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.

Alpha-synuclein is a presynaptic protein strongly implicated in Parkinson's disease (PD). Because dopamine neurons are invariably compromised during pathogenesis in PD, we have been exploring the functions of alpha-synuclein with particular relevance to dopaminergic neuronal cells. We previously discovered reduced tyrosine hydroxylase (TH) activity and minimal dopamine synthesis in stably-transfected MN9D cells overexpressing either wild-type or A53T mutant (alanine to threonine at amino acid 53) alpha-synuclein. TH, the ratelimiting enzyme in dopamine synthesis, converts tyrosine to L-dihydroxyphenylalanine (L-DOPA), which is then converted to dopamine by the enzyme, aromatic amino acid decarboxylase (AADC). We confirmed an interaction between alphasynuclein and AADC in striatum. We then sought to determine whether wild-type or A53T mutant alpha-synuclein might have affected AADC activity in dopaminergic cells. Using HPLC with electrochemical detection, we measured dopamine and related catechols after L-DOPA treatments to bypass the TH step. We discovered that while alpha-synuclein did not reduce AADC protein levels, it significantly reduced AADC activity and phosphorylation in our cells. These novel findings further support a role for alpha-synuclein in dopamine homeostasis and may explain, at least in part, the selective vulnerability of dopamine neurons that occurs in PD.

Bleomycin hydrolase is a multifaceted neutral cysteine protease with a suggested role in antigen presentation, homocysteine-thiolactone metabolism, and Alzheimer's disease pathogenesis. Deletion of the protease in mice results in increased neonatal mortality and dermatopathology. Immunohistochemical and behavioral studies of BLMH knockout mice were undertaken to further evaluate the role of the protease in the brain. No gross abnormalities in the central nervous system were observed upon preliminary histological examination of B6.129Blmh tm1Geh /J null animals. However, glial fibrillary acid protein immunohistochemistry revealed a global reactive astrogliosis in the aged null animals, indicative of undefined brain pathology. The role of BLMH in the brain was further explored by characterizing the behavioral phenotype of hybrid [129S6-Blmh tm1Geh /J X B6.129 Blmh tm1Geh /J]F1 null and littermate controls using multiple behavioral paradigms. In the water maze, deletion of BLMH resulted in poorer performance during water maze probe trials without detectable effect of the mutation on sensorimotor function. In addition, no age-dependent decline in discriminative performance on probe trials was observed in null animals. These data suggest a physiological non-redundant function for BLMH in the central nervous system. Keywords knockout; hippocampus; learning; memory; aging; Alzheimer's disease Bleomycin hydrolase (BLMH) is a highly conserved and unusual aminopeptidase with suggested importance in antigen presentation, homocysteine-thiolactone metabolism, and Alzheimer's disease (AD). A genetic polymorphism within the conserved carboxyterminal region of BLMH is a susceptibility locus for sporadic AD and has been reported by us (Montoya et al., 1998) and others (Papassotiropoulos et al., 2000) to be associated with an approximate two-fold increased risk for development of the disease, although this effect has not been replicated in all studies. Accumulation of β-amyloid peptide derived from proteolytic cleavage of the amyloid precursor protein is believed to be a critical event in the early pathogenesis of Correspondence should be addressed to: John S. Lazo, Department of Pharmacology, University of Pittsburgh, Biomedical Science Tower 3, Suite 10040, 3501 Fifth Avenue, Pittsburgh, PA 15260, E-mail: lazo@pitt.edu. 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. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2008 November 22. Published in final edited form as:Neuroscience. AD (Hardy and Allsop, 1991;Hardy and Higgins, 1992). In vitro st...

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