Biomedical Sciences ETDs

Publication Date

Fall 11-7-2019

Abstract

Multiple sclerosis (MS) is a complex neurological disorder characterized by the interactions between heightened inflammation, oxidative stress and neurodegeneration. We and others have previously demonstrated that proteasome dysfunction and its consequences are also important factors in the pathology of both MS and its rodent model, experimental autoimmune encephalomyelitis (EAE). While proteasome subunit alterations in EAE have been observed, the underlying mechanisms are poorly understood. The first goal of this dissertation was to characterize the mechanisms that regulate proteasome expression and composition in neural cells in EAE and in vitro.

Immunohistochemical analysis of the EAE spinal cord shows changes in proteasome composition in neurons and astrocytes. Molecular and biochemical methods revealed that constitutive proteasome (c-20S) mRNA and protein levels are reduced while immune-proteasome (i-20S) and 11S regulator expression is augmented in EAE. This altered composition is most likely due to two processes, the first of which is a decline in two transcription factors upstream of c-20S subunits: nuclear factor (erythroid-derived 2) like-1 (Nfe2l1/Nrf1) – which regulates proteasome gene expression – and one of its regulators, pre-B cell leukemia homeobox domain 1 (PBX1). The second is a heightened signal transducer and activator of transcription 1 (STAT-1) / interferon regulatory factor 1 (IRF-1) signaling, which mediates the displacement of constitutive subunits for inducible subunits. Differentiated N2a neurons and C6 astrocytes in vitro display similarly elevated i-20S and 11S levels when exposed to CIII, a pro-inflammatory cytokine cocktail (i.e., interferon-gamma, interleukin-1 beta and tumor necrosis factor-alpha). Surprisingly, neither exhibit changes in PBX1 / Nrf1 or in c-20S-specific subunit mRNA. Altogether, these data imply that proteasome subunit displacement caused by elevated STAT-1 / IRF-1 is the main mechanism dictating proteasome composition in cultured neurons and astrocytes.

The second goal of this dissertation was to understand how reactive astrocytes participate in neuroinflammation – recent evidence suggests that there are different neurotoxic (A1) and neurotrophic (A2) reactivities. Because A1 astrocytes have been found in the lesions of MS patients, we expanded this idea to our own models. We found A1-reactive astrocytes in the EAE spinal cord, and two pro-inflammatory cytokine-induced reactivities in differentiated C6 astrocytes. The first type (A1-reactive) express heightened NF-kappa B, complement C3 and Nrf2, while the levels of these constituents in the second (pan-reactive) type are reduced. Furthermore, inhibition of active NF-kappa B in A1-reactive astrocytes results in abrogated expression of Nrf2 and complement C3, suggesting that both the astrocytic response to inflammation and Nrf2 expression are connected to NF-kappa B signaling.

The findings presented in this dissertation shed light on the signaling mechanisms responsible for proteasome regulation in an animal model of MS. They also reveal the differences in these mechanisms and proteasome composition in neurons and astrocytes, as well as diverse reactions by astrocytes during inflammation. We hope that these studies will not only increase our understanding of the pathophysiological processes underlying MS, but also provide the basis for developing new therapeutic approaches to treat this debilitating neurological disorder.

Keywords

Experimental autoimmune encephalomyelitis, astrocyte, neuron, proteasome, Nrf1/Nrf2, astrocyte reactivity

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Oscar Bizzozero

Second Committee Member

Kevin Caldwell

Third Committee Member

Rebecca Hartley

Fourth Committee Member

Nora Perrone-Bizzozero

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