Date of Award

2024

Document Type

Thesis (Ph.D.)

Department or Program

Molecular and Systems Biology

First Advisor

Matthew Havrda

Second Advisor

Arminja Kettenbach

Abstract

Microglia are subject to chronic proteinaceous and environmental stress during the progression of prevalent neurodegenerative diseases such as Parkinson’s Disease (PD). Chronic stress can activate the NLR family pyrin domain containing 3 (NLRP3) pattern recognition receptor resulting in the formation of the NLRP3 inflammasome, proinflammatory signaling, and pyroptotic cell death. A distinct subset of extracellular vesicles (EVs) are released from cells upon NLRP3 activation. We studied WT and Nlrp3-/- primary microglia exposed to bacterial stimulating agents and alpha synuclein preformed fibrils (αsyn PFFs), which model Parkinson’s disease and associated synucleinopathies. We identified the NLRP3-dependent release of the endosome fate regulator Coronin1A (Coro1A) using quantitative tandem mass tag (TMT) proteomics of EVs released from primary microglia subject to conditions mimicking bacterial stress. We discovered that the dependence of Coro1A packaging into EVs under αsyn PFF treatment was not dependent on having NLRP3 but was instead dependent on whether the microglia were exposed to αsyn PFFs. We investigated the role of Coro1A in microglia attempting to clear toxic αsyn species and found that Coro1A loss significantly increases lysosomal damage and reduces the amount of αsyn packaged and into EVs. NLRP3 activation and cell death in response to αsyn was increased by Coro1A inactivation. In parallel studies, we sought to characterize microglia secretion in response to diseaserelevant pesticides using mass spectrometry. To this end, we optimized proteomics methods to probe the secreted proteome from microglia and applied this to WT and Nlrp3-/- primary microglia exposed the organophosphate chlorpyriphos and its metabolite TCPy. We discovered that chlorpyrifos and TCPy were each capable of inducing distinct iii changes in the EV proteome. We identified the protein haptoglobin as a component of a potentially compensatory response in Nlrp3-/- cultures under this pesticide exposure paradigm. Our data shed light on cellular mechanisms underlying how microglia attempt to clear toxic protein aggregates and how NLRP3 integrates into the microglial response to diverse stressors.

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