Hypothesis-based RNAi screening identifies neuroprotective genes in a Parkinson's disease model

Abstract

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

A Predictable Worm: Application of Caenorhabditis elegans for Mechanistic Investigation of Movement Disorders

“…Expression of human α-syn under the DA transporter (dat-1) promoter results in age-and dose-dependent neurodegeneration of DA neurons (Fig. 1); α-syn-induced degeneration of neurons in C. elegans has been reported by several laboratories [14][15][16][17]. For example, when α-syn mRNA levels are expressed at lower levels in 1 transgenic line (Fig.…”

“…Interestingly, a physical association between torsinA and the α-syn aggregates of Lewy bodies in PD patients further suggests a role for the gene in misfolded protein management [70]. This is further supported by coexpression of worm TOR-2 ameliorating α-syn::GFP misfolding in the body wall muscles of C. elegans [14]. The absence of neurodegeneration in dystonia suggests that more subtle causes of cellular dysfunction underlie the disease state.…”

“…We first overexpressed an α-syn::GFP fusion in the body wall muscles of the worm, whereby visible cytoplasmic aggregation of α-syn was observed. Co-expression of a worm chaperone-like protein (TOR-2) attenuated this effect, leading to a diffuse presence of α-syn in the cytoplasm [14]. Using this genetic background, our laboratory performed a hypothesis-based RNAi screen of~900 potential α-syn interactors, ultimately identifying 20 genes that enhance misfolding of α-syn in the body wall muscles of the worm.…”

“…Seven positive hits were further characterized through functional analyses, whereby 5 genes were found to protect against α-syn neurodegeneration when co-expressed with α-syn in the worm DA neurons. Candidates identified through this RNAi screen included DJ-1 and PINK-1, worm orthologs of known recessive PD genes, as well as C. elegans homologs of the human genes encoding VPS41, ULK2, and ATG7 [14] (Fig. 3c).…”

“…Candidates that reduce toxicity in both yeast and invertebrate neuronal systems also protected rat primary neurons from degeneration following transfection with A53T α-synuclein [24,28]. (c) A C. elegans hypothesis-based RNA interference (RNAi) screen for effectors of α-synuclein protein misfolding [14] identified candidates that were reported to cause neurodegeneration in a mouse knockout model (ATG7 [37]), as a risk factor for PD (ULK2 [36]), or neuroprotective in human cells when overexpressed (VPS41 [33]). MPP01-methyl-4-phenylpyridine and this effect may be too strong to accurately evaluate the neuroprotective influence of acetaminophen.…”