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Genomic multiplication of the locus-encoding human alpha-synuclein (alpha-syn), a polypeptide with a propensity toward intracellular misfolding, results in Parkinson's disease (PD). Here we report the results from systematic screening of nearly 900 candidate genetic targets, prioritized by bioinformatic associations to existing PD genes and pathways, via RNAi knockdown. Depletion of 20 gene products reproducibly enhanced misfolding of alpha-syn over the course of aging in the nematode Caenorhabditis elegans. Subsequent functional analysis of seven positive targets revealed five previously unreported gene products that significantly protect against age- and dose-dependent alpha-syn-induced degeneration in the dopamine neurons of transgenic worms. These include two trafficking proteins, a conserved cellular scaffold-type protein that modulates G protein signaling, a protein of unknown function, and one gene reported to cause neurodegeneration in knockout mice. These data represent putative genetic susceptibility loci and potential therapeutic targets for PD, a movement disorder affecting approximately 2% of the population over 65 years of age.

alpha-Synuclein (alpha-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast alpha-syn inhibits endoplasmic reticulum (ER)-to-Golgi (ER-->Golgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER-->Golgi trafficking. The homologous Rab1 rescues alpha-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of alpha-syn pathobiology. In a cell-free system with purified transport factors alpha-syn inhibited ER-->Golgi trafficking in an alpha-syn dose-dependent manner. Vesicles budded efficiently from the ER, but their docking or fusion to Golgi membranes was inhibited. Thus, the in vivo trafficking problem is due to a direct effect of alpha-syn on the transport machinery. By ultrastructural analysis the earliest in vivo defect was an accumulation of morphologically undocked vesicles, starting near the plasma membrane and growing into massive intracellular vesicular clusters in a dose-dependent manner. By immunofluorescence/immunoelectron microscopy, these clusters were associated both with alpha-syn and with diverse vesicle markers, suggesting that alpha-syn can impair multiple trafficking steps. Other Rabs did not ameliorate alpha-syn toxicity in yeast, but RAB3A, which is highly expressed in neurons and localized to presynaptic termini, and RAB8A, which is localized to post-Golgi vesicles, suppressed toxicity in neuronal models of PD. Thus, alpha-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive.

Alpha-synuclein (alphaSyn) misfolding is associated with several devastating neurodegenerative disorders, including Parkinson's disease (PD). In yeast cells and in neurons alphaSyn accumulation is cytotoxic, but little is known about its normal function or pathobiology. The earliest defect following alphaSyn expression in yeast was a block in endoplasmic reticulum (ER)-to-Golgi vesicular trafficking. In a genomewide screen, the largest class of toxicity modifiers were proteins functioning at this same step, including the Rab guanosine triphosphatase Ypt1p, which associated with cytoplasmic alphaSyn inclusions. Elevated expression of Rab1, the mammalian YPT1 homolog, protected against alphaSyn-induced dopaminergic neuron loss in animal models of PD. Thus, synucleinopathies may result from disruptions in basic cellular functions that interface with the unique biology of particular neurons to make them especially vulnerable.

Parkinson's disease (PD) is linked genetically to proteins that function in the management of cellular stress resulting from protein misfolding and oxidative damage. Overexpression or mutation of alpha-synuclein results in the formation of Lewy bodies and neurodegeneration of dopaminergic (DA) neurons. Human torsinA, mutations in which cause another movement disorder termed early-onset torsion dystonia, is highly expressed in DA neurons and is also a component of Lewy bodies. Previous work has established torsins as having molecular chaperone activity. Thus, we examined the ability of torsinA to manage cellular stress within DA neurons of the nematode Caenorhabditis elegans. Worm DA neurons undergo a reproducible pattern of neurodegeneration after treatment with 6-hydroxydopamine (6-OHDA), a neurotoxin commonly used to model PD. Overexpression of torsins in C. elegans DA neurons results in dramatic suppression of neurodegeneration after 6-OHDA treatment. In contrast, expression of either dystonia-associated mutant torsinA or combined overexpression of wild-type and mutant torsinA yielded greatly diminished neuroprotection against 6-OHDA. We further demonstrated that torsins seem to protect DA neurons from 6-OHDA through downregulating protein levels of the dopamine transporter (DAT-1) in vivo. Additionally, we determined that torsins protect robustly against DA neurodegeneration caused by overexpression of alpha-synuclein. Using mutant nematodes lacking DAT-1 function, we also showed that torsin neuroprotection from alpha-synuclein-induced degeneration occurs in a manner independent of this transporter. Together, these data have mechanistic implications for movement disorders, because our results demonstrate that torsin proteins have the capacity to manage sources of cellular stress within DA neurons.