A new in vivo zebrafish model to study alpha-synuclein aggregation in Lewy Body Disease

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PROJECT SUMMARY Protein aggregation plays a critical role in many important neurodegenerative diseases such as beta-amyloid & tau in Alzheimer's disease, tau in Frontotemporal dementia, huntingtin in Huntington's disease, and alpha- synuclein in Lewy body dementia (LBD) & Parkinson's disease (PD). Treatments do not currently exist to halt disease progression, in part because the underlying cell biological mechanisms driving protein aggregation and clearance are poorly understood. A growing body of evidence in DLB & PD suggests that alpha-synuclein protein aggregation within neurons causes dysfunction in the neuron's ability to communicate signals across the synapse, giving rise to the cognitive and movement symptoms found in patients. Alpha-synuclein protein is the major aggregated component of Lewy bodies, the hallmark pathological lesion within neurons that defines these “synucleinopathy” diseases. Intriguing evidence suggests that aggregation begins in synaptic terminals and may spread unidirectionally to the cell body. Specific point mutations or post-translational modifications associated with disease might play a critical role in alpha-synuclein's aggregation, and in turn influence axonal and terminal function, including axonal transport. Understanding the mechanisms involved in alpha-synuclein aggregation requires dissecting the role/s of different disease-relevant point mutations and phosphorylation events, and/or potential combinations of them, and determining the pattern/s of aggregation formation and spread, ideally in an experimental paradigm that allows for study in real time in the living nervous system. The Weissman Lab has developed a new zebrafish model and experimental approaches to study alpha-synuclein aggregation and function within individual neurons in vivo. The lab can readily express different forms of alpha- synuclein with specific point mutations, visualize whole axonal arbors and parent cell bodies in vivo, and measure protein mobility, protein aggregation, and axonal function. Recently published preliminary data from the lab strongly suggest that what was previously thought to be the critical phosphorylation event in Parkinson's disease pathogenesis (serine-129 phosphorylation) does not appear to act by itself to drive aggregation. Rather, a more elaborate “phosphorylation code” may exist, where multiple phosphorylation events at several residues must occur simultaneously. Investigating this mechanism, the complex involvement of various critical residues, and patterns of aggregation within the cell requires an easily manipulatable system for visualizing and detecting the effects of multiple simultaneous changes to the alpha-synuclein protein. The lab's approach using the living, transparent zebrafish nervous system is ideally suited for these questions. The proposed project will use a combination of in vivo fluorescence imaging, site-directed point mutants, treatment with small molecule kinase inhibitors, immunohistochemistry, and three-dimensional image reconstruction. This work, from experimental design to hands-on experimentation, data analysis and writing, is done primarily by undergraduate students in the Weissman Lab at Lewis & Clark College.