Degree Type

Dissertation

Date of Award

2017

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

Major

Biomedical Sciences

First Advisor

Arthi Kanthasamy

Abstract

Tweak and TNF family members are novel regulators of acute and chronic inflammation. Tweak / Fn14 interaction appears to be involved in angiogenesis, inflammation, proliferation, migration, cytokine production, cytotoxicity and apoptosis. Thus, TWEAK can induce both cell death and proliferation. Recently, TWEAK has been shown to be associated with neurodegenerative effects in an MPTP mouse model of PD. Despite numerous studies demonstrating TWEAK’s ability to cause cell death in diverse cell types the mechanism(s) by which TWEAK-induce dopaminergic cell death remain poorly defined. Therefore in an attempt to better understand the molecular basis of TWEAK-induced dopaminergic neurotoxicity we evaluated the apoptotic effect of TWEAK and associated molecular mechanisms using N27 dopaminergic neuronal cells. TWEAK-induced a time dependent increase in ROS generation, mitochondrial dysfunction, caspase activation, and NFkB activation. Additionally, a concurrent activation of SYK and proteolytic cleavage of PKC delta was evidenced in TWEAK treated cells. In contrast a marked down regulation of p-GSK 3b (Ser 9) and Akt activity was evidenced in TWEAK treated cells. Intriguingly, inhibition of SYK activity via R708 attenuated TWEAK-induced loss of dopaminergic cell viability. Likewise, SN50, NFkB inhibitor and Quercetin, a ubiquitous bioactive plant flavonoid attenuated TWEAK-induced apoptotic cell death further highlighting the pivotal role of NFkB and mitochondria dependent oxidative stress signaling events in the mechanism of dopaminergic neurodegeneration. Taken together, our studies demonstrate the involvement SYK/NFkB signaling axis and mitochondrial dysfunction in TWEAK-induced apoptotic cell death.

In the next series of studies we determined the effects of TWEAK on microglia. A growing body of evidence suggests that persistent microglial activation and accompanying oxidative stress may act as co-conspirators in mediating dopaminergic neurodegeneration in the substantia nigra in PD pathogenesis. Previously we demonstrated that PKC delta a redox sensitive kinase is a critical determinant of microglial activation response in response to diverse inflammagens. More recently, SYK, has been implicated in the activation of inflammatory cells in response to infection. Therefore, in the current study we hypothesized that SYK may act as an upstream regulator of NLRP3 inflammasome thereby leading to a heightened microglial activation response in TWEAK stimulated cells. In the present study we systematically elucidated the signaling network underlyingTWEAK-induced microglial activation response. A concentration dependent increase in SYK activation and accompanying increase in kinase activity was evidenced in TWEAK treated cells. Our results with pharmacological inhibitors and siRNA mediated gene silencing revealed the regulatory role of SYK in ER stress response (ERS), NOX2 upregulation, GSK 3β activation as well as autophagolysosomal system (ALS) and mitochondrial dysfunction. Taken together our findings demonstrate a role for SYK signaling network in mediating TWEAK triggered inflammatory response by positively regulating NLRP3 inflammasome activation and ERS in an autophagy dependent manner. We have discovered the pivotal role of SYK in mediating dopaminergic neurodegeneration as well as heightened microglial activation response upon TWEAK treatment. By inhibiting SYK activation we can limit dopaminergic neuronal degeneration as well as microglial activation response in response to diverse inflammagens including TWEAK. Our studies further highlight the therapeutic advantage of targeting SYK for the treatment of inflammation related disorders including PD.

DOI

https://doi.org/10.31274/etd-180810-6019

Copyright Owner

Sri Harsha Kanuri

Language

en

File Format

application/pdf

File Size

314 pages

Included in

Physiology Commons

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