Selective loss of dopaminergic neurons and formation of Lewy body‐like aggregations in α‐synuclein transgenic fly neuronal cultures

Abstract

A key pathological feature of Parkinson's disease (PD) is the selective loss of dopaminergic neurons accompanied by the formation of Lewy bodies (LB). Given the complex nature of the disease, it is imperative to develop a model system suitable for molecular and cellular manipulation in order to study the mechanisms underlying the pathogenesis of PD. Here, we report that a new in vitro model of PD has been developed by using Drosophila melanogaster primary neuronal cultures expressing a human mutant alpha-synuclein (alpha-Syn; A30P). The selective loss of dopaminergic (DA) neurons was observed when alpha-Syn was pan-neuronally expressed while non-dopaminergic neurons (e.g. GABAergic) were not influenced. This degeneration was also observed even when alpha-Syn was specifically expressed in DA neurons, demonstrating alpha-Syn toxicity is DA cell-autonomous. In all experiments, cultures 5 days or older showed clear degeneration of DA neurons whereas this degeneration was not significant in 3-day-old cultures. In addition, there were intracellular aggregations in 5-day or older alpha-Syn neurons that were recognized by anti-alpha-Syn or ubiquitin antibodies, demonstrating the formation of LB-like inclusions. By contrast, no such aggregations were found in 3-day-old neurons. The results demonstrate that mutated human alpha-Syn expressed in Drosophila primary neuronal cultures causes the selective loss of DA neurons and the formation of cellular aggregations. Therefore, this is one of the first in vitro models recapitulating two important cellular features of PD and will be useful in examining mechanisms underlying selective neurodegeneration mediated by alpha-Syn.

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).…”

Parkinson's disease as a member of Prion-like disorders

“…These primary cultures have been derived from rat, mouse, flies and even from human embryonic stem cells (Park and Lee, 2006;Rideout et al, 2004;Zhou et al, 2002Zhou et al, , 2000. Besides the species, also the region of the nervous systems from which the cell derive can vary from study to study, cerebellar granular cells (Monti et al, 2007), dorsal root ganglia , cortical and midbrain neurons (Rideout et al, 2003) and enteric neurons (Paillusson et al, 2013) just to name a few.…”

Rapamycin protects dopaminergic neurons against rotenone-induced cell death in primary mesencephalic cell culture

“…The disease is characterized clinically by a triad of cardinal motor symptoms including bradykinesia, tremors and rigidity [13], and pathologically by the selective loss of dopamine neurons in the substantia nigra pars compacta (SNpc) and the formation of Lewy bodies [27]. Although the etiology of PD is still unclear, there is growing evidence indicating that intracellularly oxidative stress, mitochondrial damage, lysosomal dysfunction, neuroinflammatory changes and formation of pathologic inclusions contribute to the pathology of the disease [9].…”