Objective: In order to see possible involvement of tyrosine hydroxylase on the etiology of Parkinson's disease, we examined the effect of proteasomal inhibition on the tyrosine hydroxylase (TH) protein in NGF-differentiated PC12D cells.
Background: Recent researches clarified that mutations in the genes related to ubiquitin-proteasome system (UPS) cause familial Parkinson's disease (PD). It suggests that cytoplasmic protein aggregates, which can be formed by dysfunction of UPS, may be involved in etiology of PD. However, there is no sufficient evidence for presence of inclusion bodies only in monoaminergic neurons in PD.
Methods: PC12D cells were cultured in the DMEM containing 250nM MG-132, a 26S proteasomal inhibitor, or vehicle for 12, 24 and 48 hours. For double-labeling immunofluorescent analyses, cells were fixed and stained with each antibody. For the Western blot analyses, cells were lysed and ultracentrifuged to separate soluble and insoluble components. For immunohistochemistry in human tissue, formalin-fixed brain of PD patient was stained.
Results: We observed that TH phosphorylated at Ser40 formed cytoplasmic aggregates, while choline acetyltransferase (ChAT), an enzyme for biosynthesis of acetylcholine, did not form cytoplasmic aggregates as observed in TH. The TH positive signal was also immunopositive to ubiquitin. Phosphorylated TH was colocalized with ubiquitin. In the Western blot analysis, phosphorylated TH was accumulated in the insoluble fractions, but not in -Actin. In order to examine the presence of TH protein in Lewy body, we stained Lewy body with anti-TH antibody. We found that Lewy body was immunoreactive to TH.
Conclusions: We showed for the first time that phosphorylated and ubiquitinated TH readily make insoluble aggregates after proteasomal inhibition in NGF-differentiated PC12D cells. We suggest that insoluble aggregates of phosphorylated TH may be involved in formation of Lewy bodies in dopaminergic neurons in the brain of PD patients.
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