Degree Type

Dissertation

Date of Award

2016

Degree Name

Doctor of Philosophy

Department

Biomedical Sciences

Major

Molecular, Cellular and Developmental Biology

First Advisor

Anumantha Kanthasamy

Abstract

Disparate lines of evidence including in vitro cell culture, ex vivo brain slice culture, and virtually every in vivo model system have clearly implicated microglial activation and neuroinflammation in the pathophysiology of the dopaminergic neuronal cell death in Parkinson’s disease (PD). The signaling pathways that lead to this chronic activation are still being elucidated. We show herein, the role of the non-receptor Src family tyrosine kinase Fyn in mediating pro-inflammatory signaling in microglia cells in response to various inflammogens. Our results from cell and animal models as well as postmortem brain tissues conclusively demonstrate that Fyn is preferentially activated in microglia post-stimulation with either Lipopolysaccharide (LPS) or Tumor necrosis factor alpha (TNFα). Activated Fyn then tyrosine-phosphorylates the known pro-inflammatory kinase PKCδ, mediating PKCδ-dependent activation of the NF-κB pathway, leading to pro-inflammatory cytokine and nitrite production. Both Fyn-/- and PKCδ-/- mice were remarkably resistant to LPS-mediated neuroinflammation, as well as neuroinflammation and dopaminergic neuronal loss induced by the Parkinsonian toxicant 6-hydroxydopamine (6-OHDA). Activation of the NLRP3 inflammasome, mediated by fibrillar amyloid-β, the major component of Alzheimer’s disease-associated senile plaques, has recently been shown to contribute to disease progression. Thus, we sought to validate whether the aggregated form of the PD-associated protein α-synuclein could activate the NLRP3 inflammasome within microglia. Our results conclusively demonstrate that α-synuclein can elicit the NF-κBdependent induction of the inflammasome components pro-IL-1β and NLRP3, as well as the Caspase-1- and ASC-dependent processing of pro-IL-1β to mature IL-1β. Remarkably, Fyn kinase was shown to contribute to both processes, via PKCδ-dependent NF-κB pathway activation to prime the NLRP3 inflammasome as well as to the uptake of α-synuclein into the cell, which leads to the assembly and activation of the inflammasome complex. Lastly, we demonstrate activation of the NLRP3 inflammasome in various α-synuclein-overexpressing PD model systems, as well as in post-mortem PD patient tissues. Fyn also contributes to microglial ASC speck formation in the adenoviral α-synuclein overexpression system. Overall, we identify Fyn kinase as a key upstream regulator of the microglia-mediated chronic neuroinflammatory cascade that is central to the pathophysiological process of nigral dopaminergic degeneration in PD.

DOI

https://doi.org/10.31274/etd-180810-4640

Copyright Owner

Nikhil S. Panicker

Language

en

File Format

application/pdf

File Size

248 pages

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