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  • Enhanced tyrosine hydroxylase activity induces oxidative stress, causes accumulation of autotoxic catecholamine metabolites, and augments amphetamine effects in vivo.

Enhanced tyrosine hydroxylase activity induces oxidative stress, causes accumulation of autotoxic catecholamine metabolites, and augments amphetamine effects in vivo.

Journal of neurochemistry (2021-05-16)
Laura M Vecchio, Patricia Sullivan, Amy R Dunn, Marie Kristel Bermejo, Rong Fu, Shababa T Masoud, Emil Gregersen, Nikhil M Urs, Reza Nazari, Poul Henning Jensen, Amy Ramsey, David S Goldstein, Gary W Miller, Ali Salahpour
ABSTRACT

In Parkinson's disease, dopamine-containing nigrostriatal neurons undergo profound degeneration. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis. TH increases in vitro formation of reactive oxygen species, and previous animal studies have reported links between cytosolic dopamine build-up and oxidative stress. To examine effects of increased TH activity in catecholaminergic neurons in vivo, we generated TH-over-expressing mice (TH-HI) using a BAC-transgenic approach that results in over-expression of TH with endogenous patterns of expression. The transgenic mice were characterized by western blot, qPCR, and immunohistochemistry. Tissue contents of dopamine, its metabolites, and markers of oxidative stress were evaluated. TH-HI mice had a 3-fold increase in total and phosphorylated TH levels and an increased rate of dopamine synthesis. Coincident with elevated dopamine turnover, TH-HI mice showed increased striatal production of H2 O2 and reduced glutathione levels. In addition, TH-HI mice had elevated striatal levels of the neurotoxic dopamine metabolites 3,4-dihydroxyphenylacetaldehyde and 5-S-cysteinyl-dopamine and were more susceptible than wild-type mice to the effects of amphetamine and methamphetamine. These results demonstrate that increased TH alone is sufficient to produce oxidative stress in vivo, build up autotoxic dopamine metabolites, and augment toxicity.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
2,2,2-Tribromoethanol, 97%
Sigma-Aldrich
Anti-Tyrosine Hydroxylase Antibody, Chemicon®, from rabbit
Sigma-Aldrich
Monoclonal Anti-Tyrosine Hydroxylase antibody produced in mouse, clone TH-16, ascites fluid
Sigma-Aldrich
3,4-Dihydroxy-L-phenylalanine, ≥98% (TLC)
Sigma-Aldrich
2-Methyl-2-butanol, ReagentPlus®, 99%
Sigma-Aldrich
Anti-Dopamine Transporter Antibody, NT, clone DAT-Nt, culture supernatant, clone DAT-Nt, Chemicon®
Sigma-Aldrich
Anti-GAPDH antibody, Mouse monoclonal, clone GAPDH-71.1, purified from hybridoma cell culture