Identification and Analysis of Gene Product Modifiers of α-Synuclein Toxicity in the Fruit Fly (D. Melanogaster).

Abstract

Parkinson s disease (PD) is a progressive neurodegenerative disorder, pathologically characterized by loss of dopaminergic neurons in the substantia nigra pars compacta. Various mutations in a-Synuclein (SNCA, PARK1/4) result in autosomal dominant, inherited PD. Overexpression or mutation of a-Synuclein is associated with protein aggregation and results in disturbance to a number of cell systems, including the ubiquitin-proteasome system and mitochondria. To identify new genetic modifiers of α-Synuclein neurotoxicity, fruit flies (Drosophila melanogaster) expressing human a-Synuclein with the familial PD mutation [A53T] in dopaminergic neurons, in combination with a specific chromosomal deletion, were screened for aging-dependent loss of brain dopamine (DA) using high performance liquid chromatography. Sub-screening within deficiencies for individual genes using P-element gene disruption and RNA interference fly lines revealed that decreased expression of the mitochondrial chaperone protein, tumor necrosis factor receptor associated protein-1 (TRAP1), enhanced a-Synuclein[A53T] neurotoxicity. Flies showed an enhanced loss of brain DA, dopaminergic neurons and climbing ability with time. In addition, sensitivity to oxidative stress treatment (paraquat or hydrogen peroxide) was enhanced. Overexpression of human TRAP1 rescued these phenotypes. Similarly, coincident overexpression of both human TRAP1 and a-Synuclein[A53T] in rat primary cortical neurons rescued a-Synuclein-induced sensitivity to rotenone treatment. In human embryonic kidney- 293 (HEK293) cells, small interfering RNA directed against TRAP1 enhanced a-Synuclein[A53T] induced sensitivity to hydrogen peroxide or rotenone. TRAP1 overexpression provided rescue. a-Synuclein[A53T] was localized to the mitochondria using cell fractionation and co-localization of fluorescent signals. a-Synuclein[A53T] can directly interfere with mitochondrial function, as it was found to inhibit Complex I activity and when in combination with siTRAP1, decreased mitochon drial membrane potential. These effects could be blocked by TRAP1 overexpression. A direct protein-protein interaction between TRAP1 and a-Synuclein[A53T] was found using immunoprecipitation. In addition, data from the primary deficiency screen was cross referenced with candidate gene data from a previously published Drosophila mitochondrial modifier screen. Seventeen additional candidate genes modifying a-Synuclein[A53T] toxicity in the fly, including Tiny Tim 50, bellweather and p70 S6k, were identified. These results show that a-Synuclein[A53T] toxicity is intimately connected to that of mitochondrial function and that rescue from toxicity in fly, rat primary neurons and human cell line can be achieved using overexpression of the mitochondrial chaperone TRAP1.