Ziram Causes Dopaminergic Cell Damage by Inhibiting E1 Ligase of the Proteasome

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 ␣-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. Parkinson disease (PD)2 is a common neurodegenerative disease characterized by relatively selective degeneration of dopaminergic (DA) neurons in the substantia nigra (nigrostriatal neurons). The etiology probably involves both environmental and genetic factors including pesticide exposure (1-3). Hundreds of pesticides are used alone or in combinations making it difficult to separate individual effects. 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 PD. Impaired UPS activity has been reported in the brains of patients with PD, and mutations in two UPS genes, Parkin and UCHL-1, cause rare genetic forms of PD (4). Although these results are not universally reproduced (5-7), in some studies administration of UPS inhibitors to rodents recapitulates many of the clinical and pathological aspects of PD (8 -10). We hypothesized that chronic pesticide exposure may increase the risk of developing PD by inhibiting the UPS. We screened several pesticides for their ability to inhibit the UPS and found a number of toxicants that can lower activity at relevant concentrations (11). We then focused on dithiocarbamate fungicides because they were found to be one of the most potent UPS inhibitors and are widely used in crop protection.In the present study, zinc dimethyldithiocarbamate (ziram) was one of several dimethyl-and diethyldithiocarbamates found to inhibit the UPS at 0.15-1 M. Furthermore, ziram increased ␣-syn...

Pesticides and Parkinson’s Disease: is It in Your genes?

“…Using this approach, we confirmed the association of paraquat with PD (especially if co-exposed to maneb or ziram) and identified previously unrecognized pesticides that increase the risk of PD [11][12][13][14][15]. For example, ziram, a dithiocarbamate fungicide, was found to inhibit E1 ligase of the UPS and selectively injure dopaminergic neurons in primary cultures and in mice [16]. Exposure to ziram in our PEG study subjects led to a 300% increase (odds ratio: 3) in risk of developing PD [12].…”

A Novel “Molecular Tweezer” Inhibitor of α-Synuclein Neurotoxicity in Vitro and in Vivo

“…Rare mutations or duplication of the SNCA gene, which encodes α-syn, cause dominantly inherited PD, and α-syn is the major component of Lewy bodies and Lewy neurites, the hallmark lesions in the brain of patients with PD [1][2][3][4][5]. Increased concentration of wild-type (WT) α-syn due to genetic or environmental insults appears to be sufficient for increasing the risk of developing PD, and in high enough levels it can cause PD [5][6][7][8].…”

Aldehyde dehydrogenase inhibition as a pathogenic mechanism in Parkinson disease

“…Primary Neuronal Cultures. Primary neuronal cultures were prepared using a protocol adapted from Rayport et al and previously described (35,47). Briefly, mesencephalic cells containing the substantia nigra pars compacta (excluding the ventral tegmental area) were dissected from coronal sections of brains from postnatal day 1 or 2 rat pups, dissociated in papain, and plated onto glial cells at densities of 4 × 10 5 per coverslip.…”

“…Some cultures were incubated with antibodies against TH and α-synuclein (1:500, anti-mouse, BD Biosciences). Relative levels of α-synuclein in TH + cells were determined as previously described (35), using the GNU Image Manipulation Program (GIMP 2.6).…”

“…It is remarkable that the environmental impact of benomyl exposure in the present work yielded similar results in vitro and in vivo as their targeted genetic approach. This is in contrast to another fungicide, ziram, which increases α-synuclein levels and damages dopaminergic cells by inhibiting E1 ligase activity in the UPS (35). Benomyl appears to act via a novel mechanism (ALDH inhibition), damaging dopaminergic cells via alterations in DA homeostasis, independent of α-synuclein.…”