Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy, collectively referred to as synucleinopathies, are associated with a diverse group of genetic and environmental susceptibilities. The best studied of these is PD. α-Synuclein (α-syn) plays a key role in the pathogenesis of both familial and sporadic PD, but evidence linking it to other predisposition factors is limited. Here we report a strong genetic interaction between α-syn and the yeast orthologue of the PD-linked gene PARK9 (ATP13A2). Dopaminergic neuron loss caused by α-syn overexpression in animal and neuronal PD models is rescued by co-expression of PARK9. Further, knockdown of the PARK9 orthologue in C. elegans enhances α-syn misfolding. These data provide a direct functional connection between α-syn and another PD susceptibility locus. Manganese exposure is an environmental risk factor linked to PD and PD-like syndromes. Remarkably, we discovered that yeast PARK9 helps to protect cells from manganese toxicity. Our studies reveal a striking connection between PD genetics (α-syn and PARK9) and an environmental risk factor (PARK9 and manganese). Finally, we show that additional genes from our yeast screen, with diverse functions, are potent modifiers of α-syn-induced neuron loss in animals, establishing a diverse, highly conserved interaction network for α-syn.

␣-Synuclein (␣-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast ␣-syn inhibits endoplasmic reticulum (ER)-to-Golgi (ER3 Golgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER3 Golgi trafficking. The homologous Rab1 rescues ␣-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of ␣-syn pathobiology. In a cell-free system with purified transport factors ␣-syn inhibited ER3 Golgi trafficking in an ␣-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 ␣-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 ␣-syn and with diverse vesicle markers, suggesting that ␣-syn can impair multiple trafficking steps. Other Rabs did not ameliorate ␣-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, ␣-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive. endoplasmic reticulum ͉ Rab GTPase ͉ yeasts ͉ vesicle trafficking ͉ Golgi

Aβ (amyloid beta peptide) is an important contributor to Alzheimer’s disease (AD). We modeled Aβ toxicity in yeast by directing the peptide to the secretory pathway. A genome-wide screen for toxicity modifiers identified the yeast homolog of phosphatidylinositol binding clathrin assembly protein (PICALM) and other endocytic factors connected to AD whose relationship to Aβ was previously unknown. The factors identified in yeast modified Aβ toxicity in glutamatergic neurons of Caenorhabditis elegans and in primary rat cortical neurons. In yeast, Aβ impaired the endocytic trafficking of a plasma membrane receptor, which was ameliorated by endocytic pathway factors identified in the yeast screen. These links between Aβ, endocytosis, and human AD risk factors can be ascertained using yeast as a model system.

Genomic multiplication of the locus-encoding human ␣-synuclein (␣-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 ␣-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 ␣-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 Ϸ2% of the population over 65 years of age.Caenorhabditis elegans ͉ neuroprotection ͉ synuclein