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 characterized by the loss of dopaminergic neurons resulting in bradykinesia, tremor, gait abnormalities, and numerous non-motor complications. Knowledge of the molecular mechanisms underlying dopaminergic neuron death remains limited, partially due to the lack of accurate disease models. Here, we report the development of a high-throughput assay for monitoring dopaminergic neurodegeneration in Caenorhabditis elegans (C. elegans). Two transgenic 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 were crossed into a neuronal-RNAi sensitive background expressing mCherry under a pharyngeal promoter. This allows for the accurate measurement of neuronal viability (via GFP) and a normalization and sorting control of the total number of worms plated (via mCherry). Daily laser cytometry readings of GFP/mCherry fluorescence intensity revealed that robust temporal degeneration occurs within the first eight days of adulthood in both PD models, but not in a wild-type control strain. We determined a signal (cell loss) to baseline ratio of approximately 4-fold, sufficient for screening applications. Particularly in the case of the mutant LRRK2 model, neurodegeneration is severe: between day 3 and day 8 of adulthood, total GFP fluorescence intensity drops by 75-85%. In the LRRK2 mutants, administration of selective LRRK2 inhibitors (LRRK2-IN-1 and CZC 25146) confers neuroprotection in a dose-dependent manner. Similarly, using high content imaging, we found that the LRRK2 mutant-expressing worms displayed susceptibility to RNA interference. Administration of RNAi targeting LRRK2 slowed the course of the neurodegeneration in these worms. Similarly, administration of RNAi targeting GFP resulted in about 50% reduction of the mean GFP intensity at day 5. 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 that inhibit neurodegeneration or use RNAi libraries to identify genes mediating the neurodegenerative response, and hence new drug targets for PD treatment.