Abstract
alpha-Synuclein is a major protein component deposited in Lewy bodies and Lewy neurites that is extensively phosphorylated at Ser(129), although its role in neuronal degeneration is still elusive. In this study, several apoptotic pathways were examined in alpha-synuclein-overexpressing SH-SY5Y cells. Following the treatment with rotenone, a mitochondrial complex I inhibitor, wild type alpha-synuclein-overexpressing cells demonstrated intracellular aggregations, which shared a number of features with Lewy bodies, although cells overexpressing the S129A mutant, in which phosphorylation at Ser(129) was blocked, showed few aggregations. In wild typealpha-synuclein cells treated with rotenone, the proportion of phosphorylated alpha-synuclein was about 1.6 times higher than that of untreated cells. Moreover, induction of unfolded protein response (UPR) markers was evident several hours before the induction of mitochondrial disruption and caspase-3 activation. Eukaryotic initiation factor 2alpha, a member of the PERK pathway family, was remarkably activated at early phases. On the other hand, the S129A mutant failed to activate UPR. Casein kinase 2 inhibitor, which decreased alpha-synuclein phosphorylation, also reduced UPR activation. The alpha-synuclein aggregations were colocalized with a marker for the endoplasmic reticulum-Golgi intermediate compartment. Taken together, it seems plausible that alpha-synuclein toxicity is dependent on the phosphorylation at Ser(129) that induces the UPRs, possibly triggered by the disturbed endoplasmic reticulum-Golgi trafficking.
Highlights
Ing neurons, the mechanism that underlies LB biogenesis is poorly understood [2, 3]. ␣-Synuclein is a 140-amino acid protein physiologically localized in presynaptic terminals [4, 5] and pathologically aggregated in hallmark inclusions, such as LB and Lewy neurites [6]
Serine 129 phosphorylation is thought to be one of the most important events [12,13,14,15], because it has been reported that almost 90% of ␣-synuclein in LB is phosphorylated at serine 129 [12] and that the serine 129 phosphorylation is closely associated with aggregate formation in cellular models [16]
In this study using a cellular model overexpressing wild type (WT) ␣-synuclein, we showed that the appearance of unfolded protein response (UPR) markers, especially activation of the PERK (PKR-like ER kinase)
Summary
Expression Construct and Cell Culture—The WT ␣-synuclein cDNA was subcloned into the pUC18 vector at SalI and SphI sites and the S129A mutant was generated by site-directed mutagenesis (Takara LA PCRTM in vitro mutagenesis kit; Takara Biomedicals, Tokyo, Japan). To examine the effect of CK2 inhibitor on eIF2␣ phosphorylation, SH-SY5Y cell lines were incubated with 10 g/l of tunicamycin (Calbiochem) and 0.2–5 M of DMAT Protein samples from these cells were analyzed by Western blots. The cells were washed three times Induced Intracellular Aggregates in ␣-Synuclein-overexpressing with PBS, resuspended into a buffer containing 50 mM Tris/ Cells—Following the transfection of WT or S129A mutant. The rela- expressing cells, the latter failed to show positive tive activity was standardized by the protein concentrations bands for anti-phosphorylated ␣-synuclein antibody (Fig. 1A). In WT ␣-synuclein-overexpressing cells, activated caspase-3-positive cells were increased after the exposure to rotenone (Fig. 3B). The incidence of activated caspase-3-positive cells was significantly higher in the WT than in the CAT and S129A mutants after 72 h of exposure of rotenone (Fig. 3B). The three target genes were MDG1/Erdj, a specific target of the Xbp-1 pathway
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