Study Rationale:
Alpha-synuclein (aSYN) is a critical protein in health and Parkinson’s disease (PD). aSYN has a soluble form and a series of increasingly aggregated forms. These aggregated forms are known to heavily contribute to PD toxicity, but their precise functions are not understood. Here we create tools that allow researchers to directly visualize, in living cells and animals, where each type of aggregate is — which is not possible with existing tools. Having these new tools and the ability to follow aggregate formation, movement, and dissolution in cells and animals will transform PD research — both in terms of basic science and for screening drugs that disrupt the aggregation process.
Hypothesis:
We hypothesize that genetically encoded fluorescent probes will enable real-time, non-invasive, fast, and high-contrast visualization of distinct alpha-synuclein species with subcellular resolution in living cells — and will directly enable discoveries not possible with existing reagents.
Study Design:
We will design and engineer novel fluorescent probes based on fluorescent proteins — like green fluorescent protein (GFP) from jellyfish – and a variety of alpha-synuclein-binding motifs. Then, we will leverage the power of molecular “evolution” — an iterative process of mutagenesis performed in test tubes and cultured cells — to isolate sensors that become highly fluorescent only in the presence of specific alpha-synuclein species. Lastly, we will express these sensors in cells to assess if they can faithfully monitor dynamic transitions and protein interactions of alpha-synuclein conformers under various cellular stressors relevant to PD, and several potential helpful interventions.
Impact on Diagnosis/Treatment of Parkinson’s disease:
Genetically encoded fluorescent sensors for directly visualizing distinct alpha-synuclein species will help establish cell- and animal-based assays to evaluate environmental/genetic factors and drug candidates influencing and even reversing alpha-synuclein aggregation.
Next Steps for Development:
Full validation of the engineered sensors will lay the groundwork for establishing a high-throughout, cell-based platform to screen pharmaceutical/genetic modulators of alpha-synuclein aggregation for drug discovery and target identification.