Inactivation of tyrosine hydroxylase by nitration following exposure to peroxynitrite and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Abstract

The decrement in dopamine levels exceeds the loss of dopaminergic neurons in Parkinson's disease (PD) patients and experimental models of PD. This discrepancy is poorly understood and may represent an important event in the pathogenesis of PD. Herein, we report that the rate-limiting enzyme in dopamine synthesis, tyrosine hydroxylase (TH), is a selective target for nitration following exposure of PC12 cells to either peroxynitrite or 1-methyl-4-phenylpyridiniun ion (MPP+). Nitration of TH also occurs in mouse striatum after MPTP administration. Nitration of tyrosine residues in TH results in loss of enzymatic activity. In the mouse striatum, tyrosine nitration-mediated loss in TH activity parallels the decline in dopamine levels whereas the levels of TH protein remain unchanged for the first 6 hr post MPTP injection. Striatal TH was not nitrated in mice overexpressing copper/zinc superoxide dismutase after MPTP administration, supporting a critical role for superoxide in TH tyrosine nitration. These results indicate that tyrosine nitration-induced TH inactivation and consequently dopamine synthesis failure, represents an early and thus far unidentified biochemical event in MPTP neurotoxic process. The resemblance of the MPTP model with PD suggests that a similar phenomenon may occur in PD, influencing the severity of parkisonian symptoms.

Figure 1

Nitration of TH molecules results in proportional loss of activity. (A) Levels of immunoprecipitated TH in PC12 cell lysates treated with different concentrations of peroxynitirite. (B) Nitrated TH. The level of nitration was evaluated after staining with affinity purified polyclonal anti-nitrotyrosine antibodies and by amino acid analysis. (C) TH activity determined under optimal conditions.

Figure 2

(A) Immunoprecipitation of TH after 1-hr treatment of PC12 cells with different concentrations of MPP⁺. (B) Nitration of tyrosine residues in the immunoprecipitated TH was evaluated by reaction with the anti-nitrotyrosine antibodies. Representative data from three independent experiments.

Figure 3

MPTP treatment leads to protein tyrosine nitration of specific proteins in the mouse midbrain. Two-dimensional, chemiluminescence-enhanced anti-nitrotyrosine immunoblots of ventral midbrain tissue of C57/bl mice 3 hr after injection of either saline (A) or MPTP (B). MPTP injection resulted in nitration of selective proteins. Indicated by the arrow is a protein with the physical characteristics of mouse tyrosine hydroxylase listed in protein databases.

Figure 4

(A) Immunoprecipitation of striatal TH from MPTP-exposed mice immediately after exposure (time 0), 3- and 6-hr post exposure. TH was visualized with polyclonal anti-tyrosine hydorxylase antibodies. Representative data from four different striatal preparations. (B) Nitration of tyrosine residues in the immunoprecipitated TH was evaluated by reaction with the anti-nitrotyrosine antibodies. As a positive control (C), TH was laso immunoprecipitated from peroxynitrite treated PC12 cell lysates as described in Fig. 1. (C) Time course of striatal dopamine, tyrosine hydroxylase activity and TH protein levels after MPTP administration (n = 4–6).