Identification of Small Molecule Therapeutics and Neuroprotective Gene Targets via High Throughput Screening and RNA Interference inC. elegansParkinson’s Disease Models

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the necrosis of midbrain dopaminergic neurons and subsequent deficiencies of dopamine (DA) signaling, resulting in tremors, rigidity, and bradykinesia among a range of other motor and non-motor complications. Despite its high prevalence and considerable economic burden, driven by a rapidly growing aging population, the underlying molecular mechanisms of PD remain poorly understood, and robust, translational models of the disease have yet to be fully established. These limitations in our collective understanding of PD warrant an urgent need for discovering and interrogating PD-associated druggable targets to address the lack of effective, neuroprotective therapeutics with minimal side effect profiles. Here, we report the development of two high throughput in vivo assays for the discovery of small molecule compounds with therapeutic potential and for probing genes potentially involved in dopaminergic neuroprotection. Transgenic mutant Caenorhabditis elegans (C. elegans) carrying human PD-linked genes were used, one expressing mutant (G2019S) leucine-rich repeat kinase 2 (LRRK2), and the other expressing mutant (A53T) a-synuclein (SNCA). Both strains express GFP exclusively within their dopaminergic neurons allowing for fluorescent signal intensity to serve as a proxy for monitoring dopaminergic neurodegeneration. A control strain (BY250) was used that expressed only dopaminergic neuronal GFP in the absence of PDlinked transgenes. Daily laser cytometry and high-content imaging readings of GFP intensity revealed a robust temporal dopaminergic neurodegeneration in both PD strains, mirroring that which is seen in human PD, within the first seven days of adulthood. By day seven, GFP fluorescent intensity had decreased by 30-50% and 75-85% in the SNCA and LRRK2 worms, respectively; such an effect was not observed in the wild-type control worms. In the LRRK2 mutant worms, we have identified a set of selective LRRK2 kinase inhibitors that may serve as positive controls for neuroprotection. Assay validation and optimization studies are ongoing with the goal of conducting a high throughput screen of small molecules that may confer neuroprotection in our LRRK2 model and serve as scaffolds for the development of drug leads. We have also established effective knockdown of GFP fluorescent signal intensity by the administration of RNA interference (RNAi) via engineered vector bacterial feeding. Due to the low penetrance of RNAi in neurons, we crossed our control and mutant worms into three RNAi hypersensitive backgrounds carrying eri-1, eri-1; lin-15B, and rrf-3 mutations. In a preliminary screen of RNAi vectors, three (ceh-43, unc-62, and ama-1) conferred robust knockdown of GFP signal in both the control and mutant a-synuclein-expressing strains, averaging $75% knockdown by day seven of treatment when compared to an empty vector control RNAi. In the future, we hope to use this assay to screen C. elegans RNAi libraries to elucidate genes that may be neurop...