Serine 129 phosphorylation reduces the ability of alpha-synuclein to regulate tyrosine hydroxylase and protein phosphatase 2A in vitro and in vivo.

Alpha-synuclein (a-Syn), a protein implicated in Parkinson disease, contributes significantly to dopamine metabolism. a-Syn binding inhibits the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Phosphorylation of TH stimulates its activity, an effect that is reversed by protein phosphatase 2A (PP2A). In cells, a-Syn overexpression activates PP2A. Here we demonstrate that a-Syn significantly inhibited TH activity in vitro and in vivo and that phosphorylation of a-Syn serine 129 (Ser-129) modulated this effect. In MN9D cells, a-Syn overexpression reduced TH serine 19 phosphorylation (Ser(P)-19). In dopaminergic tissues from mice overexpressing human a-Syn in catecholamine neurons only, TH-Ser-19 and TH-Ser-40 phosphorylation and activity were also reduced, whereas PP2A was more active. Cerebellum, which lacks excess a-Syn, had PP2A activity identical to controls. Conversely, a-Syn knock-out mice had elevated TH-Ser-19 phosphorylation and activity and less active PP2A in dopaminergic tissues. Using an a-Syn Ser-129 dephosphorylation mimic, with serine mutated to alanine, TH was more inhibited, whereas PP2A was more active in vitro and in vivo. Phosphorylation of a-Syn Ser-129 by Polo-like-kinase 2 in vitro reduced the ability of a-Syn to inhibit TH or activate PP2A, identifying a novel regulatory role for Ser-129 on a-Syn. These findings extend our understanding of normal a-Syn biology and have implications for the dopamine dysfunction of Parkinson disease.

Alpha-synuclein aggregation alters tyrosine hydroxylase phosphorylation and immunoreactivity: lessons from viral transduction of knockout mice.

Tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis, is frequently used as a marker of dopaminergic neuronal loss in animal models of Parkinson's disease (PD). We have been exploring the normal function of the PD-related protein alpha-synuclein (alpha-Syn) with regard to dopamine synthesis. TH is activated by the phosphorylation of key seryl residues in the TH regulatory domain. Using in vitro models, our laboratory discovered that alpha-Syn inhibits TH by acting to reduce TH phosphorylation, which then reduces dopamine synthesis [X.-M. Peng, R. Tehranian, P. Dietrich, L. Stefanis, R.G. Perez, Alpha-synuclein activation of protein phosphatase 2A reduces tyrosine hydroxylase phosphorylation in dopaminergic cells, J. Cell. Sci. 118 (2005) 3523-3530; R.G. Perez, J.C. Waymire, E. Lin, J.J. Liu, F. Guo, M.J. Zigmond, A role for alpha-synuclein in the regulation of dopamine biosynthesis, J. Neurosci. 22 (2002) 3090-3099]. We recently began exploring the impact of alpha-Syn on TH in vivo, by transducing dopaminergic neurons in alpha-Syn knockout mouse (ASKO) olfactory bulb using wild type human alpha-Syn lentivirus. At 3.5-21 days after viral delivery, alpha-Syn expression was transduced primarily in periglomerular dopaminergic neurons. Cells with modest levels of alpha-Syn consistently co-labeled for Total-TH. However, cells bearing aggregated alpha-Syn, as revealed by proteinase K or Thioflavin-S treatment had significantly reduced Total-TH immunoreactivity, but high phosphoserine-TH labeling. On immunoblots, we noted that Total-TH immunoreactivity was equivalent in all conditions, although tissues with alpha-Syn aggregates again had higher phosphoserine-TH levels. This suggests that aggregated alpha-Syn is no longer able to inhibit TH. Although the reason(s) underlying reduced Total-TH immunoreactivity on tissue sections await(s) confirmation, the dopaminergic phenotype was easily verified using phosphorylation-state-specific TH antibodies. These findings have implications not only for normal alpha-Syn function in TH regulation, but also for measuring cell loss that is associated with synucleinopathy.