05; ANOVA and Fisher's PLSD test.<p><b>Copyright information:</b></p><p>Taken from "Dopamine determines the vulnerability of striatal neurons to the N-terminal fragment of mutant huntingtin through the regulation of mitochondrial complex II"</p><p></p><p>Human Molecular Genetics 2008;17(10):1446-1456.</p><p>Published online 11 Feb 2008</p><p>PMCID:PMC2367694.</p><p>© 2008 The Author(s)</p

Microscopic analysis of the effects of Ip mCII subunit overexpression on DA-induced neuronal death

Cultures of striatal neurons expressing the reporter gene GFP, were transduced with a lentiviral vector coding the Ip subunit of mCII (lenti-Ip) or the DsRed reporter protein (lenti-DsR) as a control of transduction. () Representative field of view using phase contrast imaging (upper images) and fluorescence imaging of GFP (lower images) (Scale bar, 20 µm). () Histogram of GFP-positive cell counts in cultures transduced with lenti-Ds or lenti-Ip after a 24 h treatment with DA. Note the severity of neurodegeneration in cultures treated with DA, and the rescuing effect of Ip overexpression. * < 0.005; ANOVA and Fisher's PLSD test.<p><b>Copyright information:</b></p><p>Taken from "Dopamine determines the vulnerability of striatal neurons to the N-terminal fragment of mutant huntingtin through the regulation of mitochondrial complex II"</p><p></p><p>Human Molecular Genetics 2008;17(10):1446-1456.</p><p>Published online 11 Feb 2008</p><p>PMCID:PMC2367694.</p><p>© 2008 The Author(s)</p

Cumulative effects of dopamine and mutated huntingtin on mCII expression and catalytic activity

Measurement of Ip and Fp protein expression levels as indicated by western blotting, and mCII activity in striatal cultures treated for 24 h with increasing concentrations of DA. () Representative western blot showing the reduction of Ip and Fp expression, while levels of the alpha-subunit of complex V (C-V) remain essentially unchanged. () Quantification of protein levels for Ip and Fp after western blotting. () Dose-dependent effect of a 24 h DA treatment on the catalytic activity (succinate dehydrogenase) of mCII. () Quantification of protein levels for Bcl, the alpha-subunit of complex V (C-V), subunit 4 of complex IV (C-IV) and the 39 kDa subunit of complex I (C-I) after 100 µ DA treatment. () Changes in Ip and Fp mRNA levels over time during a 100 µ DA treatment, using quantitative RT–PCR. Note the transient down-regulation of both transcripts. () mCII activity in striatal neurons transduced with lentiviral Htt171-82Q (mutant) or Htt171-19Q (wild-type), before treatment with 100 µ DA or vehicle. The effects of DA and mutated Htt on the reduction of mCII activity are cumulative, and correspond to the synergistic effects on neuronal degeneration seen in Figure . * < 0.05; ** < 0.001; < 0.01; ANOVA and Fisher's PLSD test.<p><b>Copyright information:</b></p><p>Taken from "Dopamine determines the vulnerability of striatal neurons to the N-terminal fragment of mutant huntingtin through the regulation of mitochondrial complex II"</p><p></p><p>Human Molecular Genetics 2008;17(10):1446-1456.</p><p>Published online 11 Feb 2008</p><p>PMCID:PMC2367694.</p><p>© 2008 The Author(s)</p