Neuroprotection in C. Elegans Parkinson’s Disease Models: Discovering Therapeutic Insights via RNAi and Small Molecule High Throughput Screening

Authors

  • MA Martinez
  • AA Surtel
  • EO Akano
  • PK Dranchak
  • CT Ash
  • EE Anderson
  • AE Moritz
  • RB Free
  • JP Steiner
  • J Inglese
  • DR Sibley

Abstract

Parkinson's disease (PD) is a neurodegenerative condition characterized by loss of dopaminergic neurons in the midbrain substantia nigra. This results in bradykinesia, tremors, and other motor and non-motor deficits. Disease pathobiology is poorly understood, and current therapeutics merely provide temporary relief of motor symptoms. The discovery of novel druggable targets remains a priority. Herein, we describe the development and optimization of a high throughput phenotypic screen in the roundworm Caenorhabditis elegans for discovery of druggable targets and genetic influencers of PD neurodegeneration and neuroprotection. We utilized two transgenic C. elegans strains expressing human PD-linked genes—mutant (A53T) alpha-synuclein (SNCA) and mutant (G2019S) leucine-rich repeat kinase 2 (LRRK2). These strains express green fluorescent protein (GFP) in their dopaminergic neurons, allowing for the monitoring of the health of dopamine neurons via laser cytometry and high content imaging. We observed a 30-50% and 75-85% decrease in GFP intensity in SNCA and LRRK2 worms, respectively, by day seven compared to wild-type controls. We crossed the worms to RNAi hypersensitive backgrounds carrying eri-1, rrf-3, and eri-1;lin-15B mutations to overcome resistance to neuronal RNAi knockdown. As a proof of concept, we performed RNAi knockdown of selected genes known to be involved in dopamine neuron development and survival in the control and mutant worms, with a 50-75% knockdown on day seven. RNAi specifically engineered against the LRRK2 transgene slows dopamine neuron loss in the LRRK2 mutant worms. Additionally, we identified LRRK2 kinase inhibitors that confer neuroprotection in LRRK2-carrying worms to serve as controls for molecular library screening.

Scientific Focus Area: Molecular Pharmacology

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