Abstract
The etiology of Parkinson disease (PD) is unclear but may involve environmental toxins such as pesticides leading to dysfunction of the ubiquitin proteasome system (UPS). Here, we measured the relative toxicity of ziram (a UPS inhibitor) and analogs to dopaminergic neurons and examined the mechanism of cell death. UPS (26 S) activity was measured in cell lines after exposure to ziram and related compounds. Dimethyl- and diethyldithiocarbamates including ziram were potent UPS inhibitors. Primary ventral mesencephalic cultures were exposed to ziram, and cell toxicity was assessed by staining for tyrosine hydroxylase (TH) and NeuN antigen. Ziram caused a preferential damage to TH+ neurons and elevated alpha-synuclein levels but did not increase aggregate formation. Mechanistically, ziram altered UPS function through interfering with the targeting of substrates by inhibiting ubiquitin E1 ligase. Sodium dimethyldithiocarbamate administered to mice for 2 weeks resulted in persistent motor deficits and a mild reduction in striatal TH staining but no nigral cell loss. These results demonstrate that ziram causes selective dopaminergic cell damage in vitro by inhibiting an important degradative pathway implicated in the etiology of PD. Chronic exposure to widely used dithiocarbamate fungicides may contribute to the development of PD, and elucidation of its mechanism would identify a new potential therapeutic target.
Highlights
Minergic (DA) neurons in the substantia nigra
Because no individual pesticide has been established by epidemiologic studies, we chose to perform an unbiased screen of potential toxicants for their ability to interfere with the ubiquitin-proteasome system (UPS), a biological pathway implicated in the etiology of Parkinson disease (PD)
We focused on dithiocarbamate fungicides because they were found to be one of the most potent UPS inhibitors and are widely used in crop protection
Summary
Chemicals—The test compounds (see Table 1) were from Chem Service (West Chester, PA), Sigma-Aldrich, or other commercial sources as the highest available purity, except for 7 and 8, which were synthesized by Karl Fisher in the Casida laboratory. Cortical glial feeder cells were established on polyornithine/laminin-coated coverslips, which formed the base of a 10-mm-diameter well cut into 35-mm culture dishes, until they reached confluency in ϳ6 days. To determine whether ziram treatment leads to high molecular weight ␣-synuclein species, we performed Western blots on the detergent-soluble fractions of culture lysates. Evaluation of E1 Ligase Activity—The effects of ziram on E1 ligase activity were investigated using Western blot analysis of treated cellular extracts to determine E1/E1-ubiquitin ratios and using purified enzyme preparations. The proteins were electrophoretically transblotted onto nitrocellulose paper, and immunoblots were performed as previously described [16] using anti-E1 ligase antibody (BIOMOL, Plymouth Meeting, PA). Fiber density was measured with a computer-assisted image analysis system as previously described [18]. The level of significance was set at p Ͻ 0.05
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