Development of a dopaminergic neurodegeneration assay using laser cytometry of Caenorhabditis elegans models of Parkinson’s disease

Abstract

Parkinson’s disease (PD) is a neurodegenerative disorder that is characterized by loss of dopaminergic neurons resulting in bradykinesia, tremor, gait abnormalities, and numerous non‐motor complications. The prevalence and economic burden of PD is increasing due to an aging population. However, there are currently no drugs to halt the progression of this disease. Although loss of dopamine in the basal ganglia is recognized as the hallmark of PD, the molecular mechanisms underlying this loss and subsequent brain dysfunction remain poorly characterized. The majority of PD animal models involve the administration of neurotoxins that target dopaminergic neurons leading to their degeneration, however these models correlate poorly with the disease progression in humans. More accurate models may utilize genetic modulation of PD‐related genes and exhibit progressive neurodegeneration. Here, we report the development of a high‐throughput assay for monitoring dopaminergic neurodegeneration in Caenorhabditis elegans (C. elegans). Two strains of C. elegans containing human PD‐linked genes were used, one expressing mutant (A53T) alpha‐synuclein, and the other expressing mutant (G2019S) leucine‐rich repeat kinase 2 (LRRK2). Both strains express GFP in their dopaminergic neurons and the lines were further crossed into a neuronal RNAi‐sensitive background strain expressing mCherry under a pharyngeal promoter. This allows for the accurate measurement of dopaminergic neurons (via GFP) and a normalization and sorting control of the total number of worms plated (via mCherry). Daily measurements of GFP/mCherry fluorescence intensity were performed using laser cytometry of worms sorted into 384‐well microplates. Robust temporal degeneration of dopaminergic neurons was found to occur within the first eight days of adulthood in the C. elegans models of PD, but not in a wild‐type control strain. The LRRK2 model was particularly severe as total GFP intensity dropped by 75–85% during the time of assay. We determined a signal (cell loss) to baseline ratio of approximately 4‐fold, sufficient for screening applications. Our results indicate that this assay provides a reproducible high‐throughput measurement of dopaminergic neurodegeneration using an in vivo model. Future studies may exploit this model to conduct quantitative high‐throughput screens to identify small molecules capable of inhibiting this neurodegeneration or to use RNAi libraries to identify genes mediating the neurodegenerative response, and hence new drug targets for the treatment of PD.Support or Funding InformationThis study is supported by the intramural program of the NINDS/NIH