Doctor of Philosophy, Case Western Reserve University, 2024, Pathology

The Unfolded Protein Response In T Cell Thymic Development And Peripheral Differentiation Abstract by BRENDAN M. BARTON Effector T cell differentiation involves coordination of transcription factor networks reinforced by signals integrated from their microenvironment. Herein, I demonstrate that the tripartite unfolded protein response (UPR) integrated signals in vitro downstream of T cell receptor signaling and co-stimulation and in vivo in response to viral infection. While mice with T cell specific deficiency of individual UPR enzymes mounted normal anti-viral responses, mice containing T cells devoid of all three UPR enzymes markedly failed to generate anti-viral T cell responses and exhibited early exhaustion. Transcriptome and epigenetic analysis revealed UPR-deficient CD4+ and CD8+ antiviral T cells failed to upregulate the Ets1-Id2 transcription factor network to promote chromatin accessibility at multiple effector gene loci. Re-expression of the transcription factor XBP1s downstream of UPR sensor IRE1α was sufficient to rescue these defects. Thus, I demonstrate the underlying compensation of UPR enzymes and an interdependent role of UPR sensors in T cell effector differentiation.

Committee: Brian Cobb (Committee Chair); Derek Abbott (Committee Member); Stanley Adoro (Advisor); Clive Hamlin (Committee Member); John Tilton (Committee Member) Subjects: Immunology

Doctor of Philosophy, Case Western Reserve University, 2023, Biochemistry

Endoplasmic Reticulum (ER) stress, caused by the accumulation of misfolded proteins in the ER, elicits a homeostatic mechanism known as the Unfolded Protein Response (UPR). The UPR reprograms gene expression to promote adaptation to chronic ER stress. The UPR comprises an acute phase involving inhibition of bulk protein synthesis and a chronic phase of transcriptional induction coupled with the partial recovery of protein synthesis. However, the role of transcriptional regulation during the acute phase of the UPR is not well understood. In this study (Alzahrani et al., 2022), I analyzed the fate of newly synthesized mRNA encoding the protective and homeostatic transcription factor X-box binding protein 1 (XBP1) during this acute phase of UPR. Global translational repression induced during the acute UPR was documented and characterized by decreased translation and increased stability of XBP1 mRNA. My data suggest this stabilization of XBP1 mRNA is independent of new transcription. In contrast, newly synthesized XBP1 mRNA is shown to accumulate with long poly(A) tails and escapes translational repression during the acute phase of UPR. Inhibition of nascent RNA polyadenylation during the acute phase decreased cell survival with no effect in unstressed cells. During the chronic phase of the UPR, XBP1 mRNA abundance and long poly(A) tails decreased in a manner consistent with co-translational deadenylation. Finally, additional pro-survival, transcriptionally-induced genes show similar regulation, supporting the broad significance of the pre-steady state UPR in translational control during ER stress. I conclude that the biphasic regulation of poly(A) tail length during the UPR represents a previously unrecognized pro-survival mechanism of mammalian gene regulation.

Committee: Maria Hatzoglou (Advisor) Subjects: Molecular Biology

PhD, University of Cincinnati, 0, Medicine: Pathobiology and Molecular Medicine

Invasive aspergillosis (IA) is an often fatal infection most frequently caused by the opportunistic mold pathogen, Aspergillus fumigatus. Current treatment options for IA rely on a limited number of antifungal targets and are plagued by low efficacy and host toxicity. Improving treatment options for IA requires expanding our understanding of the pathways that support A. fumigatus growth and virulence in order to identify points of vulnerability that could be targeted with novel antifungal drug therapies. Because of its important role in A. fumigatus nutrient acquisition and filamentous growth, it is thought that the secretory pathway contributes to A. fumigatus pathogenesis. Homeostasis within the secretory pathway is maintained through four broad mechanisms: ER quality control (ERQC), vesicle transport, ER associated degradation (ERAD), and the unfolded protein response (UPR). ERQC, vesicle transport, and ERAD are basal functions of the secretory pathway that provide the processing capacity required to meet the secretory demands of the organism. When this balance of capacity versus demand is disrupted, the secretory system can become overwhelmed and levels of misfolded proteins within the ER increase. In order to adapt, the cell utilizes an ER stress response pathway known as the UPR. As a result of UPR induction, components of homeostasis pathways are up-regulated in order to restore secretory homeostasis. Previously, our laboratory demonstrated that the UPR contributes to the virulence of A. fumigatus. However, very little is known about how the other secretory homeostasis pathways, ERQC, vesicle transport, and ERAD, contribute to fungal pathogenesis. Additionally, the UPR regulatory mechanisms contributing to pathogenesis are not fully understood. Therefore, the purpose of this dissertation was to characterize for the first time the contribution of key proteins involved in these homeostasis pathways, including the chaperone calnexin (ERQC), the Rab GTPas (open full item for complete abstract)

Committee: David Askew Ph.D. (Committee Chair); Melanie Cushion Ph.D. (Committee Member); Linda Parysek Ph.D. (Committee Member); Judith Rhodes Ph.D. (Committee Member); Alan Smulian M.D. (Committee Member) Subjects: Microbiology

Master of Science, The Ohio State University, 2013, Medical Science

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder. Approximately 90% of ALS cases are sporadic, while the remaining 10% of ALS cases are familial and can be traced to a specific gene defect. Sporadic and familial ALS has been found to be both pathologically and phenotypically similar. In 25% of familial ALS cases, mutations in the superoxide dismutase (SOD1) gene have been found. It is suggested from mouse models of ALS that SOD1 has a dominant gain-of-function that is independent of SOD1 enzymatic activity. However, the mechanism of SOD1 toxicity remains unknown. To aid in elucidating markers and mechanisms of SOD1 toxicity, a transgenic zebrafish model expressing mutant Sod1 was generated. This model has been shown to replicate many of the pathological features observed in other ALS animal models. Initially, in mutant lines DsRed expression was induced in the nervous system while transgenic wild-type Sod fish demonstrated no induction of DsRed without heat shock. However, the heat shock response changed over time and the wild-type fish began to display non-heat shock induced DsRed expression. We investigated whether a mycobacterium infection may have initiated the induction of DsRed in the wild-type lines. We also considered the possibility that DsRed induction was initiated by a stress response such as UPR in the mutant line. We were not able to find consistent changes in monitored immune or stress response genes in either fish line. To look beyond DsRed as marker of disease, we investigated other initial mechanisms of dysfunction. We hypothesized that mutated SOD1 through AMPA receptor excitotoxicity disrupts calcium homeostasis between the ER-mitochondria in motoneurons leading to UPR and motoneuron death. We were able to successfully use the drug thapsigargin as a positive control of ER stress, and to show that mutant larvae are exhibiting the UPR response during late larval stage. We discovered that mutant larvae display a heig (open full item for complete abstract)

Committee: Christine Beattie PhD (Advisor); John Oberdick PhD (Committee Member) Subjects: Neurosciences; Pathology

MS, University of Cincinnati, 2009, Medicine : Immunobiology

The unfolded protein response (UPR) is a conserved mechanism by which cells cope with endogenous threats result in endoplasmic reticulum (ER) stress. Our laboratory has shown that HLA-B27, a human MHC-encoded class I molecule, has a tendency to misfold and accumulate in the ER resulting in UPR activation. HLA-B27 is highly associated with ankylosing spondylitis (AS) and other spondyloarthropathies. Despite long-standing recognition of a striking relationship between HLA-B27 and spondyloarthritis, the mechanism of disease remains unclear.Results from our laboratory have revealed that IL-17 production is increased in HLA-B27 transgenic rats that serve as a spondyloarthritis model. In these rats it is known that CD4+ T cells are critical for pathogenesis while CD8 T cells are dispensable, and that IL-17-producing T cells are increased in the colon tissue. Based on these results, and evidence that IL-17 production can be driven by IL-23, we focused on the question of whether macrophages exhibiting UPR activation produce elevated levels of IL-23. Here, we show that IL-23p19 mRNA is highly upregulated (90-fold) in macrophages experiencing ER stress when further stimulated with LPS. This response is robust and seen at several concentrations of LPS. Furthermore IL-12p35 mRNA is increased 4-fold and IL-12/23p40 mRNA is decreased 3-fold over LPS alone. These mRNA inductions correspond to a 23-fold increase in production of immunoreactive IL-23, while IL-12 is increased 3-fold. Our results suggest that ER stress may enhance production of cytokines that can drive the activation of critical T cell populations. Moreover, there may be a greater polarization toward Th17 IL-23 production, suggesting that Th17 T cells might be preferentially activated. These data raise the possibility that at least one role of HLA-B27 in disease might be to activate ER stress pathways secondary to misfolding, and promote IL-23 production which in turn may activate the IL-23/17 axis and promote inflam (open full item for complete abstract)

Committee: Robert Colbert MD, PhD (Advisor); Chris Karp MD (Committee Chair); Fred Finkelman MD (Committee Member); Kim Risma MD, PhD (Committee Member); Dave Hildeman PhD (Committee Member); Matt Flick PhD (Committee Member) Subjects: Biomedical Research; Cellular Biology; Immunology

PhD, University of Cincinnati, 2007, Medicine : Molecular and Developmental Biology

The Human Leukocyte Antigen (HLA)-B27 is a Major Histocompatibility Complex (MHC) Class I allele that is highly associated with spondyloarthropathy. Biochemical studies have shown that HLA-B27 heavy chains are prone to misfold in the endoplasmic reticulum (ER). Here we provide evidence that misfolding of HLA-B27 generates ER-stress, resulting in activation of the unfolded protein response (UPR) in cells from HLA-B27 transgenic rats, an animal model of spondyloarthropathy. This response is specific for HLA-B27 and strongly correlated with the magnitude of HLA-B27 expression, which could account for the lack of UPR activity in some cells and robust activation of this pathway in others. Strong UPR activation occurs in HLA-B27 transgenic rat macrophages. This, in turn, augments the expression of the inflammatory cytokines, IL-23 p19 and IFN-beta, when these cells are stimulated with LPS or other TLR ligands. IL-23 and its downstream target, IL-17, were both shown to be elevated, concurrent with the development of colitis in HLA-B27 transgenic rats. These findings suggest a novel pathogenic mechanism of disease, with HLA-B27 misfolding as the root cause of inflammation. Our studies also identify a novel immunologic effect of the UPR that could provide the critical link to explain how protein misfolding can cause inflammatory disease. Future studies will focus on testing the model set forth by this work and the relevance of our findings to human disease.

Committee: Dr. Robert Colbert (Advisor) Subjects:

Doctor of Philosophy, Case Western Reserve University, 2010, Nutrition

Expression of the arginine/lysine transporter Cat-1 is highly induced in proliferating and stressed cells via mechanisms that include transcriptional activation. It is shown here that basal expression of the Cat-1 gene is controlled by an Sp1-binding sequence (TCCCCGCCCACAGGGG) within a GC-rich region of the Cat-1 gene. The activity of the promoter is also positively regulated by a stress-response element, AARE, within the first exon of the gene, which binds ATF4, the master regulator of the transcriptional response to stress. A bifunctional DNA element (INE) within the first intron of the Cat-1 gene was identified and characterized in this study. The INE had high sequence homology to an AARE and was shown to act as a transcriptional enhancer in unstressed cells by binding the transcription factor Purine-rich element binding protein A (Purα). During endoplasmic reticulum stress, binding of Purα to the element decreased and the INE acted as both a positive regulator by binding ATF4 and as a negative regulator by binding the stress-induced C/EBP family member, CHOP. We conclude that transcriptional control of the Cat-1 gene is tightly controlled by multiple cis-DNA elements, contributing to regulation of cationic amino acid transport for cell growth and proliferation. In addition, we propose that genes may use stress-response elements such as the INE, to support basal expression in the absence of stress.

Committee: Edith Lerner (Committee Chair); Danny Manor (Committee Member); Martin Snider (Committee Member); Jonathan whittaker (Committee Member); Maria Hatzoglou (Advisor) Subjects: Nutrition

Master of Sciences, Case Western Reserve University, 2008, Physiology and Biophysics

The Unfolded Protein Response (UPR) within the Endoplasmic Reticulum (ER) is a quality control mechanism ensuring properly folded proteins. OASIS and XBP-1 are two signal carriers of the UPR. The UPR is linked to tissue and cancer development. Elevated OASIS and XBP-1 activity are observed within developing osteoblasts. Additionally, heightened OASIS activity is present within osteosarcoma while sustained XBP-1 activity, unreported in osteosarcoma, is observed in other cancers. These signaling proteins may mediate development of mature osteoblasts and osteosarcomas. Therefore, evidence is presented with supporting mechanistic hypotheses indicating unique functions for OASIS and XBP-1 in osteoblast differentiation and osteosarcoma. These functions include, but are not limited to, enlarging the ER, buffering unfolded protein accumulation, mitigating UPR-induced cell death, and processing soluble ER proteins destined for secretion.

Committee: Edward Greenfield (Advisor) Subjects:

PhD, University of Cincinnati, 2012, Medicine: Immunology

The ER-localized DnaJ homologue 4 (ERdj4) is a soluble ER chaperone induced by the unfolded protein response (UPR) to assist in the removal of unfolded/misfolded proteins from the ER lumen for proteasomal degradation. To elucidate the function of ERdj4 in vivo, ERdj4 gene trap mice were generated from embryonic stem cells harboring a gene trap cassette inserted into the ERdj4 locus. ERdj4 gene trap mice expressed hypomorphic levels of ERdj4 with a 10-100 fold reduction in all tissues and cell types examined. Approximately 30-50% of ERdj4 gene trap mice died perinatally in association with growth retardation and hypoglycemia. ERdj4 gene trap neonates exhibited signs of delayed pancreatic development, including abnormal distribution of pancreatic α- and β-cells and reduced insulin and glucagon in the pancreas. Surviving adult ERdj4 gene trap mice were glucose intolerant, resulting from hypoinsulinemia rather than insulin resistance. Pancreatic β-cells exhibited increased ER stress, including ER dilation and upregulation of the UPR. Proinsulin accumulated in the ER of β-cells from ERdj4 gene trap mice consistent with our previous finding that proinsulin is a substrate for ERdj4. The insulin processing enzymes, including Pcsk1, Pcsk2 and CPE, also associated with ERdj4, contributing to defects in proinsulin biosynthesis in ERdj4 gene trap mice. ERdj4 deficiency also resulted in pancreatic a-cell hyperplasia in association with increased ER stress. Since previous studies had demonstrated that the UPR is required for both early and late B lymphopoiesis, we hypothesized that ERdj4 deficiency would affect B cell development. Pro-B, pre-B, immature and mature B cell populations were significantly reduced in the bone marrow of ERdj4 gene trap mice in association with increased pro-B cell death. Further, mature B cell populations were reduced in the spleen and peritoneal cavity. ERdj4 gene trap donor cells transplanted into wildtype recipients rescued all stages of B cell deve (open full item for complete abstract)

Committee: Timothy Weaver Ph.D. (Committee Chair); H. Leighton Grimes Ph.D. (Committee Member); George Deepe M.D. (Committee Member); Fred Finkelman M.D. (Committee Member); Christopher Karp M.D. (Committee Member); Francis McCormack M.D. (Committee Member) Subjects: Molecular Biology