Degree Type

Dissertation

Date of Award

2011

Degree Name

Doctor of Philosophy

Department

Biomedical Sciences

Major

Immunobiology

First Advisor

Anumantha G. Kanthasamy

Abstract

Parkinson's disease (PD) is a progressive neurodegenerative disorder that was first described by the physician James Parkinson in 1817 as the shaking palsy. PD is now recognized as the most common neurodegenerative movement disorder affecting over 2% of the population and placing a severe economic burden on societies, particularly those with aging populations. The pathological hallmark of PD is a progressive loss of dopaminergic neurons in the substantia nigra of the midbrain resulting in striatal dopamine depletion which manifests as a range of motor and non-motor deficits. Progressive nigral dopaminergic degeneration is also accompanied by a chronic neuroinflammatory response that is characterized by increased microgliosis and the presence of dystrophic astrocytes. Research over the last decade has provided compelling evidence that the sustained microglial neuroinflammatory response can be neurotoxic and exacerbate dopaminergic degeneration. Therapeutic targeting of microglial activation has also been shown to mitigate dopaminergic degeneration in pre-clinical models of PD. However, the mechanisms by which microglial activation and neuroinflammation lead to dopaminergic neurotoxicity remain poorly understood. A major objective of this work was to characterize important signaling pathways activated during neuroinflammation, which could be therapeutically targeted to achieve neuroprotection in PD. Protein Kinase C delta (PKC-delta), a novel member of the PKC family, has been shown to be an oxidative stress sensitive kinase that is proteolytically activated by caspase-3 following dopaminergic insults and mediates apoptosis of dopaminergic neurons. Significantly, blocking PKC-delta using small molecule kinase inhibitors is neuroprotective in animal models of PD making it a potentially important therapeutic target for PD. However, the role of PKC-delta; in regulating microglial activation and neurotoxicity has not been studied. Since the PKC-delta; signaling pathway is a crucial regulator of innate immune cell activation and proinflammatory responses outside the central nervous system (CNS), we hypothesized that PKC-delta; could be also be involved in regulating microglial activation and dopaminergic neurotoxicity during sustained neuroinflammation in PD. We tested this hypothesis using cell culture and animal models of neuroinflammation and PD. Herein, we demonstrate for the first time that PKC-delta; is proteolytically activated in dopaminergic neurons by tumor necrosis factor alpha (TNF-alpha), an important proinflammatory cytokine, via the extrinsic cell death pathway to mediate dopaminergic degeneration. We also demonstrate that PKC-delta is proteolytically activated in vivo using the intranigral LPS injection model of neuroinflammation and that targeting PKC-delta protects against dopaminergic neurotoxicity induced by TNF-alpha. In microglial cells, we show that PKC-delta is highly induced during microglial activation and is a crucial regulator of microglial proinflammatory responses. Importantly, we demonstrate that microglia from PKC-delta knockout mice have reduced proinflammatory responses and that PKC-delta; knockout mice are protected from dopaminergic degeneration in the MPTP model of PD. We also describe a novel method for isolation of pure primary microglia from postnatal mice by magnetic separation which provides the highest yield of microglial cells to date. While characterizing the effect of TNF alpha; on dopaminergic degeneration, we serendipitously discovered that Prokineticin-2 (PK2), a novel member of the AVIT family, is highly induced in dopaminergic cells following neurotoxic insults. The final chapter of this work characterizes a novel and clinically relevant function for PK2 in the pathophysiology of PD and demonstrates for the first time that PK2 protects dopaminergic cells from oxidative stress and neurotoxicity in vitro. These findings have immense implications for prokineticin-2 signaling in the pathophysiology of Parkinson's disease and establish a novel paradigm for prokineticin signaling during neurodegeneration and neuroinflammation. Collectively, the research described herein provides novel and important insight into PKC-delta and Prokineticin-2 signaling during neuroinflammation and the pathophysiology of Parkinson's disease. These results have fundamental implications for therapeutic targeting of these crucial signaling pathways to mitigate progressive dopaminergic neuron loss in Parkinson's disease.

DOI

https://doi.org/10.31274/etd-180810-5890

Copyright Owner

Richard Gordon

Language

en

File Format

application/pdf

File Size

318 pages

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