α-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.

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 α-synuclein (α-Syn) with regard to dopamine synthesis. TH is activated by the phosphorylation of key seryl residues in the THregulatory-domain. Using in vitro models, our laboratory discovered that α-Syn inhibits TH by acting to reduce TH phosphorylation, which then reduces dopamine synthesis [31,33]. We recently began exploring the impact of α-Syn on TH in vivo, by transducing dopaminergic neurons in α-Syn knockout mouse (ASKO) olfactory bulb using wild type human α-Syn lentivirus. At 3.5 -21 days after viral delivery, α-Syn expression was transduced in periglomerular dopaminergic neurons. Cells with modest levels of α-Syn consistently co-labeled for Total-TH. However, cells bearing aggregated α-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 α-Syn aggregates again had higher phosphoserine-TH levels. This suggests that aggregated α-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 α-Syn function in TH regulation, but also for measuring cell loss that is associated with synucleinopathy. KeywordsParkinson's disease; lentivirus; knockout mice; transduction Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Short term regulation of TH depends on the phosphorylation of seryl residues, Ser19, Ser31, and Ser40, in the TH regulatory domain [5,14]. We previously demonstrated significant reduction in TH phosphorylation in dopaminergic cells that overexpress α-Syn [31,33]. The serine that regulates 14-3-3 binding to TH is phospho-Ser19 (PSer19), a site that is highly phosphorylated in dopaminergic neurons throughout the brain [36]. To evaluate the impact of α-Syn on TH in vivo, in the absence of endogenous α-Syn, we obtained ASKO mice [1] and generated wild type human α-Syn lentivirus using established methodologies [16]. Herein, we share our novel findings revealing that when α-Syn becomes aggregated, immunoreactivity (ir) for Total-TH appears to be reduced in dopaminergic...

α-Synuclein (α-Syn) is a chaperone-like protein that is highly implicated in Parkinson’s disease (PD) as well as in Dementia with Lewy Bodies (DLB). Rare forms of PD occur in individuals with mutations of α-Syn or triplication of wild type α-Syn, and in both PD and DLB the intraneuronal inclusions known as Lewy bodies contain aggregated α-Syn that is highly phosphorylated on serine 129. In neuronal cells and in the brains of α-Syn overexpressing transgenic mice, soluble α-Syn stimulates the activity of protein phosphatase 2A (PP2A), a major serine/threonine phosphatase. Serine 129 phosphorylation of α-Syn attenuates its stimulatory effects on PP2A and also accelerates α-Syn aggregation, however, it is unknown if aggregation of α-Syn into Lewy–bodies impairs PP2A activity. To assess for this, we measured the impact of α-Syn aggregation on PP2A activity in vitro and in vivo. In cell free assays, aggregated α-Syn had ~50 % less PP2A-stimulatory-effects than soluble recombinant α-Syn. Similarly in DLB and α-Syn triplication brains, which contain robust α-Syn aggregation with high levels of serine 129 phosphorylation, PP2A activity was also ~50% attenuated. As α-Syn normally stimulates PP2A activity, our data suggest that overexpression of α-Syn or sequestration of α-Syn into Lewy bodies has the potential to alter the phosphorylation state of key PP2A substrates; raising the possibility that all forms of synucleinopathy will benefit from treatments aimed at optimizing PP2A activity.