Dopamine determines the vulnerability of striatal neurons to the N-terminal fragment of mutant huntingtin through the regulation of mitochondrial complex II.

In neurodegenerative disorders associated with primary or secondary mitochondrial defects such as Huntington's disease (HD), cells of the striatum are particularly vulnerable to cell death, although the mechanisms by which this cell death is induced are unclear. Dopamine, found in high concentrations in the striatum, may play a role in striatal cell death. We show that in primary striatal cultures, dopamine increases the toxicity of an N-terminal fragment of mutated huntingtin (Htt-171-82Q). Mitochondrial complex II protein (mCII) levels are reduced in HD striatum, indicating that this protein may be important for dopamine-mediated striatal cell death. We found that dopamine enhances the toxicity of the selective mCII inhibitor, 3-nitropropionic acid. We also demonstrated that dopamine doses that are insufficient to produce cell loss regulate mCII expression at the mRNA, protein and catalytic activity level. We also show that dopamine-induced down-regulation of mCII levels can be blocked by several dopamine D2 receptor antagonists. Sustained overexpression of mCII subunits using lentiviral vectors abrogated the effects of dopamine, both by high dopamine concentrations alone and neuronal death induced by low dopamine concentrations together with Htt-171-82Q. This novel pathway links dopamine signaling and regulation of mCII activity and could play a key role in oxidative energy metabolism and explain the vulnerability of the striatum in neurodegenerative diseases.

BH3-only proteins Bid and Bim(EL) are differentially involved in neuronal dysfunction in mouse models of Huntington's disease.

Apoptosis, a cell death mechanism regulated by Bcl-2 family members, has been proposed as one of the mechanisms leading to neuronal loss in Huntington's disease (HD). Here we examined the regulation of Bcl-2 family proteins in three different mouse models of HD with exon 1 mutant huntingtin: the R6/1, the R6/1:BDNF+/-, and the Tet/HD94 in which the huntingtin transgene is controlled by the tetracycline-inducible system. Our results disclosed an increase in the levels of the BH3-only proteins Bid and Bim(EL) in the striatum of HD mouse models that was different depending on the stage of the disease. At 16 weeks of age, Bid was similarly enhanced in the striatum of R6/1 and R6/1:BDNF+/- mice, whereas Bim(EL) protein levels were enhanced only in R6/1:BDNF+/- mice. In contrast, at later stages of the disease, both genotypes displayed increased levels of Bid and Bim(EL) proteins. Furthermore, Bax, Bak, Bad, Bcl-2, and Bcl-x(L) proteins were not modified in any of the points analyzed. We next explored the potential reversibility of this phenomenon by analyzing conditional Tet/HD94 mice. Constitutive expression of the transgene resulted in increased levels of Bid and Bim(EL) proteins, and only the Bid protein returned to wild-type levels 5 months after mutant huntingtin shutdown. In conclusion, our results show that enhanced Bid protein levels represent an early mechanism linked to the continuous expression of mutant huntingtin that, together with enhanced Bim(EL), may be a reporter of the progress and severity of neuronal dysfunction.

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

Numerous 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.

Ciliary neurotrophic factor activates astrocytes, redistributes their glutamate transporters GLAST and GLT-1 to raft microdomains, and improves glutamate handling in vivo.

To study the functional role of activated astrocytes in glutamate homeostasis in vivo, we used a model of sustained astrocytic activation in the rat striatum through lentiviral-mediated gene delivery of ciliary neurotrophic factor (CNTF). CNTF-activated astrocytes were hypertrophic, expressed immature intermediate filament proteins and highly glycosylated forms of their glutamate transporters GLAST and GLT-1. CNTF overexpression produced a redistribution of GLAST and GLT-1 into raft functional membrane microdomains, which are important for glutamate uptake. In contrast, CNTF had no detectable effect on the expression of a number of neuronal proteins and on the spontaneous glutamatergic transmission recorded from striatal medium spiny neurons. These results were replicated in vitro by application of recombinant CNTF on a mixed neuron/astrocyte striatal culture. Using microdialysis in the rat striatum, we found that the accumulation of extracellular glutamate induced by quinolinate (QA) was reduced threefold with CNTF. In line with this result, CNTF significantly increased QA-induced [(18)F]-fluoro-2-deoxyglucose uptake, an indirect index of glutamate uptake by astrocytes. Together, these data demonstrate that CNTF activation of astrocytes in vivo is associated with marked phenotypic and molecular changes leading to a better handling of increased levels of extracellular glutamate. Activated astrocytes may therefore be important prosurvival agents in pathological conditions involving defects in glutamate homeostasis.

Brain mitochondrial defects amplify intracellular [Ca2+] rise and neurodegeneration but not Ca2+ entry during NMDA receptor activation.

According to the "indirect" excitotoxicity hypothesis, mitochondrial defects increase Ca2+ entry into neurons by rendering NMDA-R hypersensitive to glutamate. We tested this hypothesis by investigating in the rat striatum and cultured striatal cells how partial mitochondrial complex II inhibition produced by 3-nitropropionic acid (3NP) modifies the toxicity of the NMDA-R agonist quinolinate (QA). We showed that nontoxic 3NP treatment, leading to partial inhibition of complex II activity, greatly exacerbated striatal degeneration produced by slightly toxic QA treatment through an "all-or-nothing" process. The potentiation of QA-induced cell death by 3NP was associated with increased calpain activity and massive calpain-mediated cleavage of several postsynaptic proteins, suggesting major neuronal Ca2+ deregulation in the striatum. However, Ca2+ anomalies probably do not result from NMDA-R hypersensitivity. Indeed, brain imaging experiments using [(18)F]fluorodeoxyglucose indirectly showed that 3NP did not increase QA-induced ionic perturbations at the striatal glutamatergic synapses in vivo. Consistent with this, the exacerbation of QA toxicity by 3NP was not related to an increase in the QA-induced entry of 45Ca2+ into striatal neurons. The present results demonstrate that the potentiation of NMDA-R-mediated excitotoxicity by mitochondrial defects involves primarily intracellular Ca2+ deregulation, in the absence of NMDA-R hypersensitivity.

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.

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

Alix/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.