Establishing an RNA interference (RNAi) screen for neuroprotection of dopaminergic neurons in Caenorhabditis elegans models of Parkinson’s disease

Abstract

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the selective death of dopaminergic neurons in the substantia nigra of the midbrain, resulting in bradykinesia, tremors, muscle rigidity, and other motor and non‐motor deficits. The prevalence and economic burden of Parkinson’s disease are increasing due to the aging population. Despite this increasing burden, the discovery of clinically useful therapies to halt the progression of the disease remains an unmet need. This is partly because of gaps in our knowledge of the underlying molecular mechanisms of the disease and the lack of accurate disease models. Therefore, understanding the pathobiology and discovery of potential druggable targets remain a priority in PD research. In the current study, we report the development of a high‐throughput assay in the nematode, Caenorhabditis elegans, to monitor neurodegeneration and to probe for genes potentially involved in mediating neuroprotection of dopaminergic neurons using RNA interference (RNAi) technology. Two transgenic strains of C. elegans carrying human PD‐linked genes were used, one expressing mutant (A53T) alpha‐synuclein, and the other expressing mutant (G2019S) leucine‐rich repeat kinase 2 (LRRK2). These strains express the PD‐transgenes and GFP exclusively in their dopaminergic neurons allowing for the monitoring of neurodegeneration via changes in GFP signal intensity. A third control strain was used that lacked the PD‐transgenes genes but expressed GFP in their dopaminergic neurons. We crossed these three strains of worms into neuronal RNAi‐sensitive backgrounds that express mCherry under a pharyngeal promoter thus allowing for accurate quantitation of the worms in liquid cultures within 384‐well microtiter plates. Using a combination of laser cytometry and high content imaging, we have established that these models recapitulate the age‐dependent degeneration of dopaminergic neurons observed in human PD. Neurodegeneration was seen in the SNCA (A53T) and LRRK2 (G2019S) transgenic models, as evidenced by a 30‐50% and 75‐85% loss of GFP intensity, respectively, after 7 days in culture. In preliminary studies, we have found that these worms show susceptibility to RNAi via bacterial feeding as evidenced by a loss of GFP signal from RNAi against GFP or from RNAi against growth factors supporting the differentiation of dopaminergic neurons. Further optimization studies of this RNAi screening assay are currently ongoing. The demonstrated susceptibility of C. elegans to neuronal RNAi in our screening assay, and the availability of chromosomal and genome‐wide RNAi libraries will allow for the interrogation of the entire C. elegans genome in these models. This line of inquiry, if successful, will improve our knowledge of the underlying mechanisms of neurodegeneration and may also identify genes encoding druggable targets for therapeutics directed at slowing the progression of PD.