Doctor of Philosophy
Michelle L Block
NF-κB p50: THE ROLE OF REDOX SIGNALING AND LOSS OF FUNCTION IN DYSREGULATED MICROGLIAL ACTIVATION
By Thomas J Taetzsch B.S.
A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University.
Virginia Commonwealth University, 2014
Major Director: Michelle L Block PhD,
IUPUI Associate Professor
Department of Anatomy and Neurobiology
Oxidative stress and chronic microglia-mediated inflammation are well characterized underlying factors in neurodegenerative disease. While it is clear that increased levels of inflammation and augmented oxidative stress in the aged brain create an environment that is particularly harmful to neurons, little is known about how oxidative stress impacts the molecular mechanisms that regulate microglial activation or how healthy microglia transform into an enduring state of damaging pro-inflammatory activation that is characteristic of neurodegenerative disease. Here we identify the NF-κB p50 subunit as an important redox sensitive regulator of microglial M1 (pro-inflammatory) polarization whose function becomes increasingly important in the aged brain. Microglia exposed to steady fluxes of H2O2 showed altered NF-κB p50 protein-protein interactions, decreased NF-κB p50 DNA binding, and loss of LPS tolerance, indicating that H2O2 impairs NF-κB p50 function and disrupts transcriptional regulation of M1 activation. Mice and cell cultures lacking NF-κB p50 exhibited a muted M2 (alternative) response and impaired resolution of the M1 response. Treatment with the radical scavenger DMPO, reduced the initial LPS-induced TNFα response in the midbrain (3h post-injection) of NF-κB p50-/- mice. Yet during the resolution phase of the central nervous system (CNS) inflammatory response (22 h post-LPS injection), DMPO treatment mildly reduced TNFα levels in NF-κB p50+/+ mice but significantly augmented TNFα expression in NF-κB p50-/- mice, emphasizing a key role for NF-κB p50 in redox regulation of CNS inflammation resolution. LPS treatment of aged (16-18 months old) NF-κB p50-/- mice resulted in significantly elevated pro-inflammatory markers in serum, brain and microglia isolated from the brain that was not observed in NF-κB p50+/+ mice and was not concomitant with an increase in NF-κB p65 activation. In a study of the long-term effects of CNS inflammation on dopaminergic neuropathology, LPS-injected NF-κB p50-/- mice showed loss of motor function without significant loss of dopaminergic neuron cell bodies in the substantia nigra. These data identify NF-κB p50 as a key redox-signaling mechanism regulating the M1/M2 balance in microglia where loss of function leads to augmented age-related microglial reactivity and a CNS-specific vulnerability to chronic inflammation.
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Available for download on Sunday, December 15, 2024