Accumulation of phosphorylated α‐synuclein in dopaminergic neurons of transgenic mice that express human α‐synuclein

Abstract

Parkinson's disease is neuropathologically characterized by the presence of Lewy bodies, whose major component is alpha-synuclein. We had previously generated transgenic mice that expressed human alpha-synuclein carrying an Ala53Thr point mutation (halpha-syn140m) under the control of the rat tyrosine hydroxylase promoter and found that halpha-syn140m was localized not only in the cytoplasm but also in the nuclei of mesencephalic dopaminergic neurons. In the present study, we carried out immunohistochemical analysis of the brain of Tg mice using anti-PSer129, an antibody that specifically recognizes alpha-synuclein phosphorylated at Ser129. The antibody detected only phosphorylated halpha-syn140m, whereas phosphorylation of endogenous alpha-synuclein, if any, was below the detection limit of the method employed. The analysis showed that approximately one-third of the halpha-syn140m-positive neurons in the midbrain of heterozygous Tg mice were concomitantly reactive to anti-PSer129. The ratio almost doubled in homozygotes, indicating that the phosphorylation level depends directly on the amount of substrate. In addition, the ratio did not change at least up to 48 weeks of age. These data strongly suggest that halpha-syn140m underwent constitutive phosphorylation and that the phosphorylation level was maintained to a certain level until the aged stages. Remarkably, halpha-syn140m localized in the nuclei seemed to be preferentially phosphorylated compared with that in the cytoplasm. Among kinases that have been reported to be involved in the phosphorylation of alpha-synuclein, the beta subunit of casein kinase-2 was detected in the nuclei by immunohistochemistry. These data imply that at least casein kinase-2 is involved in the phosphorylation of halpha-syn140m in the Tg mice.

The phosphorylation state of Ser-129 in human α-synuclein determines neurodegeneration in a rat model of Parkinson disease

“…Phosphorylated S129 was also found to accumulate in a transgenic mouse model expressing the A53T mutant of human ␣-syn (20). In this study, phosphorylation of endogenous ␣-syn was not detected but the human mutant ␣-syn was phosphorylated on Ser-129 and, in contrast to our study, seemed to preferentially localize to the nucleus.…”

“…In this study, phosphorylation of endogenous ␣-syn was not detected but the human mutant ␣-syn was phosphorylated on Ser-129 and, in contrast to our study, seemed to preferentially localize to the nucleus. However, like our study, the accumulation of phosphorylated ␣-syn was not associated with toxicity (20).…”

“…2A). This was Ϸ3.9-fold higher than values reported for endogenous ␣-syn in normal mice [36 ng/100 g (20)]. As expected, no human ␣-syn expression was detected on the uninjected left side (Fig.…”

“…Several groups have reported that Lewy bodies predominately consist of ␣-syn that is phosphorylated at Ser-129 (16,18), whereas soluble, monomeric ␣-syn contains relatively little of the S129-phosphorylated form (15). In addition, coexpression of human ␣-syn with G proteinreceptor kinase 5 (GRK5), one of several kinases that is capable of phosphorylating Ser-129 (14,20), increased the formation of ␣-syn aggregates in cultured cells (21). In another study, coexpression of synphilin 1 with the S129A mutation of ␣-syn in cultured cells reduced aggregation and interaction with synphilin 1 (22), another component found in Lewy bodies.…”

“…Casein kinase 1 and 2 and GRK2 and 5 are all capable of phosphorylating S129 (14,15,17,20,21), and no specific phosphatase has yet been identified. Given the multiple cellular locations of ␣-syn, it is possible that several kinases are functional in vivo.…”