Causes and Downstream Effects of 14-3-3 Phosphorylation in Synucleinopathies - Project Summary/Abstract This NIH F31 application describes a four-year plan for mentored research and career development for the PI. This proposal is focused on understanding the mechanisms of 14-3-3 phosphorylation in synucleinopathies. Parkinson’s Disease (PD) and Dementia with Lewy bodies (DLB) are synucleinopathies characterized by their aggregation of alpha-synuclein (αsyn). These diseases are characterized by a mix of progressive motor, cognitive, and autonomic symptoms. Our lab studies the role 14-3-3 proteins play in these diseases. 14-3-3s are a ubiquitously expressed family of proteins representing nearly 1% of all soluble protein in the brain that mainly exert their functions through protein-protein interactions (PPIs). We have found 14-3-3 proteins, in particular the 14-3-3θ isoform, are protective against αsyn while inhibition of these proteins leads to increased toxicity. Furthermore, we found that human cortical lysates of PD and DLB patients showed increased 14-3-3θ phosphorylation at S232 compared to healthy, age-matched controls. Testing the impact of 14-3-3θ phosphorylation, we found that the non-phosphorylatable S232A mutant is protective in PD models, while the phosphomimetic S232D mutant showed no protection or accelerated toxicity. These results lead us to hypothesize S232 phosphorylation plays a role in 14-3-3θ’s loss of protective functions. The goal of this project is to understand the causes and downstream effects of S232 phosphorylation. In Aim 1 I propose to identify what kinases cause this phosphorylation. I will work with Dr. Christopher Willey, who is in charge of the UAB Kinome Core. I will use both a candidate approach and non-biased approach to identify key kinases that increase 14-3-3 phosphorylation by PD-associated toxicants, trichloroethylene (TCE) and rotenone. For an unbiased approach, I will use the PamStation Kinomics chip to identify kinases activated by rotenone and TCE. I will validate these results in-vitro utilizing kinase inhibitors and will evaluate the biological relevance of identified kinases by genetic knockdown as well as pharmacological inhibitors of the kinases. Dr. Willey will guide me on the bioinformatic techniques as well as selection of candidate kinases for further study. In Aim 2 I want to understand what the downstream consequences are for the hundreds of 14-3-3 PPIs. I will work with Dr. James Mobley, head of UAB’s Mass spectrometry and proteomics core, to perform mass spectrometry of co-immunoprecipitates from wildtype, S232A or S232D knock-in cortical brain lysates to understand how protein networks binding to 14-3-3θ change in response to 14-3-3θ phosphorylation. The experiments in addition to the input from my mentor, Dr. Talene Yacoubian as well as my committee will help me meet my goals of increasing understanding of neurodegeneration, develop lab and bioinformatic skills, communication skills, teaching and mentorship skills, and clinical skills necessary for future patient care. All these will help me become an excellent physician-scientist focused on neurodegenerative disorders.
