Zonisamide attenuates α-synuclein neurotoxicity by an aggregation-independent mechanism in a rat model of familial Parkinson's disease.

The anti-epileptic agent zonisamide (ZNS) has been shown to exert protective effects in neurotoxin-based mouse models of Parkinson disease. However, it is unknown whether ZNS can attenuate toxicity of familial Parkinson's disease-causing gene products. In this study, we investigated the effects of ZNS on neurodegeneration induced by expression of A53T α-synuclein in the rat substantia nigra using a recombinant adeno-associated virus vector. Expression of A53T α-synuclein yielded severe loss of nigral dopamine neurons and striatal dopamine nerve terminals from 2 weeks to 4 weeks after viral injection. Oral administration of ZNS (40 mg/kg/day) significantly delayed the pace of degeneration at 4 weeks after viral injection as compared with the vehicle group. This effect lasted until 8 weeks after viral injection, the final point of observation. ZNS treatment had no impact on the survival of nigrostriatal dopamine neurons in rats expressing green fluorescent protein. Quantification of striatal Ser129-phosphorylated α-synuclein-positive aggregates showed that these aggregates rapidly formed from 2 weeks to 4 weeks after viral injection. This increase was closely correlated with loss of nigrostriatal dopamine neurons. However, ZNS treatment failed to alter the number of all striatal Ser129-phosphorylated α-synuclein-positive aggregates, including small dot-like and large round structures. The number of these aggregates was almost constant at 4 weeks and 8 weeks after viral injection, although ZNS persistently prevented loss of nigrostriatal dopamine neurons during this period. Also, ZNS treatment did not affect the number of striatal aggregates larger than 10 µm in diameter. These data show that ZNS attenuates α-synuclein-induced toxicity in a manner that is independent of the formation and maturation of α-synuclein aggregates in an in vivo model of familial Parkinson's disease, suggesting that ZNS may protect nigrostriatal dopamine neurons by modulating cellular damage or a cell death pathway commonly caused by neurotoxins and α-synuclein.

Authentically phosphorylated α-synuclein at Ser129 accelerates neurodegeneration in a rat model of familial Parkinson's disease.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra (SN) and the appearance of fibrillar aggregates of insoluble α-synuclein (α-syn) called Lewy bodies (LBs). Approximately 90% of α-syn deposited in LBs is phosphorylated at serine 129 (Ser129). In contrast, only 4% of total α-syn is phosphorylated in normal brain, suggesting that accumulation of Ser129-phosphorylated α-syn is involved in the pathogenesis of PD. However, the role of Ser129 phosphorylation in α-syn neurotoxicity remains unclear. In this study, we coexpressed familial PD-linked A53T α-syn and G-protein-coupled receptor kinase 6 (GRK6) in the rat SN pars compacta using recombinant adeno-associated virus 2. Coexpression of these proteins yielded abundant Ser129-phosphorylated α-syn and significantly exacerbated degeneration of dopaminergic neurons when compared with coexpression of A53T α-syn and GFP. Immunohistochemical analysis revealed that Ser129-phosphorylated α-syn was preferentially distributed to swollen neurites. However, biochemical analysis showed that the increased expression of Ser129-phosphorylated α-syn did not promote accumulation of detergent-insoluble α-syn. Coexpression of catalytically inactive K215R mutant GRK6 failed to accelerate A53T α-syn-induced degeneration. Furthermore, introducing a phosphorylation-incompetent mutation, S129A, into A53T α-syn did not alter the pace of degeneration, even when GRK6 was coexpressed. Our study demonstrates that authentically Ser129-phosphorylated α-syn accelerates A53T α-syn neurotoxicity without the formation of detergent-insoluble α-syn, and suggests that the degenerative process could be constrained by inhibiting the kinase that phosphorylates α-syn at Ser129.

Phosphorylated alpha-synuclein at Ser-129 is targeted to the proteasome pathway in a ubiquitin-independent manner.

α-Synuclein (a-Syn) is a major component of fibrillar aggregates in Lewy bodies (LBs), a characteristic hallmark of Parkinson disease. Almost 90% of a-Syn deposited in LBs is phosphorylated at Ser-129. However, the role of Ser-129-phosphorylated a-Syn in the biogenesis of LBs remains unclear. Here, we investigated the metabolism of Ser-129-phosphorylated a-Syn. In SH-SY5Y cells, inhibition of protein phosphatase 2A/1 by okadaic acid, and inhibition of the proteasome pathway by MG132 or lactacystin accumulated Ser-129-phosphorylated a-Syn. However, these inhibitions did not alter the amounts of total a-Syn within the observation time. Inhibition of the autophagy-lysosome pathway by 3-methyladenine or chloroquine accumulated Ser-129-phosphorylated a-Syn in parallel to total a-Syn during longer incubations. Experiments using cycloheximide showed that Ser-129-phosphorylated a-Syn diminished rapidly (t(½) = 54.9 ± 6.4 min), in contrast to the stably expressed total a-Syn. The short half-life of Ser-129-phosphorylated a-Syn was blocked by MG132 to a greater extent than okadaic acid. In rat primary cortical neurons, either MG132, lactacystin, or okadaic acid accumulated Ser-129-phosphorylated a-Syn. Additionally, we did not find that phosphorylated a-Syn was ubiquitinated in the presence of proteasome inhibitors. These data show that Ser-129-phosphorylated a-Syn is targeted to the proteasome pathway in a ubiquitin-independent manner, in addition to undergoing dephosphorylation. The proteasome pathway may play a role in the biogenesis of Ser-129-phosphorylated a-Syn-rich LBs.