Author ORCID Identifier

https://orcid.org/0000-0002-8798-5484

Semester

Fall

Date of Graduation

2023

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Gordon P Meares

Committee Member

Randy Nelson

Committee Member

Candice Brown

Committee Member

Edwin Wan

Committee Member

Timothy Eubank

Abstract

Neurological disorders such as multiple sclerosis (MS) are a major public health concern in the US, with no available therapeutic cure. Chronic neuroinflammation and aberrant proteostasis in the central nervous system (CNS) are the major hallmarks of neurological diseases. Endoplasmic Reticulum (ER) is a major cellular organelle involved in protein synthesis, folding and maturation of various secretory and transmembrane proteins. Pathophysiological stressors such as trauma and infection result in misfolded protein accumulation in the endoplasmic reticulum (ER) lumen, which results in ER stress. To regain proteostasis (protein homeostasis), cells activate the unfolded protein response (UPR). UPR is an evolutionarily conserved stress response pathway that is vital for maintaining proteostasis and cell survival. UPR signaling is partly driven by the activation of ER-resident transmembrane Se/Thr Kinase protein double-stranded RNA-activated protein kinase (PKR)–like ER kinase (PERK). PERK phosphorylates eukaryotic initiation factor (eIF)2α which inhibits global translation to reduce cellular protein folding load. Chronic UPR activation promotes inflammation and apoptotic cell death. Astrocytes and microglia are the major drivers of neuroinflammation in response to CNS injury and infection. Astrocytes and microglia are relatively resistant to ER stress induced cell death. Our lab and others have observed increased expression of proinflammatory mediators such as tumor necrosis factor-α (TNF-α) and PERK activation in the glial cells from MS patients and experimental Autoimmune Encephalomyelitis (EAE), a model of MS. The contribution of glial PERK signaling in driving neuroinflammation is not well known. Previously, we have demonstrated that ER stress induces PERK dependent proinflammatory gene expression in primary astrocytes. In this study, we have reported that, ER stress globally alters TNF-α induced astrocytic transcription and translational program. ER stress also synergizes with TNF-α induced astrocytic interleukin-6 (IL-6) gene expression in a PERK dependent manner. Increased IL-6 expression contributes to the pathogenesis of MS and EAE. Therefore, we have examined the molecular mechanism of astrocytic IL-6 synergistic gene expression. Our results showed that IL-6 synergy in astrocytes is dependent on nuclear factor kappa B (NF-κB), phosphoinositide-3-kinase–protein kinase B/Akt (PI3K-PKB/Akt) signaling pathways. Our results also showed that IL-6 synergy is dependent on cellular protein synthesis and secretion, which indicated possible regulation of IL-6 synergy by autocrine signaling. Our in vitro studies have demonstrated that astrocytic PERK signaling is a critical regulator neuroinflammation in vitro. Previous studies have defined the role of oligodendrocyte and neuron specific PERK signaling in driving neuroinflammation and neural injury in vivo. We have bred astrocyte and microglia specific PERK knockout mice and examined the contribution of glial PERK signaling in EAE model. Both astrocytic and microglial PERK knockout mice did not show any changes in general locomotion. Our in vivo EAE studies demonstrated that astrocytes and microglia PERK deletion in vivo does not affect EAE disease onset and progression. However, astrocytic PERK have a modest beneficial neuroprotective effect in chronic phase of EAE whereas microglial PERK does not have any effect in disease severity. We have also found iii | P a g e that in middle cerebral artery occlusion (MCAO) model, an ER stress inducing CNS injury model, astrocyte specific PERK knockout mice had significantly more death and worse functional impairment compared to sham. These data indicate that astrocytic PERK might confer beneficial neuroprotection in the in vivo models of neuroinflammation and neural injury. Aging is associated with proteostasis impairment and resultant cognitive decline. Phosphorylation of eIF2α is critical in the regulation of learning and memory. PERK signaling in neuron was examined mostly in the regulation of memory. Although astrocytes and microglia significantly modulate learning and memory function, the role of glial cell specific PERK signaling in learning and memory function is not known. Our behavioral studies have demonstrated that astrocytic PERK knockout in middle and old aged mice does not affect learning and memory function. We have also observed that microglial PERK does not affect short-term memory. To summarize, this work has addressed a substantial knowledge gap in cell specific function of ER stress signaling in driving neuroinflammatory response and cognitive processes. Our findings have demonstrated that glial cell specific PERK signaling is a critical regulator of neuroinflammation in vitro and in vivo.

Embargo Reason

Publication Pending

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