Disruption of dopamine homeostasis underlies selective neurodegeneration mediated by α‐synuclein

Abstract

A key challenge in Parkinson's disease research is to understand mechanisms underlying selective degeneration of dopaminergic neurons mediated by genetic factors such as alpha-synuclein (alpha-Syn). The present study examined whether dopamine (DA)-dependent oxidative stress underlies alpha-Syn-mediated neurodegeneration using Drosophila primary neuronal cultures. Green fluorescent protein (GFP) was used to identify live dopaminergic neurons in primary cultures prepared on a marked photoetched coverslip, which allowed us to repeatedly access preidentified dopaminergic neurons at different time points in a non-invasive manner. This live tracking of GFP-marked dopaminergic neurons revealed age-dependent neurodegeneration mediated by a mutant human alpha-Syn (A30P). Degeneration was rescued when alpha-Syn neuronal cultures were incubated with 1 mm glutathione from Day 3 after culturing. Furthermore, depletion of cytoplasmic DA by 100 microm alpha-methyl-p-tyrosine completely rescued the early stage of alpha-Syn-mediated dopaminergic cell loss, demonstrating that DA plays a major role in oxidative stress-dependent neurodegeneration mediated by alpha-Syn. In contrast, overexpression of a Drosophila tyrosine hydroxylase gene (dTH1) alone caused DA neurodegeneration by enhanced DA synthesis in the cytoplasm. Age-dependent dopaminergic cell loss was comparable in alpha-Syn vs dTH1-overexpressed neuronal cultures, indicating that increased DA levels in the cytoplasm is a critical change downstream of mutant alpha-Syn function. Finally, overexpression of a Drosophila vesicular monoamine transporter rescued alpha-Syn-mediated neurodegeneration through enhanced sequestration of cytoplasmic DA into synaptic vesicles, further indicating that a main cause of selective neurodegeneration is alpha-Syn-induced disruption of DA homeostasis. All of these results demonstrate that elevated cytoplasmic DA is a main factor underlying the early stage of alpha-Syn-mediated neurodegeneration.

Decaffeinated Coffee and Nicotine-Free Tobacco Provide Neuroprotection in Drosophila Models of Parkinson's Disease through an NRF2-Dependent Mechanism

“…Previous work has shown that the expression of human ␣-synuclein in primary neuronal cultures from Drosophila embryos induces a progressive and selective loss of dopamine neurons over a period of several days (Park and Lee, 2006;Park et al, 2007). However, we found that supplementing the culture media with either a coffee extract or a tobacco extract completely suppressed the loss of THpositive neurons induced by ␣-synuclein expression in this cell culture system (Fig.…”

“…Primary neuron culture. Primary DA neuron cultures were prepared as described previously (Park and Lee, 2006;Park et al, 2007) and grown for 3 d. Decaffeinated coffee and nicotine-free tobacco extracts were added to cultures at a final dilution of 1:2000. After 6 d of exposure to coffee and tobacco extracts, DA neurons were detected and quantified relative to 4Ј,6Ј-diamidino-2-phenylindole-positive cells, as described previously (Park and Lee, 2006;Park et al, 2007).…”

Targeting α-Synuclein as a Parkinson’s Disease Therapeutic

“…Indeed, early reports demonstrated that oxidative conditions in vitro [153] and in cultured cells [154] promote α-synuclein aggregation to amyloid-like structures. Moreover, using primary neurons from transgenic Drosophila expressing A30P mutant α-synuclein, it was shown that lessening dopamine-dependent oxidative stress with glutathione treatment or by sequestering dopamine in vesicles reduced toxicity [155]. In addition to iron, metals such as and copper, calcium, aluminum, and zinc have been shown to promote α-synuclein aggregation, perhaps by neutralizing the negative C-terminal charge, thereby promoting a partially folded amyloidogenic intermediate [156,157], or through other redox-dependent interactions.…”

Modelling Parkinson’s Disease in Drosophila

“…DJ-1 and dLRRK function seem to provide essential protection against oxidative stress also in Drosophila but the mechanisms involved remain to be elucidated (dashed arrow represents possible association with mitochondria). Elevating the levels of glutathione-S-transferase or SOD protects DA neurons in parkin mutants or in a-synuclein-expressing flies suggesting a central role of oxidative stress in those fly models for PD a-synuclein (Park et al 2007). Therefore, the increased toxicity of a-synuclein seen in flies under hyperoxia might be due to an accelerated auto-oxidation of dopamine, a process that normally takes place slowly during ageing under normal oxygen conditions.…”

“…Second, in different experiments aimed to counteract oxidative damage on a-synuclein transgenic flies, both, the histological and the locomotor phenotypes could be rescued (Feany and Bender;2000;Yang et al 2003;Pendleton et al 2002;Wassef et al 2007;Botella et al 2008). Third, Park et al (2007) have assessed the toxicity of a-synuclein using Drosophila primary neuronal cultures in an in vitro model for PD. In this work, the decrease in GFP intensity of traced cultivated neurons was used as a sign of neurodegeneration.…”