An expeditious route toward discerning genetic contributors to PD involves the use of animal systems amenable to screening methods to identify potential genetic modifiers. In this regard, the use of C. elegans has the potential to greatly accelerate the discovery of neuroprotective factors for PD (23) and other diseases of protein misfolding. These assays consist of forward and reverse genetic screens, as well as chemical screens. An example of a relevant forward genetic screen in C. elegans was performed for enhancers of polyglutamine aggregation in muscle cells, whereby it was determined that a general imbalance in protein homeostasis in post-synaptic muscle cells can occur if there is an increase in acetylcholine (ACh) signaling, or defective GABA signaling (24). Another example of a forward genetic analysis involved screening C. elegans DA neurons for insensitivity to 6-hydroxydopamine (6-OHDA), whereby new alleles of the DA transporter (DAT-1) were identified (25).

There are many examples of reverse genetic, RNAi, screens using the full genome in C. elegans. Several of these screens scored phenotypes related to protein misfolding. For example, worms expressing α-syn::YFP in body wall muscle cells were screened for a modulation in protein misfolding and 80 candidates were identified (18). The candidates clustered into a few biological categories. Notably, quality control and ER/Golgi vesicle trafficking gene products were identified as those that, when knocked down, increased protein aggregation. Another category of gene products identified were aging related, but these were suppressors of protein aggregation.

Recently, candidate RNAi screens, based on the mature bioinformatics available within the C. elegans field, have become more common. Our laboratory performed a hypothesis-based screen for genetic modifiers of α-syn::GFP misfolding in body wall muscle cells and identified 20 candidate genes. Five of these candidates were subsequently validated as having a protective role in DA neurons as well (17). The candidates identified from this screen included orthologs of known recessive PD genes, DJ-1 and PINK1, VPS41, a vesicular protein necessary for lysosomal trafficking and biogenesis, ATG7 an autophagy-associated regulatory gene, and ULK2, a conserved serine-threonine kinase also related to the yeast autophagy protein, Atg1p.

The outcome of this screen represented an exciting group of gene products with implications for PD, both as potential susceptibility markers and novel targets for therapeutic development. For example, knockout of the lysosomal protein ATG7 has been reported to produce a neurodegenerative phenotype in mice (26). Another neuroprotective gene product identified from our screen, VPS-41, is the nematode homolog of mammalian and yeast VPS41, a protein implicated in trafficking from the Golgi to the vacuole/lysosome in yeast (27). Little was known about the function of VPS41 in mammalian systems except that it is strongly expressed within DA neurons of the substantia nigra pars compacta. However, subsequent mammalian studies have since revealed that VPS41 over-expression is protective against rotenone- and 6-OHDA-induced toxicity in SHSY5Y cells (28). Finally, ULK2 was one of six genes significantly associated with single nucleotide polymorphisms in a genome-wide association study (GWAS) of PD patients (29). Therefore, it is clear that establishing a functional screening paradigm for modifiers of PD-related phenotypes in C. elegans has successfully yielded an intriguing collection of effectors that demonstrate the predictability of the nematode model to identify targets with high translational potential.

Our ability to successfully predict the probability of PD among individuals is dependent upon knowledge about genetic susceptibility factors that render certain populations at risk. In attempting to discern genetic factors associated with PD, scientists working with a variety of different organisms have generated many lists of candidate genes and proteins, but most of these leads remain mechanistically undefined. In this regard, application of C. elegans in a directed manner can facilitate the functional evaluation of leads originally identified in other species (yeast, cell culture, mouse, and human genomic studies). As described previously, a distinct advantage of using C. elegans for functional investigation of gene activity and validation is that large populations of isogenic animals can be propagated and analyzed, leading to an unequivocal level of accuracy when evaluating neurodegeneration. As shown in Fig. 2, we have established isogenic lines of transgenic C. elegans that overexpress human α-syn in DA neurons (P _dat-1 ::α-syn  +  P _dat-1 ::GFP) that enable rapid evaluation of factors (drugs and genes) that protect or enhance DA neurodegeneration. This worm strain has been successful in rapidly predicting genes that have significant consequences for neuronal survival in mammalian systems. For example, we investigated three Rab gene products, involved in regulating vesicular trafficking, that were originally identified from yeast screens as effectors of α-syn-dependent toxicity (30, 31). Suppression of cytotoxicity was recapitulated in yeast cells, Drosophila, rat neuronal cell cultures, and in C. elegans, where we showed that elevated expression of specific Rab GTPases rescue DA degeneration induced by α-syn overexpression. For example, overexpression of mammalian Rab1 successfully by-passed an α-syn-associated block of ER to Golgi trafficking (30). New molecules that impact this cellular mechanism have since been discovered in yeast and subsequently validated across multiple organisms, including C. elegans (32).

There is a growing body of literature demonstrating the utility of C. elegans for pharmacological research. A variety of compounds have been uncovered, ranging from those associated with modulating neurotransmitter activity (33, 34) anesthetics (35), lifespan extension (36), and a new calcium channel antagonist (37). Our laboratory has used C. elegans to conduct a small molecule screen to identify compounds that positively modulate the activity of the neuroprotective chaperone-like protein, torsinA, in vivo (38). Deficit in torsinA function is responsible for a human movement disorder termed early-onset torsion dystonia, and is linked to an in-frame 3-bp deletion in the human DYT1 gene. These same molecules were later shown to restore functional capacity of torsinA activity in human DYT1 dystonia patient fibroblasts and reverse behavioral abnormalities in a DYT1 knock-in mouse model of early-onset torsion dystonia (38). Of relevance to PD, worms are being exploited as a system for therapeutic drug screening (39) and functional validation of drugs with a therapeutic potential for PD that have been identified in other systems (32, 40, 41).