PINK1-induced mitophagy promotes neuroprotection in Huntington's disease.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by aberrant expansion of CAG repeat in the huntingtin gene. Mutant Huntingtin (mHtt) alters multiple cellular processes, leading to neuronal dysfunction and death. Among those alterations, impaired mitochondrial metabolism seems to have a major role in HD pathogenesis. In this study, we used the Drosophila model system to further investigate the role of mitochondrial damages in HD. We first analyzed the impact of mHtt on mitochondrial morphology, and surprisingly, we revealed the formation of abnormal ring-shaped mitochondria in photoreceptor neurons. Because such mitochondrial spheroids were previously detected in cells where mitophagy is blocked, we analyzed the effect of PTEN-induced putative kinase 1 (PINK1), which controls Parkin-mediated mitophagy. Consistently, we found that PINK1 overexpression alleviated mitochondrial spheroid formation in HD flies. More importantly, PINK1 ameliorated ATP levels, neuronal integrity and adult fly survival, demonstrating that PINK1 counteracts the neurotoxicity of mHtt. This neuroprotection was Parkin-dependent and required mitochondrial outer membrane proteins, mitofusin and the voltage-dependent anion channel. Consistent with our observations in flies, we demonstrated that the removal of defective mitochondria was impaired in HD striatal cells derived from HdhQ111 knock-in mice, and that overexpressing PINK1 in these cells partially restored mitophagy. The presence of mHtt did not affect Parkin-mediated mitochondrial ubiquitination but decreased the targeting of mitochondria to autophagosomes. Altogether, our findings suggest that mitophagy is altered in the presence of mHtt and that increasing PINK1/Parkin mitochondrial quality control pathway may improve mitochondrial integrity and neuroprotection in HD.

Nigrostriatal denervation does not affect glutamate transporter mRNA expression but subsequent levodopa treatment selectively increases GLT1 mRNA and protein expression in the rat striatum.

There is growing evidence that the loss of the nigrostriatal dopaminergic neurones induces an overactivity of the corticostriatal glutamatergic pathway which seems to be central to the physiopathology of parkinsonism. Moreover, glutamatergic mechanisms involving NMDA receptors have been shown to interfere with the therapeutical action of levodopa. Given the key role played by uptake processes in glutamate neurotransmission, this study examined the effects of nigrostriatal deafferentation and of levodopa treatment on the striatal expression of the glutamate transporters GLT1, GLAST and EAAC1 in the rat. No significant changes in striatal mRNA levels of these transporters were detected after either levodopa treatment (100 mg/kg; i.p., twice a day for 21 days) or unilateral lesion of the nigrostriatal pathway by intranigral 6-hydroxydopamine injection. In contrast, animals with the lesion subsequently treated with levodopa showed a selective increase (36%) in GLT1 mRNA levels in the denervated striatum versus controls. These animals also showed increased GLT1 protein expression, as assessed by immunostaining and western blotting. These data provide the first evidence that levodopa therapy may interfere with striatal glutamate transmission through change in expression of the primarily glial glutamate transporter GLT1. We further suggest that levodopa-induced GLT1 overexpression may represent a compensatory mechanism preventing neurotoxic accumulation of endogenous glutamate.

Impaired glutamate uptake in the R6 Huntington's disease transgenic mice.

Huntington's disease (HD) is a late-onset neurodegenerative disease for which the mutation is CAG/polyglutamine repeat expansion. The R6 mouse lines expressing the HD mutation develop a movement disorder that is preceded by the formation of neuronal polyglutamine aggregates. The phenotype is likely caused by a widespread neuronal dysfunction, whereas neuronal cell death occurs late and is very selective. We show that a decreased mRNA level of the major astroglial glutamate transporter (GLT1) in the striatum and cortex of these mice is accompanied by a concomitant decrease in glutamate uptake. In contrast, the expression of the glutamate transporters, GLAST and EAAC1, remain unchanged. The mRNA level of the astroglial enzyme glutamine synthetase is also decreased. These changes in expression occur prior to any evidence of neurodegeneration and suggest that a defect in astrocytic glutamate uptake may contribute to the phenotype and neuronal cell death in HD.

Serotonin depletion produces long lasting increase in striatal glutamatergic transmission.

The ability of serotonin (5-HT) to influence striatal glutamatergic transmission was examined by determining changes over time in glutamate extracellular levels, transporter expression and synaptosomal uptake in rats with lesion of serotonergic neurones. By 8 days after intraraphe injections of 5,7-dihydroxytryptamine, producing 80% decreases in striatal tissue 5-HT levels, no changes were observed in the glutamatergic transmission. When 5-HT depletion was almost complete (21 days post-lesion), high affinity glutamate uptake in striatal synaptosomal preparations was significantly increased (156% of control), although no changes in striatal GLT1, GLAST and EAAC1 mRNAs, and GLT1 protein were detected by in situ hybridization and immunohistochemistry. Meanwhile, the serotonin lesion produced large increases in basal extracellular levels of glutamate and glutamine (364% and 259%, respectively) determined in awake rats by in vivo microdialysis, whereas no change was observed in dopamine levels as compared with control rats. High potassium depolarization as well as L-trans-pyrrolidine-2,4-dicarboxylate, also induced larger increases in extracellular levels of glutamate in lesioned rats than in controls. Finally, similar changes in glutamate transmission were observed by 3 months post-lesion. These results suggest that 5-HT has a long lasting and tonic inhibitory influence on the striatal glutamatergic input, without affecting the basal dopaminergic transmission.

Differential effects of corticostriatal and thalamostriatal deafferentation on expression of the glutamate transporter GLT1 in the rat striatum.

This study compared the effects of the disruption of the two main presumably glutamatergic striatal inputs, the corticostriatal and thalamostriatal pathways, on GLT1 expression in the rat striatum, using in situ hybridization and immunohistochemistry. Unilateral ibotenate-induced thalamic lesion produced no significant changes in striatal GLT1 mRNA labeling and immunostaining as assessed at 5 and 12 days postlesion. In contrast, significant increases in both parameters were measured after bilateral cortical lesion by superficial thermocoagulation. GLT1 mRNA levels increased predominantly in the dorsolateral part of the striatum; there, the increases were significant at 5 (+84%), 12 (+101%), and 21 (+45%) but not at 35 days postlesion. GLT1 immunostaining increased significantly and homogeneously by 17-26% at 12 and 21 days postlesion. The increase in GLT1 expression at 12 days postlesion was further confirmed by western blot analysis; in contrast, a 36% decrease in glutamate uptake activity was measured at the same time point. These data indicate that striatal GLT1 expression depends on corticostriatal but not thalamostriatal innervation. Comparison of our results with previous data showing that cortical lesion by aspiration downregulates striatal GLT1 expression further suggests that differential changes in GLT1 expression, and thus presumably in glial cell function, may occur in the target striatum depending on the way the cortical neurons degenerate.

Characterization of striatal lesions produced by glutamate uptake alteration: cell death, reactive gliosis, and changes in GLT1 and GADD45 mRNA expression.

This study investigated the time course of the striatal lesions produced by continuous local injection of the glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylate (PDC) at the rate of 25 nmol/h in rats. The extent of the neurodegeneration area (defined as the lesion area) did not significantly vary with the duration of the PDC treatment between 3 and 14 days, but was markedly reduced 3 months after cessation of the 14-day treatment, probably reflecting striatal atrophy. After the 3-day treatment, the lesion zone showed calcium precipitates and marked microglial reaction contrasting with the reduction of astroglial labeling and loss of the glutamate transporter GLT1 mRNA expression; however reactive astrocytes were observed around the lesion. After the 14-day treatment, the lesion zone presented reactive astrocytes and microglia without calcification, and a partial recovery of GLT1 mRNA expression. Interestingly, the growth arrest DNA damage-inducible GADD45 mRNA expression was induced around the lesion after 3 days but inside the lesion after 14 days of treatment. Three months after the 14-day treatment, the astroglial reactivity persisted within the lesion whereas most of the other markers examined tended to normalize. These data suggest that defective glutamate transport induces primary death of neurons and dysfunction of astrocytes. They strongly implicate reactive astrocytes with GLT1 and GADD45 transcripts in preventing secondary neuronal death.

Continuous administration of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate produces striatal lesion.

This study examined the effects of chronic intrastriatal infusion of L-trans-pyrrolidine-2,4-dicarboxylate (PDC), a selective competitive inhibitor of high affinity glutamate transport systems, via osmotic minipumps in rats. Injection of PDC at the rate of 25 nmol/h for 14 days caused striatal lesion. Histological evaluation on frontal striatal sections showed that the lesion was circumscribed to a circular area showing a dramatic neuronal loss accompanied by gliosis and representing 30% of the whole striatal surface at the level of the injection site. A total loss of neurons expressing glutamate decarboxylase (GAD67), enkephalin or substance P mRNA was observed on a similar circular area, suggesting degeneration of the two populations of striatal efferent neurons. In the whole striatum outside the region devoided of hybridization signal, a selective 27% decrease in enkephalin mRNA expression occurred, suggesting a higher sensitivity of enkephalin neurons versus substance P neurons to glutamate uptake-mediated alterations. Injection of PDC at the rate of 25 nmol/h for 3 days produced striatal lesion of similar extent. In contrast, PDC at the rate of 5 nmol/h did not produce neuronal damage when administered over 14 days. This study provides new in vivo evidence that defective glutamate transport is one of the critical conditions that may give rise to toxicity of an endogenous transmitter system in the striatum, and may underlie neuronal death in neurodegenerative diseases.