α-Synuclein (α-Syn), one of the most abundant proteins in the CNS, is known to be a major player in the neurodegeneration observed in Parkinson's disease. We currently report that transient focal ischemia upregulates α-Syn protein expression and nuclear translocation in neurons of the adult rodent brain. We further show that knockdown or knock-out of α-Syn significantly decreases the infarction and promotes better neurological recovery in rodents subjected to focal ischemia. Furthermore, α-Syn knockdown significantly reduced postischemic induction of phospho-Drp1, 3-nitrotyrosine, cleaved caspase-3, and LC-3 II/I, indicating its role in modulating mitochondrial fragmentation, oxidative stress, apoptosis, and autophagy, which are known to mediate poststroke neuronal death. Transient focal ischemia also significantly upregulated serine-129 (S129) phosphorylation (pα-Syn) of α-Syn and nuclear translocation of pα-Syn. Furthermore, knock-out mice that lack PLK2 (the predominant kinase that mediates S129 phosphorylation) showed better functional recovery and smaller infarcts when subjected to transient focal ischemia, indicating a detrimental role of S129 phosphorylation of α-Syn. In conclusion, our studies indicate that α-Syn is a potential therapeutic target to minimize poststroke brain damage.

MicroRNAs (miRNAs) are small non-coding RNAs that are known to control mRNA translation. Most miRNAs are transcribed from specific genes with well-defined promoters located throughout the genome. The mechanisms that control miRNA expression under normal and pathological conditions are not yet understood clearly. Peroxisome proliferator-activated receptor (PPAR) c is a ligand-activated transcription factor that is extensively distributed in the CNS. PPARc activation induces neuroprotection by modulating genes that contain peroxisome proliferator response elements (PPREs) in their promoters. We presently evaluated if PPARc modulates miRNA expression. When adult rats were treated with PPARc agonist rosiglitazone, expression of 28 miRNAs altered significantly (12 up-and 16 down-regulated; 3-119 fold) in the cerebral cortex compared to vehicle-treated controls. In silico analysis showed 1-5 PPREs in the putative promoter regions (within 1 Kb upstream of the transcription start site) of these miRNA genes. Cotransfection with a PPARc constitutively expressing vector significantly induced the miR-145 and miR-329 promoter vectors (each have four PPREs), which was curtailed by point mutations of PPREs in their promoters. Interestingly, the PPARc promoter has binding sites for both these miRNAs and transfection with miR-329 mimic and miR-145 mimic induced the PPARc expression. Thus, these studies show a cyclical induction of miRNAs and PPARc, indicating that the pleiotropic beneficial effects of PPARc agonists might be modulated in part by miRNAs and their down-stream mRNAs.

α-synuclein (α-syn) is one of the most abundant proteins in mammalian brain that is known to be a major player in the neurodegeneration observed in chronic conditions like Parkinson’s disease (PD). Several mechanisms were proposed for α-syn-induced neuronal death in PD including inflammation, oxidative stress, mitochondrial fission, and autophagy. Interestingly, all these pathophysiologic mechanisms also mediate neuronal death after acute CNS insults like stroke. Therefore, we examined whether α-syn contributes to post-stroke neuronal death and neurological dysfunction. Rodents were subjected to transient middle cerebral artery occlusion (tMCAO) and α-syn induction was silenced with a cocktail of α-syn-specific siRNAs. The levels of α-syn were estimated with qPCR and Western Blots. Post-ischemic motor deficit was evaluated with rotarod, beam walk and adhesive removal test 1 to 7 days after ischemia, and infarct volume was measured on cresyl violet stained brain sections. Cellular changes after ischemia were examined using immunofluorescence staining. Following tMCAO, α-syn levels were significantly up-regulated and phosphorylated at serine-129 in ischemic penumbra. Interestingly, in the ischemic brain non-phosphorylated and phosphorylated α-syn species translocated into the neuronal nuclei. We also observed that silencing α-syn by α-syn siRNA cocktail before tMCAO significantly decreased the infarction and improved the motor function. The neuroprotective effects were still observed when α-syn siRNAs were administered 30 min after the tMCAO. We found that the rate of survival at 7 days after a 90 min tMCAO was significantly higher in α-syn KO mice compared to wild-type control mice. Furthermore, α-syn suppression significantly mitigated the post-ischemic oxidative stress (8-OHdG and 3-NT), apoptosis (cleaved Caspase-3) and mitochondrial dysfunction (phospho-Drp1). Thus, we show that α-syn plays a critical role in neuronal death following stroke. Furthermore, phospho-S129 α-syn in the neuronal nuclei might be a potential mediator of the induction of ischemic brain damage. Preventing α-syn expression is a potential therapeutic target to minimize post-stroke brain damage.