Phenotypic Screening Assays to Identify Parkinson’s Disease Drug Candidates
Parkinson’s disease (PD) is characterized by a significant loss of dopaminergic neurons and corresponding loss of dopamine. One of the primary pathological hallmarks is the presence of Lewy bodies that are largely composed of alpha-synuclein. Charles River has extensive experience with development and screening using high-content imaging-based assays measuring PD-specific readouts, including the following:
Alpha-synuclein Aggregation Assay
In collaboration with The Michael J. Fox Foundation’s Parkinson’s Disease Research Tools Consortium, Charles River has developed a robust assay to screen disease-modifying therapies for Parkinson’s disease (PD) using alpha-synuclein aggregation as a readout. Mutations in the alpha-synuclein gene were among the first mutations linked to Parkinson’s disease. The reported mutations were gene duplications/triplications or point mutations that increased the propensity of α-synuclein to form large β-sheet-rich aggregates (fibrils) that are found in Lewy bodies, a hallmark feature of neurons affected by PD. The formation of α-synuclein oligomeric species has been hypothesized to be a contributing factor to dopaminergic cell death.
The assay uses ReNcell VM cells, an immortalized human neural progenitor cell line that can rapidly differentiate into neurons expressing dopaminergic markers. Full-length alpha-synuclein is expressed in the cells via adenoviral delivery and the reduction in alpha-synuclein aggregation is measured using an aggregate-specific antibody. The reduction in alpha-synuclein aggregates is normalized against total alpha-synuclein levels and the number of cell nuclei. The alpha-synuclein aggregation assay is performed in a scalable, high-throughput plate-based format and can screen compounds that selectively reduce aggregated alpha-synuclein.
Figure: Reduction in alpha-synuclein aggregate levels with increasing levels of KU 0063794 (PI-3 kinase inhibitor)
MPP+ Neuronal Cell Death Assay
MPP+ is a neuronal toxin that induces Parkinsonism in animal models. The MPP+ neurotoxicity can be monitored using a high-content imaging assay. SH-SY5Y neuroblastoma cells are differentiated into neurons with retinoic acid and the reduction of MPP+ induced cell death by compound administration is measured by the level of propidium iodide (PI) incorporation in dead cells.
Figure: MPP+ increased the percentage of dead cells in a dose-dependent manner. Staurosporine is included as a positive control for induction of cell death.
Neuronal Mitophagy Assays
Defective mitochondrial clearance and aberrant energy metabolism have been linked to PD by both genetic association studies and preclinical disease models. Mutations in genes encoding proteins involved in targeted autosomal destruction of mitochondria (e.g., Parkin and PINK1) have been described in PD. This sparked heavy interest in therapeutic approaches that facilitate neuronal mitophagy. High-content imaging methods are used to measure different readouts of mitophagy:
- Fluorescent reporters that measure pH changes during mitophagy (mitoRosella, mtKeima)
- Mitochondrial membrane potential changes as a measure of viability
- Reduction in protein expression of Tom20, a mitochondrial marker (see figure below)
- Parkin translocation to mitochondria (see figure below)
Figure: Reduction in Tom20 expression post treatment with the mitochondrial uncoupler CCCP (10 µM for 18h). Mitochondrial damage induced a shift in GFP-Parkin localization from predominantly cytoplasmic to punctate, mitochondrial localized staining.