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Parkinson's disease (PD) is a progressive neurodegenerative disease characterised by the loss of dopaminergic (DA) neurons, causing deficits in motor function. At present, mitochondrial dysfunction and the endoplasmic reticulum (ER) unfolded protein response (UPR) are perceived to be molecular features of PD. The UPR is an evolutionarily conserved adaptive cellular response to unfolded or misfolded proteins in the ER. Three ER-resident proteins sense the UPR, one of which is the protein kinase RNA (PKR)-like ER kinase (PERK). PERK activation leads to translational repression via phosphorylation of eukaryotic translation initiation factor-2 α (eIF2α) as well as activation of activating transcription factor 4 (ATF4). This triggers a transcriptional response, initially promoting cell survival. Eventually, the sustained activation of the UPR leads to cell death. PERK has also been reported to modulate mitochondrial function. The overarching aim of this thesis was to investigate the relationship between PERK kinase and mitochondrial dysfunction in the pathogenesis of PD using the fruit fly Drosophila melanogaster as a model organism. To characterise the Drosophila PERK (dPerk) expressional landscape, I combined microarray and quantitative proteomics analysis from adult flies overexpressing dPerk. Using this approach, I identified tribbles (trbl) and Heat shock protein 22 (Hsp22) as two novel Drosophila ATF4 (dAtf4) regulated transcripts. Furthermore, I established that dPerk expression leads to translational repression of several mitochondrial proteins. The mitochondria-ER tether ATPase Family AAA Domain Containing 3A (ATAD3A) has recently been suggested to function as a negative regulator of PERK in mammalian models. I show that the ATAD3A fly orthologue Belphegor (Bor) does not act as an inhibitor of dPerk. Furthermore, I propose that this is due to the lack of the proline-rich motif in Drosophila, otherwise present in the protein structure of its mammalian orthologue. Mutations in PTEN-induced putative kinase 1 (PINK1), a mitophagy gene, cause neurodegeneration in some autosomal recessive forms of PD. In Drosophila, mutations in the pink1 gene cause mitochondrial dysfunction and the degeneration of DA neurons. Pink1 mutants also show overactivation of the dPerk/eIF2α/dAtf4 axis. Therefore, I used the pink1 PD Drosophila model to further probe the role of dPerk in the pathomechanism of PD. PD patients experience gastrointestinal issues that often precede the onset of motor symptoms, implicating the gut-brain axis in the pathogenesis of this disease. Likewise, in pink1 mutants, mitochondrial dysfunction leads to cell death and proliferation in the Drosophila midgut. Suppressing intestinal dysfunction is neuroprotective. I found that the intestinal expression of dPerk causes intestinal damage, and silencing dPerk in the pink1 intestine leads to a rescue of intestinal dysfunction and neurodegeneration. The ER stress marker Trbl also acts as an inhibitor of Akt, implicating this pseudokinase as a negative regulator of insulin signalling. The human Trbl orthologue tribbles pseudokinase 3 (TRIB3) is upregulated in insulin resistant as well as obese adults, and TRIB3 polymorphisms are associated with type 2 diabetes and insulin resistance. The fat body is a fly organ homologous to the mammalian liver and adipose tissue. It functions to synthesise and store triacylglycerol and regulate endocrine and immune signalling, implicating it in the regulation of complex behaviours such as sleep. My results show that the FB expression of Trbl leads to metabolic defects, systemic repression of insulin signalling and a reduction in night-time sleep. This thesis investigates inter-organelle as well as inter-organ signalling using Drosophila melanogaster as a model organism. My research shows that the ER UPR kinase dPerk is an important regulator of mitochondrial function and intestinal homeostasis, suggesting dPerk as a potential therapeutic target for PD. Furthermore, analysis of Trbl pseudokinase function in Drosophila melanogaster proposes Trbl as a molecular link between animal nutrient state and behaviour.

Heart failure (HF) associated with diabetes is a distinct clinical entity known as diabetic cardiomyopathy (DCM) without the prevalence of other risk factors such as hypertension. The human heart is a pool of different cell types, including cardiomyocytes, fibroblasts, endothelial cells and immune cells, interacting via various secretory factors. The cardiomyocyte acting as an active mediator of the inflammatory response is an emerging concept with limited mechanistic understanding. Superimposed effects of hyperglycaemia and hyperlipidaemia dysregulate the intercellular links, contributing to diabetes-induced cardiac dysfunction. DCM is instigated by various pathological insults, including endoplasmic reticulum (ER) stress response and ectopic fibroblast growth factor receptor 1 (FGFR1) signalling, resulting in detrimental impacts on cardiac structure and function. Hence, elucidating the paracrine and autocrine mediators of cardiac intracellular signalling can provide new mechanistic and therapeutic strategies for HF in diabetes. First, we demonstrate the opposing roles of the cardiac ER stress response in our time-course model of high fat high-sugar diet (HFHSD)-induced diabetes. Using cardiac P21-activated kinase 2 (PAK2) deficient mice, we found that PAK2, an ER-localised signalling kinase, decelerates the transition from an adaptive to a maladaptive ER response in DCM. A concurrent examination of the myocardial inflammation, particularly macrophage response, revealed chronic cardiac ER stress to be the initiating cascade of the alarmin high-mobility group box 1 (HMGB1) in DCM. HMGB1 expression and subsequent secretion into the circulation was promoted via C/EBP homologous protein (CHOP), an ER stress responder. Our in vivo DCM models of ER dysfunction, the remediated ER response and pharmacological treatments established the functional and therapeutic relevance of targeting cardiac ER stress-mediated inflammation for alleviating DM-induced cardiac complications. Secondly, we determined that the FGFR1 cascade is the upstream facilitator of the PAK2 response. In diabetes, we observed a lack of cardiac FGFR1 and beta-Klotho (β-KL) signalling. We found that restoring cardiac FGFR1/β-KL expression results in the increased secretion of anti-inflammatory and proangiogenic factors from cardiomyocytes. Using proteomic analyses, we identified key cardiac-derived signals that regulate angiogenesis and macrophage polarisation upon FGFR1/β-KL activation in diabetes. This provides a proof-of-concept to explore the novel therapeutic avenue of myocardial cell interactions to prevent cardiac dysfunction under metabolic stress. Type 2 diabetes is a chronic disorder; therefore, our time-course analyses provide a fascinating insight into the early mechanistic events in DCM. We address the knowledge gap of cardiomyocyte-mediated macrophage and endothelial cell response and reveal mechanistic links behind cardiac inflammation and vascular damage in response to diet-induced metabolic stress. Hence, further dissection of the identified molecular web between cardiomyocytes and endothelial cells or macrophages in DCM may lead to the development of efficacious treatments for diabetes-induced HF.

For centuries, the threat of prion disease has plagued populations – whether it be in the form of scrapie ravaging through the sheep populations of Spain in the eighteenth century, fatal familial insomnia afflicting families in Italy, or an outbreak of Creutzfeldt-Jakob disease in the UK triggered by the consumption of contaminated beef. The ability of prions to spread with such pathogenic intent whilst remaining incurable fuels the fire of public concern - yet delving beyond that classical view of prions has led to an enlightened appreciation of the role of such proteins throughout the biota of life. Prion-like proteins subvert the dogma of the one-to-one structure-function relationship traditionally held onto when considering the physiology of proteins. Instead, these proteins display structural polymorphism, capable of adopting different conformations under different conditions; this expands the capabilities of protein function and represents a mechanism for epigenetic coding, with protein conformation possessing the ability convey and propagate specific states for biological advantage without the need for DNA modification. The properties observed in many of these proteins, including those of multivalency and low complexity, predispose them towards phase separation, whereby localised concentration of biomolecules compartmentalise cellular activity. This ability confers upon an organism the ability to respond to stimuli in dynamic and transient fashions without needing to use cumbersome membrane-bound organelles. Using the Prion-Like Amino Acid Composition program, we have identified the stress-responsive protein ABU-13 as a potential prion in the nematode species, Caenorhabditis elegans. Phenotypic analysis of ABU-13 knockout animals has identified a non-redundant role for this protein in ER stress and innate immune responses – in keeping with previous evidence that this family of proteins is involved with a non-canonical unfolded protein response pathway of the endoplasmic reticulum. These knockout animals do not display defects in the activation of the stress-response gene hsp-4 upon tunicamycin treatment, suggesting that these effects occur independently of the canonical UPR pathway. In vivo and in vitro characterisation of ABU-13 has demonstrated a propensity for coalescence, reminiscent of a phase-separated organelle. This is further supported by the ability of this protein to bind RNA – a common feature of prion-like proteins involved with such transitions. Co-immunoprecipitation of ABU-13 confirmed an enrichment of ER and RNA-related protein interactions, supporting a role for this protein in ER stress responses. In addition to this, a number of glycosylation related terms were identified, pointing towards a role for ABU-13 in the folding quality control of such proteins. These punctated structures do, however, appear less mobile than traditional liquid-like condensates, as demonstrated by a slow FRAP recovery, indicative of a more hydrogel-like structure. To further investigate this, we extracted hydrogel-forming proteins from N2 animals using a biotinylated isoxazole precipitation, identifying a number of novel proteins that may be involved with in the formation of such solid-like structures. ABU-13, however, was not amongst this dataset, suggesting that if it were indeed involved in a physiological hydrogel, this transition is driven by another protein component. Overall, we propose that ABU-13 represents a phase-separating prion-like protein capable of modulating ER stress and immune responses, potentially via the regulation of RNA processing.

Endoplasmic Reticulum (ER) is a vital part for functional protein synthesis in eukaryotic cells. ER stress is caused by over-accumulation of unfolded or misfolded proteins, which are massively produced in dysfunctional protein synthesis in the ER lumen. In a plant cell, ER stress can be induced by various environment factors, such as saline stress and heat stress. Under ER stress, a self-defensive mechanism, unfolded protein response (UPR), can be activated to restore the homeostasis from ER stress in a systemic way. Unlike the UPR in mammalian cells, by now only two pathways have been identified: IRE1α/β pathway and bZIP28 pathway. Both IRE1α/β and bZIP28 are ER stress sensors located on the ER membrane. IRE1α/β is activated under sensing the accumulation of unfolded protein in the ER lumen and then catalyse the splicing of bzip60 mRNA. Spliced bzip60 mRNA will finally be translated to transcription factor bZIP60 . bZIP28, when activated by sensing unfolded proteins in the ER lumen, will dissociate from the ER membrane and then be further cleaved by protease at Golgi before turning to transcription factor bZIP28. The transcription factor bZIP60 and bZIP28 can then promote the transcription of a group of UPR genes. A gene of molecular chaperone, BiP, is one of the typical UPR genes and BiP protein can help alleviate ER stress by refolding the badly folded proteins in the ER lumen. In recent researches, ER stress was found to be connected with plant pro- grammed cell death (PCD). Under prolonged ER stress, plant cells would finally go to PCD and caspase-like activity before cell death can be detected by exper- imental techniques. Additionally, more evidence was presented to link ER stress and plant PCD. One important transcription factor, NAC089, was found not only to be involved in UPR signalling (controlled by both bZIP60 and bZIP28), but could also promote PCD in plant cells. The conflicting roles of NAC089 play could potentially decide the fates of cells under ER stress. In this thesis, we are going to use both experimental and computational meth- ods to further understand the UPR in plant cells based on the current knowledge. We first present the functions of cathepsin-B and proteasome under ER stress, giving more evidence of the connection between ER stress and plant PCD. Next, we hope to know more about the role that NAC089 plays under ER stress. We present a novel mathematical model on the UPR signalling, with NAC089 included. We present both algebraic and numerical analysis of the model we use. In this model, we use ordinary differential equations (ODEs) in order to make prediction of cell-fate decision in a quantitative way. To have an insight of the fate decision under ER stress in Arabidopsis, we present our data using different experimental systems, from single cell protoplast system to plant leaf tissue system. We also introduce our design of an original 'Time-Dose' matrix approach for Arabidopsis seedling system in the thesis. Our matrix approach shows clear phenotype difference in seedlings under different ER stress conditions. Finally, we try to use our UPR model to compare the transcript changes with our experimental data. We find our UPR model still needs improve- ment due to some incompatibilities between the experimental data and our UPR model, although the model makes some promising predictions at the same time.

Obesity-associated metabolic disorders are amongst the most prevalent causes of death worldwide. Understanding how obesity leads to the development of the Metabolic Syndrome (MetS) and cardiovascular disease (CVD) will enable the development of novel therapies that dissociate obesity from its cardiometabolic complications. Our laboratory views the functional capacity of white adipose tissue (WAT), the organ designed for safe lipid storage, as a key factor in the development of MetS and CVD. At a genetically-defined stage of the aberrant WAT expansion that occurs during obesity, adipocytes undergo a functional failure, resulting in an impaired control of serum free fatty acid (FFA) concentration. In such setting, FFAs and their metabolic derivatives accumulate in other organs, where they cause lipotoxicity, leading to the development of insulin resistance and CVD. We therefore aim to understand the pathophysiological mechanisms that induce adipocyte dysfunction. The past two decades of research have established the immune system as an important regulator of WAT function. The number of adipose tissue macrophages (ATMs), the most abundant immune cell type in WAT, increases during obesity, resulting in WAT inflammation. Multiple genetic and pharmacological intervention studies of murine models of obesity have assigned a causal link between ATM pro-inflammatory activation and WAT dysfunction. However, while the propagation of inflammation in ATMs during obesity has been extensively studied, factors triggering ATM inflammatory activation are less clear. Recently, our lab has observed lipid accumulation in the ATMs isolated from obese mice. Lipid-laden ATMs were pro-inflammatory, leading us to hypothesise that aberrant lipid build-up in macrophages triggers WAT inflammation during obesity. This thesis expands on the initial findings from our lab and describes two novel mechanisms that potentially contribute to lipid-induced inflammatory activation of ATMs. In chapter 3, the role of de novo phosphatidylcholine (PC) synthesis pathway during lipotoxicity in macrophages is addressed. The first part of the chapter demonstrates that lipotoxic environment increased de novo PC synthesis rate in bone marrow-derived macrophages (BMDMs) and ATMs, and that loss of rate-limiting enzyme in de novo PC synthesis pathway, CTP:phosphocholine cytidylyltransferase a (CCTa) diminished saturated FFA-induced inflammation in BMDMs. In the second part, I show that macrophage-specific CCTa deletion did not impact on the development of WAT inflammation or systemic insulin resistance, but had a minor benefitial effect on hepatic gene transcription during obesity. Chapter 4 develops on recent observations of interactions between sympathetic nerves and macrophages in WAT. In the first part of the chapter, I demonstrate that stimulating B2-adrenergic receptor (B2AR), the main receptor for sympathetic neurotransmitter norepinephrine in macrophages, enhanced intracellular triglyceride storage by up-regulating diacylglycerol O-acyltransferase 1 (Dgat1) gene expression in BMDMs. The second part of the chapter shows that macrophage-specific B2AR deletion did not modulate systemic glucose and lipid metabolism during obesity, but mice lacking B2ARs in macrophages demonstrated augmented hepatic glucose production on a chow diet. Furthermore, systemic B2AR blockade or macrophage-specific B2AR deletion in mice did not affect the thermogenic response to cold exposure. Chapter 5 includes the characterisation of B2AR stimulation-induced changes to the global cellular proteome of BMDMs, and a subsequent validation of the role of candidate transcription factors in regulating B2AR agonism-induced gene expression in BMDMs.

Approximately 340 million people worldwide have Type 2 Diabetes Mellitus (T2DM), making identification of the aetiological processes underlying this disease imperative. Endoplasmic reticulum (ER) stress has emerged as a potential mechanism driving the pathogenesis of obesity and T2DM. Palmitate, the predominant saturated fatty acid elevated in the blood plasma of obese individuals, induces ER stress and insulin resistance in skeletal muscle. However, the interaction between ER stress, lipid metabolism and T2DM remains poorly understood. This thesis uses lipidomic tools to investigate skeletal muscle ER stress in cell culture and animal models. Chronic palmitate treatment of both mouse C2C12 and human primary myotubes induced ER stress, concurrent with the cytosolic phospholipase A2-dependent release of polyunsaturated fatty acids (PUFAs) from phosphatidylcholines (PCs). This phenotype was also observed in skeletal muscle from mouse models of diet-induced obesity and T2DM patients. Palmitate-stimulated catabolism of PUFA-containing PCs was concomitant with increases in bioactive eicosanoid secretion, which was shown to be important in the control of ER stress, inflammatory signalling and macrophage activation. This provides a novel link between palmitate and the control of ER stress and inflammation in metabolic disease. Previous work has suggested that the propagation of ER stress signalling between cells may result from the secretion of a, as yet undefined, cell non-autonomous signal. In this thesis, long-chain ceramides (40:1 and 42:1) were identified as signals transmitting the induction of ER stress between myotubes via exosome-mediated transport. Long-chain ceramide concentrations were increased in skeletal muscle and blood plasma from in vivo models of obesity and were elevated in the muscle of T2DM patients. Muscle synthesis of long chain ceramides in response to palmitate was found to occur via the de novo pathway and was linked to ER stress by Perk, an important unfolded protein response kinase. This work identifies ceramides as cell non-autonomous signals that propagate ER stress activation following exposure to palmitate, providing a novel mechanism for stress signalling in obesity and insulin resistance.

Dihydronicotinamide riboside (NRH) quinone oxidoreductase 2 (NQO2) is involved in quinone metabolism reducing quinone to hydroquinone. Quinones are products of oestrogen metabolism and are responsible for the oestrogen-initiated breast carcinogenesis. It has been demonstrated that oestrogen quinones are endogenous biological substrates of NQO2 which acting as a detoxification enzyme catalyses the reduction of oestrogen quinones to hydroquinone. Hydroquinone can then be removed by conjugation to glutathione or glucuronic acid. In this study, the oestrogen dependent and oestrogen independent effects of NQO2 in a variety of networks implicated in breast tumorigenesis were investigated aiming to understand the potential role of NQO2 overexpression in mammary carcinomas. The use of NRH as a cofactor for NQO2 is being studied in parallel with the Î2-oestradiol and tamoxifen treatments. The MCF-7, T47D, MDA-MB-231 and MDA-MB-468 breast cancer cells were transfected with increasing amounts of NQO2 and its biological activity in regulating ERα transcriptional activity, reactive oxygen species (ROS) generation, cell cycle control, mitochondrial membrane potential and antioxidant activities including catalase activity, glutathione (GSH) levels and glutathione peroxidase (GPx) activity were studied. NQO2 overexpression in MDA-MB-231 and T47D cells reduced ROS generation. Increasing amounts of transfected NQO2 induced the ERα transcriptional activity in Î2-oestradiol treated MCF-7 and T47D cells and decreased cyclin D1 protein levels in these cells treated with Î2-oestradiol compared to untransfected cells. Reduction of catalase activity was detected in tamoxifen treated T47D cells overexpressing NQO2, an effect that was not evident in Î2-oestradiol treated cells, whereas NQO2 mediated reduction of GSH levels was detected in these cells treated with Î2-oestradiol but not with tamoxifen. Finally, NQO2 affected mitochondrial membrane depolarization in Î2-oestradiol treated MDA-MB-231 cells. Given the fact that NRH is not physiologically synthesized in humans, the results presented in this study are valuable from the fundamental science point of view indicating the existence of a potential link between NQO2 and estrogens affecting a number of biological pathways important for breast carcinogenesis and as such from the clinical angle it could be assumed that NQO2 effects could impact the design of personalised breast cancer treatment of oestrogen receptor positive and negative breast cancers.

Cytochrome P450 enzymes (CYP) are key oxidative enzymes that are crucial in several biological processes, such as metabolism of exogenous and endogenous substances, the biological transformation of drugs and xenobiotics and biosynthesis of steroids and fatty acid. Several CYP have been identified in extra hepatic tissues implying that these enzymes exert other biological functions, which might explain their association with a number of diseases including diabetes, obesity and cancer. Understanding of these functions may provide the platform for the development of new therapeutic approaches and this is the aim of this investigation, namely to delineate the role of CYP in breast carcinogenesis. Cancer cells exhibit high levels of glycolysis even in the presence of high oxygen concentration. Cancer cells have very high proliferating rates so they need more biosynthesis materials like nucleic acids, phospholipids, fatty acids and glycolysis is the main source of biosynthetic precursors. Energy metabolism has recently attracted the interest of several laboratories as targeting the pathways for energy production in cancer cells could be an efficient anticancer treatment. Previous studies have shown that reactive oxygen species (ROS) regulate the energy metabolism in cancer cells. CYP are one of the ROS source. Expression of CYP in extrahepatic implies that these enzymes exert other biological functions which have not yet been elucidated. These findings led us to hypothesise that cytochrome P450 enzymes might be involved in the determination of the pathway of cellular energy metabolism in breast cancer cells and in particular in directing tumour cells to produce energy through glycolysis rather than Oxidative phosphorylation (OXPHOS). To investigate the role of CYP in breast carcinogenesis, we followed the protein levels of CYP1B1, CYP1A1, CYP2E1, CYP2C8, CYP2C9 and CYP3A4 in MCF-7 (Michigan Cancer Foundation-7), T47-D, MDA-MB-231 (MD Anderson series 231 cell line) and MDA-MB-468 (MD Anderson series 468 cell line) breast cancer cells treated with glycolytic inhibitors 3-Bromopyruvate and 2-Deoxyglucose (3BP and 2DG). CYP were differentially expressed in breast cancer cells upon treatment with the glycolytic inhibitors (2DG and 3BP) in breast cancer cell lines bearing different genetic background and migratory capacity. The CYP mediated ROS generation was followed in breast cancer cells overexpressing CYP1B1, CYP2C8, CYP2C9 and CYP2E1 or treated with 3BP, 2DG and CYP1B1 specific inhibitor 2,3',4,5'-Tetramethoxystilbene (TMS) by H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) staining. The functional significance of the CYP1B1, CYP2C8, CYP2C9, CYP2E1 mediated modulation of the cellular redox state was investigated by recording changes of indicators of biological pathways known to be affected by the cellular redox state such as cell cycle, adenosine triphosphate (ATP) level, lactate level, mitochondrial potential, autophagy and endoplasmic reticulum (ER) stress. Furthermore, the effect of CYP1B1 and CYP2E1 induction by their inducers (Benzopyrene and Acetaminophen respectively) and inhibition by their specific inhibitors (TMS and chlormethiazole (CMZ) respectively) on cell survival was investigated. Migratory potential of breast cancer cells was investigated under the treatment of glycolytic inhibitors, CYP1B1 inducer and inhibitors. The results obtained provide evidence that CYP are potentially involved in the regulation of ROS, cell cycle, ATP level, lactate level, mitochondrial potential, autophagy, ER stress and migratory potential in a manner dependent on the genetic background of the cells and the stage of the breast cancer, supporting the notion that CYP are potential breast cancer biomarkers.

Mutations in the COL10A1 gene cause metaphyseal chondrodysplasia type Schmid (MCDS) by triggering ER stress and unfolded protein response (UPR). MCDS is characterised by a mild short-limb dwarfism accompanied by expansion of the cartilage growth plate hypertrophic zone (HZ) and altered differentiation of hypertrophic chondrocytes (HCs). ATF6 is one of the UPR mediators, which exists in two isoforms, ATF6α and ATF6β. Activation and up-regulation of ATF6α was a prominent biochemical sign of ER stress in a mouse model of MCDS, COL10a1 p.N617K. Although ATF6β is induced and activated in response to ER stress in a similar fashion to ATF6α, the role and significance of ATF6β in the pathology of many ER stress-associated diseases including MCDS is unknown. Here we utilized a combination of in vitro and in vivo approaches to define the precise role of each isoform of ATF6 in MCDS.To investigate the functions of ATF6α and ATF6β in vitro, we developed a MCDS cell model system (expressing either the wild type collagen X or one of the following MCDS-causing mutant forms of the protein: p.N617K, G618V, Y598D, and NC1del10) in which the expression of either ATF6α or ATF6β was efficiently silenced using siRNAs. ATF6α knockdown in HeLa cells expressing different MCDS-causing mutations suppressed the increased expression of UPR-associated genes such as BiP leading to an elevated ER stress, based on increased XBP1 splicing and/or ATF4 protein. In contrast, ATF6β knockdown did not significantly affect the mutant collagen X-induced increased expression of UPR-associated genes. Furthermore, the ER stress levels were significantly reduced in the ATF6β knockdown MCDS mutant cells based on the lower levels of XBP1 splicing and/or ATF4 protein detected. We then crossed the ATF6α/β knockout mice models with COL10a1 p.N617K mouse model of MCDS to investigate the function of ATF6α and ATF6β in vivo. Ablation of ATF6α in MCDS mice further- reduced the endochondral bone growth rate, further expanded the growth plate hypertrophic zone, and disrupted differentiation of HCs. Therefore, ATF6α appeared to play a chondroprotective role in MCDS as its deficiency caused an increase in the severity of the disease. Of particular note, the level of ER stress was further increased in the absence of ATF6α in MCDS, based on enhanced activities of PERK and IRE1 signalling pathways in compensation for the ATF6α loss. Paradoxically, ablation of ATF6β in MCDS mice reduced the intracellular retention of collagen X protein, and alleviated the ER stress as judged by the attenuated activities of PERK and IRE1 signalling pathways. The reduced ER stress resulting from deficiency for ATF6β in MCDS mice restored the expression of collagen X mRNA towards normal and improved the differentiation of HCs, causing a mark decrease in the expansion of HZ. The results presented within this thesis greatly increased our understanding of the function of ATF6α and ATF6β and their interplay in the pathogenesis of MCDS. We demonstrated an indispensable beneficiary role for ATF6α but a detrimental role for its closely related isoform, ATF6β, in pathology of MCDS. We also showed that the role of ATF6β should not be ignored. These findings may be used to develop a potential therapeutic strategy for MCDS through targeting and enhancing ATF6α-dependent and/or attenuating/blocking of ATF6β-dependent signalling pathways.

MCDS is an autosomal dominant skeletal dysplasia disorder caused by mutations in collagen X. In most cases, mutations in collagen X result in a misfolded protein which is retained within the ER of hypertrophic chondrocytes, causing increased ER stress. It has previously been demonstrated that increased ER stress causes hypertrophic chondrocytes to de-differentiate in an attempt to avoid the stress. The altered differentiation results in reduced cell hypertrophy and impaired vascular invasion accounting for reduced bone growth. The presence of increased ER stress in hypertrophic chondrocytes is sufficient to cause the MCDS pathology; therefore reducing ER stress may be beneficial in terms of improving the associated pathology. The autophagy enhancing drug carbamazepine (CBZ) has been shown to be capable of reducing ER stress in cells expressing the MCDS-causing p.N617K collagen X mutation. I show in this thesis that CBZ treatment reduced ER stress in HeLa cells transiently expressing a further 3 MCDS-causing collagen X mutations. I have also demonstrated that CBZ treatment induced the degradation of mutant collagen X proteins either through autophagy or proteasomal degradation depending on the nature of the mutation. The drug was tested in vivo using the p.N617K collagen X mouse model of MCDS. In MCDS mice, CBZ reduced the severity of the disease pathology based on histological analyses, restored hypertrophic chondrocyte differentiation toward normal, increased long bone growth rates and decreased the severity of the hip dysplasia. Gene expression analyses on RNA isolated from microdissected hypertrophic chondrocytes revealed that CBZ shifted the pattern of hypertrophic differentiation markers in MCDS mice toward the wild-type pattern, most likely through its stimulation of gene expression associated with intracellular proteolytic pathways. The results presented in this thesis have contributed to the identification of a potential treatment strategy for MCDS- the stimulation of intracellular proteolysis of mutant collagen X. CBZ is FDA approved for the use of epilepsy and bipolar disorder and has a strong safety record in humans. Therefore CBZ could be a potential treatment strategy for MCDS.

Beukes Hip Dysplasia (BHD) is an autosomal dominant disorder where the abnormal shape of the hip joint leads to secondary osteoarthritis. The locus of BHD has been previously mapped to 4q35 and screening of candidate genes within this region revealed a mutation in the gene encoding the Ubiquitin-fold modifier 1 specific protease 2 (Ufsp2). The mutation prevents Ufsp2 from cleaving its target, Ufm1. Ufsp2 and Ufm1 are both components of a novel ubiquitin-like protein modification system which involves Ufm1 being processed via the E1, E2 and E3 enzymes (Uba5, Ufc1 and Ufl1, respectively) and attachment to target protein(s) one of which has been identified (Ddrgk1). The aim of this study was to investigate the link between the UFSP2 mutation and the BHD phenotype by: (i) examining the expression of components of the Ufm1 system in vivo and in vitro; (ii) development of an in vitro Ufm1 conjugation system; and (iii) generation and analysis of transgenic mice overexpressing the Ufsp2 gene with the BHD mutation. The expression of Ufsp2 was determined by radioactive RNA in situ hybridisation of mouse tissue sections. The analysis revealed Ufsp2 expression predominantly in the bone of the hip joint and in the bone and secondary ossification centres of the knee of 10 day old mice. Real-Time PCR analysis showed increased expression of components of the Ufm1 system during in vitro osteogenic and chondrogenic differentiation which coincided with induction of ER stress evidenced by upregulation of Bip. These components were also upregulated in response to chemically induced ER stress in vitro. Analysis of the promoter regions of Ufm1 system genes identified unfolded protein response elements in the upstream sequences of Uba5, Ufl1, Ufm1 and Lzap genes and the elements in Uba5 and Lzap were found to be required and responsive to ER stress using luciferase promoter assays. A Tandem Affinity Purification method was developed for isolation and identification of Ufm1 conjugation targets from cell lines expressing modified forms of Ufm1. Mass spectrometry analysis of Ufm1 conjugates purified from HEK293T cell line identified Uba5 and Ufc1 but no new Ufm1 targets. Western blot comparison of Ufm1 conjugated proteins purified from the HEK293T and 2T3 osteoblast cell lines identified putatative Ufm1 conjugation targets and increased conjugation in osteoblasts in response to ER stress. One of these targets was identified as Ddrgk1 but the remaining putative targets remain to be identified by mass spectrometry. Transgenic mice overexpressing the mutated Ufsp2 gene were generated and subjected to phenotypic analysis. No significant differences were found between transgenic and wild type mice following X-ray, histological and weight analysis. Higher expression of Ufsp2 in bone and secondary ossification centres as well as upregulation of components of the Ufm1 system in response to ER stress suggests that the molecular pathway between the UFSP2 mutation and the BHD phenotype may relate to abnormal ER stress responses during osteoblast differentiation. Further studies are however required to determine how the Ufm1 system modulates ER stress responses and how disruption of these processes caused by the UFSP2 mutation causes BHD.

MCDS is an autosomal dominant disorder, with a mild dwarfed phenotype and is caused by mutations in collagen X. The majority of the mutations identified so far are localized almost exclusively within the NC1 domain, which is responsible for trimerization of the collagen X protein. Little is known about the onset of MCDS, but recently, up-regulation of ER stress has been suggested as an important mechanism promoting the MCDS phenotype. Several studies have shown that the mutated collagen X protein is retained within the ER triggering the UPR, which has proved to be the key pathway responsible for the pathogenesis of the MCDS phenotype. In order to study the consequences of the expressing the MCDS-causing COL10A1p.N617K mutation at the molecular level, we selected HeLa cells as an appropriate cell line for the characterisation of the UPR response, by showing that the three branches of the UPR can be activated by ER stress inducing conditions in a similar manner to that seen in vivo in the MCDS growth plate. Importantly we have also shown that HeLa cells can be transduced with the collagen X cDNA constructs and will express, fold and secrete collagen X into the supernatant.Having established the cellular model for MCDS studies we demonstrated for the first time direct evidence for the retention of mutant collagen X within the ER. Moreover, we demonstrated that the mutant collagen X was degraded via a proteasomal pathway. Nevertheless, the level of ER stress induced by expression of mutant collagen X, based on BiP induction at the protein level, was disappointingly low. We therefore directly compared the level of ER stress induced by the COL10A1p.N617K mutation with that of the chondrodysplasias-causing MATN3p.V194D mutation. The ER stress induced by the matrillin mutation was far greater than that caused by the mutant collagen X. We showed that general protein synthesis was reduced in cells expressing either of the mutant proteins, most likely by the mechanism associated with the phosphorylation of eIF2alpha. Moreover, we showed the mutant matrilin-3 protein was also retained specifically in the ER. However, we could find no evidence for either proteasomal or autophagic/lysosomal degradation of mutant matrilin 3.We tested a broad range of ER stress-relieving compounds on cells expressing mutant collagen X and matrilin 3. Carbamazepine, which was previously shown to reduce ER stress in alpha1-antitripsin deficiency, reduced ER stress in cells expressing the mutant collagen X (but not matrilin 3) by way of enhanced proteasomal degradation of the retained protein. This drug should now be tested in vivo against the MCDS mouse to determine its capacity to reduce disease severity.The results presented within this thesis have contributed to the understanding of how cells deal with mutant collagen X and matrilin-3 proteins. We have identified a potential therapeutic compound that may be of use in the treatment of MCDS. Furthermore, the data presented support the concept that generic approaches to relieving ER stress may not be suitable for treating a broad range of diseases. Treatments may need to be tailored not only in a gene-specific manner but also may need to be tailored to address the differing consequences of different mutations in the same gene.

Proteotoxic stress drives the progression of numerous degenerative diseases including Type I diabetes, Huntingtin’s Disease, Parkinson’s Disease, and retinitis pigmentosa, a degenerative blinding disease. Cells respond to misfolded proteins by activating the unfolded protein response (UPR), including endoplasmic-reticulum-associated-protein-degradation (ERAD) and proteasomal degradation, but persistent stress triggers apoptosis. Enhancing ERAD is a promising therapeutic approach for protein misfolding diseases. From plants to humans, loss of the zinc transporter ZIP7 causes ER stress; however, the mechanism is unknown. We used Drosophila melanogaster to genetically manipulate ZIP7 and investigate its function in vivo. Here, we show that ZIP7-mediated Zn2+ transport enhances ERAD and that cytosolic Zn2+ is limiting for deubiquitination of client proteins by the Rpn11 Zn2+ metalloproteinase as they enter the proteasome. We then decided to test ZIP7’s effects in a fly model of retinitis pigmentosa. Interestingly, we found ZIP7 overexpression rescues defective vision and photoreceptor degeneration caused by misfolded rhodopsin in Drosophila. We have begun a collaboration looking into whether ZIP7 can prevent or suppress mutant Rhodopsin-mediated neurodegeneration in human retinal organoids. Additionally, these results prompted a screen for potential ZIP7 rescue of other aggregate-prone proteins associated with neurodegenerative diseases. So far, we found that ZIP7 rescues photoreceptor degeneration caused by Aβ42 and Vap33. Further study of ZIP7, a novel component of ERAD, holds significant promise for therapeutic advancements in protein-folding disorders.

Type 1 diabetes mellitus (T1D) is an autoimmune disease characterized by the loss of the only insulin secreting cells in the human body, the pancreatic beta-cells. Exogenous insulin is used to control the resulting hyperglycemia and replace the lack of insulin, but it is not a cure. To cure T1D, the autoimmune destruction of beta-cells must cease, and insulin secretory capacity must be restored. We have identified a novel compound that both protects against cytokine-induced cell death and stimulates insulin secretion from mouse islets. Importantly, I demonstrate that this compound exhibits protection from overnight cytokine treatment and enhances insulin secretion from human islets. Surprisingly, acute exposure to this compound severely reduced intracellular calcium. These findings conflict with observed effects on insulin secretion. However, the onset of enhanced insulin secretion appears between 2 and 4 hours with a maximal effect around 12 hours. In addition, the insulin secretory effects are shown to remain and enhance the response to glucose stimulation even after washout as treated islets showed increased insulin secretion in high glucose compared to control and glipizide-treated islets. We also found that our compound was one out of two compounds tested that augmented overnight insulin secretion. Testing of the amplifying pathway of insulin secretion revealed that this compound does enhance insulin secretion through this pathway. Finally, our lead experimental compound shows varying protection against ER stressors thapsigargin, brefeldin A, and tunicamycin. Overall, these findings suggest the small-molecule compounds we are developing and testing may hold therapeutic potential for treating type 1 diabetes through their dual action.

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.

Overall, this review aims to outline IPF regarding macrophage and fibroblast interactions, in addition to the therapeutic roles that macrophages may play in becoming an inhibitory target for IPF. Idiopathic pulmonary fibrosis (IPF) is a chronic inflammatory disease in which the tissue surrounding the alveoli within the lungs undergoes fibrosis or permanent scarring. IPF and the consequent disruption to lung blood flow, in addition to the change in immune landscape, will cause severe impact to lung function to the point of fatalities. The estimated number of patients in the United States affected by IPF is between 35,000 and 55,000 cases, with cases expected to rise as older generations continue to grow proportional to the total size of the population.2,53,141,142 Due to a lack of effective therapies, this disease exhibits a median survival time of 3 to 5 years from the time of diagnosis53,140, much worse than many cancer types. In recent years, there has been a renewed appreciation for the role of immune cells in mediating the fibrotic process with an increased interest in the role of macrophages, a type of innate immune cell which responds to environmental stimuli by secreting a wide range of biochemical signaling compounds. Macrophages help to break down the ECM using matrix metalloproteinases (MMPs). Macrophages also produce tissue inhibitors of metalloproteinases (TIMPs) to inhibit the process of ECM breakdown. Macrophages have been shown to be increased in IPF lung tissue and have a significant and poorly appreciated role in mediating fibrosis. In general, fibrotic diseases are characterized by excessive M2-associated and Th2-driven responses. Therefore, macrophages are an attractive target for inhibiting progressive pulmonary fibrosis. Recent literature has further shown that macrophages and fibroblasts directly interact with each other to influence the tissue microenvironment to affect disease outcomes26,28. In other words, both cell types interact to maintain tissue homeostasis. Together, these cells may interact during IPF and the overall fibrotic lung process to affect pulmonary homeostasis and IPF disease progression.

Nonalcoholic Fatty Liver Disease (NAFLD) is a progressive liver disease that can lead to liver cirrhosis and hepatocellular carcinoma, eventually resulting in death. The disease is prevalent worldwide, affecting around a quarter of the population, and is associated with risk factors such as obesity and type 2 diabetes. Currently, no standard treatment exists for NAFLD, which is characterized by excessive hepatic lipid accumulation leading to ER and mitochondrial stress, impaired insulin signaling, and inflammatory response. These events form a vicious cycle and exacerbate the progression of NAFLD. Sirtuin 7 (SIRT7), an NAD+-dependent deacetylase, is a nutrient sensor that alleviates ER stress and mitochondrial protein folding stress and prevents fatty liver. The insulin/IGF-1 signaling is the first nutrient-sensing pathway reported to regulate longevity in model organisms. Inhibiting the Insulin/IGF-1 signaling extends the lifespan of mice and worms. Paradoxically, low circulating IGF-1 is linked to hepatic steatosis and severe liver fibrosis in NAFLD. It remains unclear whether the somatotroph axis, which controls the insulin/IGF-1 signaling pathway, plays a role in liver damage during the progression of NAFLD.This dissertation aimed to explore the underlying mechanisms of NAFLD and develop a novel therapeutic strategy to combat this progressive liver disease. Chapter 1 provided a comprehensive review of the current state of nutrient-sensing pathways and oxidative stress response. We also discussed the therapeutic opportunities to prevent aging- or disease-driven tissue dysfunction by targeting the nutrient-sensing pathways. In Chapter 2, we investigated the role of the somatotroph axis in NAFLD and identified a novel regulatory pathway involving hepatic ER stress and ATF3, which suppresses the somatotroph axis in hepatocytes and leads to decreased cell proliferation and ER stress- induced cell death. Our findings in genetic and diet-induced NAFLD mouse models suggest that the suppressed somatotroph axis prevents apoptosis and inflammation but decreases hepatocyte proliferation and exacerbates fibrosis in the livers. Finally, we demonstrated that pharmacological activation of SIRT7 via NAD+ boosting reduces hepatic ER stress, rescues the suppressed somatotroph axis, and ameliorates NAFLD pathogenesis, offering a promising new therapeutic approach for treating this disease.

Obesity-induced inflammation has been proposed to be a factor in onset and progression of type 2 diabetes, marked by dysfunction of pancreatic beta-cells. Pro-inflammatory cytokines, specifically the obesity-induced IL-6 and IL-1 beta, have been indicated. Zinc, calcium, and iron are essential metals for proper function of beta-cells, yet it is not well understood how obesity-induced inflammation influences metals in beta-cells. This study was undertaken to investigate the metallome of pancreatic beta-cells and compare those results to the metallome of cells exposed to obesity-induced pro-inflammatory cytokines. To study the metallome of beta-cells, synchrotron X-ray florescent (SXRF) microscopy was performed. SXRF is a technique that allows quantification and visualization of multiple metals in a single cell simultaneously. The collected data showed significant zinc depletion, a redistribution and reduction of calcium, and an accumulation of iron in unique iron structures after exposure to cytokines. Investigation of gene expression in beta-cells after exposure to cytokines revealed upregulation of zinc transporters consistent with increase in intracellular zinc and granular zinc, in addition to a significant upregulation of iron-regulating genes, which are consistent with iron accumulation. An investigation of gene expression of ER stress and unfolded protein response (UPR) markers suggests that after exposure to cytokines, beta-cells induce UPR. In summary, this work, for the first time, reported details of the beta-cell metallome and showed cytokine-induced changes are potentially adaptive.

The failure to balance protein synthesis, folding, and degradation in the endoplasmic reticulum (ER) leads to the accumulation of unfolded proteins, leading to ER stress. Cells respond to this stress by activating a response signaling pathway known as the Unfolded Protein Response (UPR). One of the branches of the UPR induces the phosphorylation of eIF2α (Eukaryotic Initiation Factor 2) to attenuate global protein synthesis, allowing for a chance to clear misfolded and unfolded proteins. This phosphorylation of eIF2α is opposed by a phosphatase, containing a catalytic subunit, Protein Phosphatase 1, and a scaffolding protein, either GADD34 or CReP. Inhibition of eIF2α phosphatases has shown to promote survival in cell types by prolonging the effects of the UPR. This research focuses on understanding the gene expression patterns and localization of UPR specific genes with the presence of constant ER stress. Zebrafish are an ideal model for this research because they are a good mimic of what happens in humans and provide the ability to study gene expression and localization patterns at different stages during ER stress and its recovery. The eIF2α phosphatases were shown to have a protective effect on apoptosis when overexpressed in acute ER stress but were shown to have a protective effect on apoptosis when knocked out in chronic ER stress. We sought to determine the flow of gene expression of these phosphatases as well as other UPR specific genes, such as BiP and CHOP, to determine the contradictory effects of acute versus chronic ER-stress induced apoptosis. We studied the changes in gene expression for these genes in zebrafish embryos by isolating RNA and performing RT-qPCR after the induction of ER stress with pharmacological drugs across multiple time points. There was increased gene upregulation and mRNA localization to the fin epidermis and eye of GADD34, CReP, and BiP in acute ER stress from 2 hours to 6 hours, and these genes steadily declined in chronic ER stress from 24 hours to 48 hours. CHOP is a late-phase pro-apoptotic protein whose gene expression was upregulated in chronic ER stress from 12 hours to 48 hours. This data was also supported by mRNA localization studies performed by conducting whole mount in-situ hybridization on zebrafish embryos treated with ER stress inducers for 4 hours and 24 hours. Our results indicate that all UPR genes examined are affected by ER stress and their expression patterns are dependent on the time length of ER stress induction, allowing us to get a more in-depth working model of this branch of the UPR signaling pathway in zebrafish.

Abstract: Cellular responses to stress are an integral part of cardiovascular physiology and pathology, and endoplasmic reticulum (ER) stress is the key component in the development and progression of various heart diseases. However, the relative contribution of ER stress pathways to muscle damage and molecular mechanisms governing muscle ER stress regulation are still unclear. The objectives of this thesis were to investigate a role and regulation of IRE1α, an ER membrane associated stress sensor, in skeletal and cardiac muscle and to determine structural and function features of catecholaminergic polymorphic ventricular tachycardia (CPVT) related Casq2 mutants and their role in heart pathology. We identified two distinct pools of IRE1α in skeletal muscle fibers and in cardiomyocytes. One pool localized at the perinuclear ER membrane system and other at the junctional sarcoplasmic reticulum (SR). We also discovered that, at the junctional SR, calsequestrin interacts directly with the ER luminal domain of IRE1α preventing its dimerization, an initial step in activation IRE1α signaling. We generated a mouse model with cardiomyocyte specific, inducible deletion of the IRE1α gene. Heart with silenced IRE1α developed dilated cardiomyopathy and impaired cardiomyocyte Ca2+ transient indicating important role of IRE1α in the heart physiology and potential functional impact on muscle excitation-contraction coupling. Mutations in the gene encoding for cardiac calsequestrin, CASQ2, cause a stress-induced arrhythmia, CPVT. We carried out functional and structural analysis of six CPVT related CASQ2 mutations (R33Q, L167H, D307H, D351G, G332R, and P329S). The six mutations are distributed in diverse locations of the calsequestrin and impact on structure and function of the protein including folding, aggregation, and impaired or reduced Ca2+ binding. Remarkably these mutations are manifested in a similar phenotype in humans. Overall, in this thesis, we show that IRE1α is a new component of the junctional SR where it interacts with calsequestrin. This novel protein-protein interaction provides new insight into muscle specific regulatory mechanisms associated with IRE1α mediated UPR. We also provide the first direct evidence that IRE1α is required to maintain health of the heart. Finally, we provide the first evolutionary insights into the calsequestrin gene and showed that different Casq2 mutations may have distinct underlying molecular mechanisms leading to CPVT.

Abstract: Eukaryotic cells are characterized by the presence of membrane-bound organelles. Close proximity between organelles has been observed for decades, but only recently we have really started to understand the importance of these contacts for cellular function. These contacts consist of areas of close apposition between the membranes of two organelles, and are the platform for the exchange of metabolites and signals which are essential for the normal function of the organelles. One of the most prominent inter-organelle contact is the one established between the endoplasmic reticulum (ER) and mitochondria. ER-mitochondria contacts are formed in a specialized region in the ER membranes known as Mitochondria-Associated Membranes (MAM), which mediates the exchange of lipids and Ca(2+) between the two organelles. Mitochondrial Ca(2+) activates several metabolic enzymes in the mitochondrial matrix, therefore the flux of Ca(2+) from ER to mitochondria is essential to maintain a normal mitochondrial metabolism and oxidative phosphorylation. Reducing Ca(2+) flux to mitochondria by increasing ER-mitochondria distance or blocking Ca(2+) channels at the MAM compromises cellular metabolism due to reduced mitochondrial energy production. However, if mitochondria are overloaded with Ca(2+), cell death pathways are activated. To regulate this exchange, the MAM is populated by proteins that control its structure and function. Some of them are tethers that form physical proteinaceous bridges between ER and mitochondria membranes; others participate in the transfer of lipids or Ca(2+), like Ca(2+) channels. One notable group of MAM regulatory proteins are ER chaperones and oxidoreductases, the proteins responsible for one of the main functions of the ER: the folding and maturation of newly synthesized proteins. Chaperones like BiP and Calnexin bind to recently synthesized peptides imported to the ER to assist on the folding and assembly of mature proteins, and prevent the aggregation of misfolded proteins. Oxidoreductases like ERp57 and TMX1 assist chaperones by catalyzing the formation, reduction and isomerization of disulfide bonds by oxidizing and reducing cysteines in the target protein. These chaperones and oxidoreductases regulate MAM function by binding and/or chemically modifying tethers and Ca(2+) channels, regulating the extent and distance of ER-mitochondria contacts, and the intensity of the release of Ca(2+) from the ER. In this work, we describe the role of the chaperone Calnexin and the oxidoreductase TMX1 in the regulation of MAM function. We found that Calnexin and TMX1 regulate the ER Ca(2+) pump SERCA, responsible for the loading of Ca(2+) in the ER, therefore controlling the amount of Ca(2+) available for Ca(2+) flux at the MAM. In addition, they regulate MAM formation, controlling the extent and distance of ER-mitochondria contacts. The effect of Calnexin and TMX1 in ER Ca(2+) loading and ER-mitochondria distance results in the regulation of Ca(2+) flux to mitochondria, mitochondrial Ca(2+) content, and mitochondrial metabolism. We also explored the role of the ER-mitochondria tether Mitofusin-2 in the regulation of MAM function. Mitofusin-2 connects ER and mitochondria together, but the loss of Mitofusin-2 activates an adaptive rescue mechanism that results in increased ER-mitochondria contacts and enhanced mitochondrial metabolism. These adaptations are characterized by the activation of ER stress signalling pathways and rearrangement of MAM protein composition. Taken together, this doctoral thesis provides new knowledge about the role of Calnexin, TMX1 and Mitofusin-2 in the regulation of MAM tethers and Ca(2+) channels, their impact on ER-mitochondria contact formation and Ca(2+) flux to mitochondria, and ultimately their role in the regulation of mitochondrial function. These results provide new links between ER protein folding and energy production in the cell.

Type 2 diabetes mellitus (T2DM) is a chronic disease characterized by impaired glucose-stimulated insulin secretion in the setting of increased insulin resistance, a condition in which the body fails to respond to insulin properly. The endoplasmic reticulum (ER) serves as an essential quality control organelle and a Ca2+ reservoir. ER stress has been proposed to cause T2DM, but the specific effects of ER stress on beta-cell function are incompletely understood. To determine the interrelationship between ER stress and beta-cell function, the work in this dissertation primarily used the well-known ER stress inducer tunicamycin (TM) to trigger ER stress in insulin-secreting INS-1(832/13) cells or isolated mouse pancreatic islets. Beta-cell function, as exemplified by the tight regulation of ER or cytosolic [Ca2+], or membrane potential oscillations and insulin secretion, and beta-cell apoptosis were quantified at various time points, in parallel with well-established ER stress response markers. We observed that the induction of ER stress resulted in decreased ER [Ca2+], increased cytosolic free Ca2+ oscillations, membrane potential oscillations and increased insulin secretion, even under sub-threshold glucose conditions (e.g. 5 mM glucose) that are typically associated with only minimal insulin release. As ER Ca2+ depletion is generally known to activate store-operated Ca2+ entry (SOCE) in many cell types, we then used YM58483, a selective SOCE blocker. We found that the cytosolic Ca2+ and membrane potential oscillations and increased insulin secretion we observed resulted from increased SOCE, as these were all acutely blocked by the application of YM58483. Further dissection of TM-triggered ER [Ca2+] loss was carried out by studying the effect of TM on the ER Ca2+ channels, inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), and how their activity was tied to subsequent alterations in beta-cell Ca2+ homeostasis. Mouse islets were treated with dantrolene (Dan, a RyR1 isoform inhibitor) or xestospongin C (XeC, an IP3Rs inhibitor) along with TM overnight. We found that TM-induced ER Ca2+ depletion, cytosolic Ca2+ and mitochondrial Ca2+ oscillations were inhibited by co-treatment with Dan, whereas XeC was without effect on the oscillations. While RyR1 transcript was increased after TM exposure, transcripts corresponding to IP3R1 and IP3R2 were decreased by TM. Taken together, TM appeared to deplete ER [Ca2+] by increasing RyR1 level and triggering the subsequent activation of SOCE, resulting in increased cytosolic Ca2+ oscillations occurring despite the presence of sub-threshold glucose concentration. Finally, we characterized the role of an inward rectifier K+ channel, Kir2.1, in the beta-cell. Our lab previously published data supporting the hypothesis that Kir2.1 channels compensated for the loss of functional KATP channels in islets from KATP (SUR1) deficient mice in the production of cytosolic Ca2+ oscillations. We tested here whether Kir2.1 channels might also be involved in ER stress-induced beta-cell dysfunction, by using the selective Kir2.1 channels inhibitor ML133. We found that overnight exposure of beta cells to ML133 suppressed TM-triggered cytosolic Ca2+ oscillations in 5 mM glucose solution, likely due to ML133’s inhibitory effect on the activation of the ER stress response marker spliced XBP1. In summary, this dissertation maps the complex process of ER stress altering beta-cell function. In the process, we identified several novel mechanisms, including SOCE, RyR1 and Kir2.1 channels, that may augment insulin secretion in T2DM patients, and their clinical potentials as drug targets.

The intracellular bacterial pathogen Legionella pneumophila (L.p.) secretes over 300 bacterial proteins (effectors) to establish its replicative niche within host cells. Through its infectious life cycle, L.p. deploys its effectors to disrupt numerous host cell processes, including endoplasmic reticulum (ER) homeostasis. My dissertation explored the interaction between L.p. and the major homeostatic response pathway in the ER, the unfolded protein response (UPR). The results from my dissertation provide evidence that L.p. infection induces cleavage of the UPR sensor, activating transcription factor-6 (ATF6). Furthermore, I show that L.p.-mediated ATF6 cleavage is independent of proteasomal processing and does not require ER-associated degradation pathways. L.p. infection caused downstream activation of ATF6 target genes which include both lipid metabolism and proteostasis related genes. Interestingly, ATF6 activation during L.p. infection bypassed the requirement of conventional ATF6 pathway components. For example, chemical inhibition of ER to Golgi translocation of ATF6 with Ceapin A7 did not block its activation during L.p. infection. Furthermore, conventional site 1 and 2 protease (S1P and S2P) activities were dispensable during L.p. infection, as were the S1P and S2P cleavage sites on the ATF6 sensor. Additionally, time-lapse and confocal microscopy revealed that L.p.-mediated ATF6 activation bypasses ER to Golgi translocation and showed a direct recruitment of ATF6 to the L.p.-containing vacuole. Next, I compared ATF6 cleavage in the L.p. Philadelphia strain to the L.p. Paris strain, and showed that the Paris strain failed to activate the ATF6 pathway. Bioinformatic comparison between the Philadelphia and Paris strains revealed 17 effectors that were unique to the Philadelphia strain. When individually expressed, L.p. effectors Lpg2131, Lpg0519, Lpg2523, and Lpg2465 could induce expression of an ATF6-specific luciferase reporter. Subsequent analysis showed that Lpg0519 could induce ATF6 processing without affecting other UPR sensors, such as PERK. Thus, my findings highlight the unique regulatory control that L.p. exerts upon the UPR sensors and discovers a novel strategy by which an intracellular bacterium can selectively perturb host homeostatic pathways.

Numerous stresses on the heart, both physiological and pathological, present challenges to ER proteostasis in the heart. Simultaneously, various cardiac cell lineages respond to injury in ways requiring increased protein flux through the ER. While cardiac myocytes react to these stresses largely through hypertrophy or cell death mechanisms, other non-myocyte cells in the heart respond in more dynamic ways, including proliferation and differentiation. Additionally, virtually all cardiac cells secrete signaling cytokines or hormones both at baseline and during injury. Thus, precisely when ER proteostasis is most critical, cells are challenged with conditions which impair ER protein folding. ER unfolded protein response signaling attempts to respond to these challenges through the three main ER stress sensors, PERK, IRE1, and ATF6α. All sense protein misfolding and induce downstream signaling which, to varying degrees, includes global translational attenuation with concurrent upregulation of chaperones, protein disulfide isomerases, and ER-associated degradation machinery. Of these pathways, ATF6α is the most extensively characterized as adaptive in the heart and other tissues. Additionally, recent studies have identified multiple noncanonical ATF6α gene programs which are only indirectly related to ER proteostasis but are nevertheless critical to the ability of ATF6α to preserve tissue function. Research on the effect of ATF6α in the heart had previously been restricted to ventricular myocytes. Though these cells are the most direct effectors of cardiac function, this ignores critical roles played by other cardiac non-myocyte cells. This work examines the effects of ATF6α gain- and loss-of-function in three model systems designed to uncover previously unknown roles for ATF6α signaling in cardiac non-myocytes. ATF6α global knockout mice were found to progress to heart failure more quickly than wild-type counterparts following permanent-occlusion myocardial infarction, an unexplored disease model in the context of ATF6α signaling. We subsequently explored the role of ATF6α in two murine cardiac non-myocyte cell types. The first, c-Kit+ cardiac stem cells, required ATF6α both for survival and stemness while ATF6α loss-of-function induced multiple lineage markers. Second, we identified roles for ATF6α in limiting fibrosis in a pressure overload injury model and found that ATF6α blunted activation of isolated adult murine ventricular fibroblasts.

The endoplasmic reticulum (ER) is the site of folding and maturation for virtually all secreted and transmembrane proteins. When the ER folding capacity is overwhelmed, conserved signaling pathways in eukaryotes, collectively termed the unfolded protein response (UPR), sense this ER stress to reestablish proteostasis or initiate apoptosis in conditions of unresolved stress. The best studied of the three known UPR sensors, IRE1α, consists of an ER lumenal sensing domain, a single transmembrane helix followed by cytosolic kinase and ribonuclease (RNAse) effector domains. During ER stress, IRE1α activates by oligomerization, trans-autophosphorylation and subsequent allosteric RNase activation. Active IRE1α RNase initiates noncanonical splicing of its XBP1 mRNA substrate to allow the translation of active XBP1s transcription factors that in turn upregulate hundreds of genes to increase ER folding capacity and reduce folding load. In solution, IRE1α forms reversible oligomers that correlate with higher enzymatic activity. In ER-stressed cells, activated IRE1α dynamically clusters as discrete foci visible by fluorescence microscopy, which dissolves when stress is mitigated. This thesis presents three complementary approaches aimed to better understand the direct relationship between IRE1α oligomeric states and activity: 1. Characterizing the dynamic behavior of IRE1α molecules in mammalian cells during different UPR phases and corresponding IRE1α activation stages, 2. Solving the in situ structure of large IRE1α oligomers observed in stressed cells, and 3. Dissecting elements controlling IRE1α oligomerization. We discovered that: 1. Cellular IRE1α foci are entities with complex morphologies and dynamic behaviors consisting of two distinct populations of IRE1α: a diffusionally constrained core and a freely exchanging periphery. 2. In cells, IRE1α oligomers form ordered double helices contained in a newly described ER structure comprising extremely narrow (~28 nm) anastomosing membrane tubes. The arrangement of IRE1α molecules in this ER subdomain creates a positive feedback loop that sustains IRE1α activation and provides the structural basis for the two dynamically distinct populations observed. 3. A flexible linker region connecting IRE1α’s transmembrane helix to the effector kinase/RNase domains plays a major role in controlling IRE1α kinase/RNase domains activation and oligomerization in cells. Mutants carrying deletions or mutations within this region fail to form foci during stress and cannot mount an effective UPR. Taken together, this work bridges previous observations of IRE1α behaviors observed in vitro and IRE1α’s dynamic properties in cells to provide a more complete picture of why IRE1α oligomerization and activation are intricately linked.

Nonsense-mediated RNA decay (NMD) is a cell surveillance system that degrades aberrantly processed RNA transcripts often contain premature termination codons (PTCs). PTCs can arise from various genetic mutations. In addition to serving as a passive fail-safe mechanism, recent studies suggest NMD is also actively involved in alternative splicing (AS), stress responses, neurogenesis, and other biological processes. This dissertation is aimed to further investigate the relationship between NMD and other stress mechanisms.To overcome some inherent problems of assaying endogenous NMD targets, Chapter 2 presents an easily and broadly applicable AS-NMD reporter assay to quantify cellular NMD activity, in real time. This new strategy reliably distinguishes NMD regulation from transcriptional control, AS regulation, and discloses a different sensitivity of NMD targets to NMD inhibition. It is then used to screen molecules for NMD inhibitors including thapsigargin in neuro-2A cells. Chapter 3 shows stresses affect NMD differently and the mechanistic discovery of NMD inhibition upon thapsigargin treatment. The endoplasmic reticulum (ER) stress and polysome disassembly caused by thapsigargin suggest that an activated unfolded protein response (UPR) pathway by protein kinase RNA-like endoplasmic reticulum kinase (PERK) is required for NMD inhibition, instead of calcium signaling pathways. Such stress induced NMD inhibition can compound TDP-43 depletion proteinopathy via upregulating NMD isoforms that had been implicated in the pathogenic mechanisms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) which could be completely blocked by PERK deactivation. In Chapter 4, a pioneer in vitro conditional NMD deficient neural progenitor cell (NPC) system is created using adeno-associated virus serotype 9 (AAV9). Significant loss in NPC viability is observed during UPF2 knockout. Candidate genes, Gadd45b and Gadd45g, knockout using CRISPR-Cas9 failed to rescue NPC survival. This finding suggests NMD activity is essential for cell viability, likely maintaining the homeostasis of NPC transcriptome during renewal processes. In summary, AS-NMD strategy screens and reveals the pathway of NMD inhibition by ER stress and the implicated ALS proteinopathy. More importantly, the research highlights NMD interaction with stress and cell survival, especially in NPCs, that could provide future therapeutic approaches toward related genetic diseases.

Entamoeba histolytica is an intestinal parasite infecting over 50 million people worldwide and is the causative agent of amebic dysentery and amoebic liver abscess. In the human host and nonhuman primates, E. histolytica experiences stress brought on by nutrient deprivation and the host immune response. To be a successful parasite, E. histolytica must counter the stress; therefore, understanding the stress response may uncover new drug targets. In many systems, the stress response includes down-regulation of general protein translation, which is regulated by phosphorylation of eukaryotic initiation factor (eIF-2α). Previous work in E. histolytica has demonstrated that EheIF-2α phosphorylation increases significantly when exposed to long-term serum starvation, oxidative stress, and long-term heat shock. However, the effects of nitrosative and endoplasmic reticulum (ER) stresses, on the eIF2α protein translation control system have yet to be evaluated. Nitrosative stress is part of the host’s immune response and ER stress can be caused by several physiological or pathological factors. We treated E. histolytica cells with different reagents to induce nitrosative stress (DPTA-NONOate and SNP) or ER stress (BFA and DTT). We examined the morphology of the ER, tracked phosphorylation of eIF2α, and assessed protein translation in the control and stressed cells. While all four stress-inducing reagents caused a global reduction in protein translation, only DTT was capable of also inducing changes in the morphology of the ER (consistent with ER stress) and phosphorylation of EheIF-2α. This suggests that DTT authentically induces ER stress in E. histolytica and that this stress is managed by the eIF2α-based system. This was supported by the observation that cells expressing a non-phosphorylatable version of eIF2α were also highly sensitive to DTT-stress. Since protein translation decreased in the absence of phosphorylation of eIF2α (after treatment with DPTA-NONOate, SNP or BFA), the data also indicate that there are alternative protein-translational control pathways in E. histolytica. Overall, our study further illuminates the nitrosative and ER stress responses in E. histolytica.

Ehlers-Danlos Syndrome (EDS) comprises a spectrum of heritable connective tissue disorders with varying genetic origins and clinical manifestations such as soft tissue fragility and skin hyperextensibility. There are multiple EDS subtypes, the first few of which were defined by collagen mutations. Many new EDS variants have been discovered involving mutations that do not necessarily implicate collagen biosynthesis but do involve extracellular matrix (ECM) proteins. One of these proteins, Aortic Carboxypeptidase-Like Protein (ACLP), is a large secreted protein encoded by the AEBP1 (adipocyte enhancer binding protein 1) gene. Previous research has shown that ACLP plays a vital role in binding collagen via its discoidin domain and therefore regulates connective tissue assembly. Thus far, individuals from 7 different families have been identified with different EDS-causing ACLP mutations. Some mutations are ACLP null whereas other mutations lead to expressed mutant ACLP. One of these mutations is characterized by a single-nucleotide deletion that causes the insertion of 40 amino acids in the discoidin domain of ACLP. It is therefore denoted “ACLP-Ins40”. The goal of this research was to characterize the ACLP-Ins40 protein and investigate how mutations in ACLP disrupt ECM homeostasis and cause EDS. We initially sought to determine if the ACLP-Ins40 mutation would alter ACLP’s ability to bind collagen. To achieve this goal we generated expression vectors of full length human ACLP carrying the Ins40 mutation. By western blot, it was determined that ACLP-Ins40 was not secreted from fibroblasts and was retained intracellularly. We then hypothesized that the retention of ACLP-Ins40 in the secretory pathway would induce ER stress due to misfolding. 3T3 fibroblasts were co-transfected with the ACLP-Ins40 expression vector and an XBP1u-EGFP sensor of ER stress. Immunofluorescence imaging revealed that in comparison to WT, fibroblasts expressing ACLP-Ins40 experienced ER stress with significantly increased spliced XBP1. This may then cause cell death, the improper secretion of other important ECM proteins, or defective collagen scaffolding, all which could contribute to symptoms of EDS. These studies contribute to our current understanding of how mutations in the AEBP1 gene and alterations in the ACLP protein cause EDS. This connection provides a framework for future research and for targeted interventions to treat EDS.

Heavy ethanol use causes neurodegeneration manifested by neuronal loss and dysfunction. It is becoming imperative to delineate the underlying mechanism to promote the treatment of ethanol-induced neurodegeneration. Endoplasmic reticulum (ER) stress is a hallmark and an underlying mechanism of many neurodegenerative diseases. This study aims to investigate the role of ER stress in ethanol-induced neurodegeneration. In experimental design, adult mice were exposed to binge ethanol drinking by daily gavage for 1, 5, or 10 days and the response of ER stress was examined. We found the induction of ER stress appeared at 5 days and remained at 10 days. Moreover, the induction of ER stress was accompanied by an increase in neurodegeneration. With cell culture, we demonstrated that ethanol exposure resulted in neuronal apoptosis and that blocking ER stress by sodium phenylbutyrate (4-PBA) abolished ethanol-induced neuronal apoptosis, suggesting that ER stress contributes to ethanol-induced neurodegeneration. Mesencephalic astrocyte-derived neurotrophic factor (MANF) responds to ER stress and has been identified as a protein upregulated in ethanol-exposed developmental mouse brains. To investigate its implication in ethanol-induced neurodegeneration, we established a central nervous system (CNS)-specific Manf knockout mouse model and examined the effects of MANF deficiency on ethanol-induced neuronal apoptosis and ER stress using a third-trimester equivalent mouse model. We found MANF deficiency worsened ethanol-induced neuronal apoptosis and ER stress and that blocking ER stress abrogated the harmful effects of MANF deficiency on ethanol-induced neuronal apoptosis. Moreover, a whole transcriptome RNA sequencing supported the involvement of MANF in ER stress modulation and revealed candidates that may mediate the ER stress-buffering capacity of MANF. Collectively, these data suggest that MANF is neuroprotective against ethanol-induced neurodegeneration via ameliorating ER stress. Because MANF is a neurotrophic factor, we also examined the effects of MANF deficiency on neurogenesis. We observed that MANF deficiency increased neurogenesis in the subgranular zone of the hippocampal dentate gyrus and subventricular zone of the lateral ventricles in the mouse brain. Mechanistically, this finding was supported by a decrease of cell cycle inhibitors (p15 and p27), an increase of G2/M marker (phospho-histone H3), and an increase of neural progenitor markers (Sox2 and NeuroD1) in the brain of conditional Manf knockout mice. Our in vitro studies demonstrated that the gain-of-function of MANF inhibited cell cycle progression, whereas the loss-of-function of MANF promoted cell cycle progression. Taken together, these data suggest that MANF may affect the process of neurogenesis through altering cell cycle progression.

In metazoans, the Unfolded Protein Response (UPR) restores homeostasis in the endoplasmic reticulum (ER) by both increasing the protein-folding capacity of the ER and by reducing the protein folding burden. The UPR in S. cerevisiae exclusively relies on this first solution to survive ER stress. Remarkably, S. pombe utilizes the second, though the UPR in both species is mediated through site-specific RNA cleavage by the evolutionarily conserved kinase/RNase transmembrane protein Ire1. While most of the S. pombe Ire1 mRNA substrates are degraded post cleavage, the mRNA encoding the ER chaperone protein BiP1 evades decay and the 5’ fragment accumulates after truncation. Though the mechanism behind this stabilization remains unresolved, we have discovered that the relevance of bip1 mRNA processing to the cell’s survival may be greater than previously estimated. We have compared the Ire1 kinase/RNase domain from each species in vitro and discovered key structural differences we can modulate to make the S. cerevisiae Ire1 behave like S. pombe Ire1. First, we characterized differences in cofactor sensitivity in the kinase domains of the two enzymes. Additionally, we exploited the toxicity of S. pombe Ire1 in bacterial cells to identify mutations in the more stringent S. cerevisiae Ire1 protein that would cause toxicity and relax substrate stringency. We identified a pair of mutations at the RNase dimer interface that expanded the S. cerevisiae enzyme’s substrate repertoire and coincided with an increase in its propensity to dimerize. This interface could represent an under-utilized target for drug design to modulate the RNase activity of metazoan Ire1.

Disruption of protein folding homeostasis in the endoplasmic reticulum activates a signaling pathway termed the unfolded protein response, or UPR, which can mediate cell recovery or apoptosis. Understanding how the decision to live or die is made by the UPR is integral to affecting the outcome of ER stress-related pathologies. In cases of unwanted cell proliferation, inhibiting the cytoprotective branches of the UPR has shown promise in controlling cell populations. However, for cases of unwanted cell death, developing strategies to inhibit pro-apoptotic signaling of the UPR requires a deep, mechanistic understanding of the molecular events that commit the cell to an untimely fate. Chapter 1 gives an overview of both cytoprotective signaling and pro-apoptotic mechanisms of the UPR that pinpoint Death Receptor 5 (DR5) as a key driver of cell death. Chapter 2 provides published experimental evidence that reaffirms the role of DR5 in ER stress-induced apoptosis. Chapter 3 describes the experiments that demonstrate that apoptosis is driven by the direct interaction between DR5 and the source of ER stress, which is misfolded protein. In this way, DR5 assumes a terminal quality control pathway to detect unresolved protein folding stress along the secretory pathway and commit apoptosis to mitigate the consequences of secreting or displaying dysfunctional proteins.

Since its discovery more than 25 years ago, great progress has been made in our understanding of the unfolded protein response (UPR), a homeostatic mechanism that adjusts endoplasmic reticulum (ER) function to satisfy the physiological demands of the cell. However, if ER homeostasis is unattainable, the UPR switches to drive cell death to remove defective cells in an effort to protect the health of the organism. This functional dichotomy places the UPR at the crossroads of the adaptation versus apoptosis decision. Here, we focus on new developments in UPR signaling mechanisms, in the interconnectivity among the signaling pathways that make up the UPR in higher eukaryotes, and in the coordination between the UPR and other fundamental cellular processes.

Skeletal muscle mass, contractile properties, and metabolic function are regulated through the coordinated activation of multiple intracellular signaling pathways and genetic reprogramming. The endoplasmic reticulum (ER) plays a pivotal role in protein folding and calcium homeostasis in many cell types, including skeletal muscle. Disruption of calcium levels or accumulation of misfolded proteins in the ER lumen leads to stress, which results in the activation of a signaling network called the unfolded protein response (UPR). Further, recent studies have suggested that in certain conditions, UPR pathways can be activated independent of ER stress. However, the role of ER stress and the UPR in the regulation of skeletal muscle mass and function had not been previously investigated. This dissertation demonstrates that the markers of ER stress are increased in skeletal muscle of mouse models of cancer cachexia. Chronic administration of 4-phenylbutyrate (4-PBA), a molecular chaperon and an inhibitor of ER stress, leads to the loss of skeletal muscle mass and function in naïve conditions and in Lewis lung carcinoma (LLC) tumor-bearing mice. 4-PBA also causes atrophy in cultured primary myotubes. Further, our results demonstrate that the targeted deletion of X-box binding protein (XBP1), a downstream target of the inositol-requiring enzyme 1α (IRE1α) arm of the UPR, attenuates the loss of skeletal muscle mass in LLC tumor-bearing mice. Overexpression of a spliced form of XBP1 causes atrophy and induces the gene expression of several proinflammatory cytokines and the components of ubiquitin proteasome system and autophagy in cultured myotubes. Our results also demonstrate that toll-like receptors-mediated signaling is responsible, at least in part, for the activation of the UPR in skeletal muscle of LLC tumor-bearing mice. Finally, the role of the XBP1 in skeletal muscle growth and regeneration was also investigated. Results showed XBP1 mediates overload-induced myofiber hypertrophy and skeletal muscle regeneration potentially through augmenting the proliferation of satellite cells in a non-cell-autonomous manner. Altogether, this dissertation provides initial evidence that while basal levels of ER stress/UPR is essential for the maintenance of skeletal muscle mass and strength, supra-physiological activation of the UPR, especially the IRE1/XBP1 arm, causes skeletal muscle wasting.

Autophagy is a major degradation mechanism, responsible for clearing damaged and dysfunctional organelles, including the endoplasmic reticulum, a structure essential for protein synthesis and myocellular hypertrophy. Alterations in autophagy throughout various tissues of the body have been linked to various negative side effects such as decreased myocellular hypertrophy and insulin resistance. High fat diets lead to changes (both increases and decreases) in autophagy in various tissues throughout the body in a tissue-specific manner. Skeletal muscle autophagy is decreased in myotubes cultured from obese women, however the mechanism by which this occurs is unknown. As the largest organ system in the human body, skeletal muscle serves an important role in overall metabolic health. Therefore, sufficient skeletal muscle autophagy is important for proper metabolic function. Moreover, a decrease in liver and pancreas autophagy has been found to lead to endoplasmic reticulum (ER) stress and the development of insulin resistance. Understanding the relationship between autophagy and ER stress in the skeletal muscle following a high fat diet may help elucidate a novel target for decreasing negative side effects. Interestingly, both acute and chronic exercise have been shown to increase skeletal muscle autophagy. This points to a potential therapeutic treatment for those suffering with decreased skeletal muscle autophagy and may help improve ER stress. The purpose of this study was to compare the in vivo and in vitro effects of high fat exposure on skeletal muscle autophagy. Additionally, the relationship of autophagy and ER stress in skeletal muscle was explored. Lastly, this project identified changes in skeletal muscle autophagy and ER stress following cyclic stretch, an in vitro model of exercise in C2C12 myotubes. Eight-week-old C57BL/6J were fed a high fat diet for 16 weeks and tibialis anterior muscle examined for changes in autophagy markers. Gene expression (mRNA content) of autophagy markers Atg3 (p=0.011, fold change 1.37), Atg12 (p=0.026, 1.38), and Atg16L (p=0.004, 1.49) were increased in skeletal muscle of obese mice. Protein content was also measured, where increases in Atg3 (p = 0.04, 1.22), Atg12 (p = 0.027, 1.21), and Atg16L1(p = 0.021, 1.59) were found. However, there was no difference in LC3 II:I ration. No changes were seen in Atg5 or LC3. Additionally, C2C12 myotubes were treated with equimolar palmitate and oleate for 24h then assessed for mRNA content of genes involved in autophagy and ER stress. Autophagy genes Atg5 (p = 0.007, fold change 1.78), Atg12 (p = 0.001, fold change 1.99), and LC3 (p = 0.01, fold change 2.02) were decreased with high fat treatment. Paradoxically, there was an increase in Atg16L (p = 0.005, fold change 1.90). There were no changes in protein content. ER stress was increased indicated by an increase of sXBP1 (p = 0.005, fold change 1.33). Furthermore, inhibition of autophagy lead to changes in ER morphology and ER stress. To identify the impact of cyclic stretch on skeletal muscle autophagy and ER stress, C2C12 myotubes were subjected to 30 minutes of equibaxial stretch and examined for changes in autophagy and ER stress. Autophagy flux, measured by tyrosine release, increased by 34% (p = 0.04) following exercise and ER stress was decreased. In conclusion, this study provides the novel finding that decreased skeletal muscle autophagy is sufficient for inducing ER stress. Additionally, cyclic stretch increases autophagy and improves ER homeostasis.

ATF6α is an endoplasmic reticulum (ER) transmembrane protein that senses the accumulation of misfolded proteins in the ER of cells during ER stress. Upon ER stress, ATF6α is proteolytically cleaved. The resulting N-terminal fragment becomes an active transcription factor, i.e. N-ATF6α, which induces genes that adaptively reprogram ER protein folding. N-ATF6α exerts potent but transient transcriptional activation of its target genes; the transient nature of its activity is due in part to its rapid degradation upon engagement in transcription. The transcriptional activation domain (TAD) and degradation domain (degron) of N-ATF6α map to the same N-terminal region of N-ATF6α, implying that transcriptional activity and degradation are linked. Moreover, mutations introduced into the TAD of N-ATF6α that decrease its activity also decrease its degradation, indicating that N-ATF6α degradation is coupled with its transcriptional activity. To elucidate how and why N-ATF6α degradation and activation are coupled, we examined the mechanism of N-ATF6α degradation, positing that ubiquitylation may target it for proteasome-mediated degradation. Thus, this study focused primarily on delineating whether N-ATF6α and ATF6α are ubiquitylated, and if so, what domain(s) of N-ATF6α and ATF6α are ubiquitylated, as well as investigating the functional consequences of such ubiquitylation.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder that results in elevated levels of low-density lipoprotein cholesterol (LDL-C). This increase in serum cholesterol level has been shown to result in premature coronary artery disease (CAD) with devastating symptoms from a young age. Today, the prevalence of heterozygous FH (HeFH) and homozygous FH (HoFH) is estimated to be 1 in 320 and 1 in 160,000 people, respectively. FH is referred to as an underdiagnosed disease due to the large number of mutations that continues to grow. These mutations often exist in one of or a combination of three genes: low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB100), or proprotein convertase subtilin/kinase 9 (PCSK9). Mutations in the LDLR adapter protein 1 (LDLRAP1) were also identified as a less common cause of FH, the autosomal recessive variety, specifically. In treating the disease, patients are prescribed various treatment protocols aimed at reducing endogenous cholesterol synthesis, removal of excess cholesterol through extracorporeal machinery, and other medications aimed at upregulating the LDL receptors in the body. To this day, liver transplants remain as the only cure for FH. FH-induced pluripotent stem cells (FH-iPSC) derived from HoFH skin fibroblasts were permanently corrected using CRISPR technology to insert the three missing base pairs and restore transport of the LDL receptor from the endoplasmic reticulum (ER) to the Golgi body. Hepatocytes are the cells primarily responsible for LDL-C uptake from the plasma, so we differentiated non-corrected familial hypercholesterolemia (NC) and CRISPR-corrected (C) iPSC to hepatocyte-like cells (HLC) to analyze restoration of cholesterol homeostasis. iPSC and HLC were treated with Rosuvastatin, excess sterols, or tunicamycin and collected for mRNA analysis and protein analysis of LDLR and ER stress markers. HLC were also treated with Rosuvastatin and immunocytochemistry and the Thermofisher Amplex Red Cholesterol Assay kit were used to analyze localization of LDLR within the cell and internalization of cholesterol, respectively. Statin-treated NC-iPSC and HLC showed predominant expression of an immature LDLR protein that was not present in C-iPSC or HLC and this upregulation was not the result of regulation at the transcriptional level. The LDLR co-localized to ER resident-protein, Calnexin, in NC-HLC whereas the LDLR co-localized to the cell-membrane in the C-HLC. Upon correcting expression of the mature LDLR, cholesterol internalization increased overtime in C-HLC in contrast to a minimal amount internalized at the 24-hour mark in the NC-HLC. Lastly, statin-treated NC-iPSC

Atherosclerosis is the main cause of coronary heart disease, the leading cause of mortality in developed countries. Development of atherosclerotic lesions is in part, associated with increased apoptosis of endothelial cells and disturbance of endothelial integrity. By virtue of its important role and strategic anatomic location in the arterial tree, the endothelium plays a major role in all phases of lesion development. In endothelial cells, a number of atherorelevant systemic and local factors induce endoplasmic reticulum (ER) stress by causing disturbance in endothelial cell ER homeostasis which activates the unfolded protein response (UPR). Although this concerted and adaptive process is initially aimed at protecting the cell, sustained and unresolved ER stress can lead to endothelial cell apoptosis. In a number of cell types, persistent and/or exacerbated Ca2+ influx has also been shown to promote ER stress. In previous studies from our laboratory it was shown that in human coronary artery endothelial cells (HCAECs) constitutive Ca2+ influx plays a critical role in inflammatory signaling, and that the Transient Receptor Potential Canonical 3 (TRPC3) channel mediates such constitutive Ca2+ entry, which is exacerbated under atherorelevant conditions. It was also observed in these studies that TRPC3 constitutive function couples to Ca2+/calmodulin-dependent protein kinase II (CAMKII) to drive inflammatory signaling. In other cell types, TRPC3 has been found to contribute to UPR signaling and apoptosis, and CAMKII is shown to act as a mediator in the signaling pathway that results in ER stress-mediated apoptotic cell death. However, whether a similar role of TRPC3 exists in HCAECs remained unknown. In this work, we first examined whether inhibition of constitutive Ca2+ influx in general and TRPC3 in particular, plays a role in ER stress-induced apoptosis in HCAECs. Our results show that global blockade of constitutive Ca2+ influx or buffering of intracellular Ca2+ changes, markedly decreased the susceptibility of HCAECs to ER stress-induced apoptosis. Notably, we found that selective inhibition of TRPC3 resulted in decreased UPR activation and reduced susceptibility of HCAECs to ER stress-induced apoptosis. Our findings provide pharmacological evidence suggesting that TRPC3, through its constitutive function, supports mechanisms of ER stress-induced apoptosis in an atherorelevant cell type. In order to examine if and to what extent this action of TRPC3 is of relevance to atherosclerotic lesion development and endothelial cell apoptosis in vivo, we used a transgenic mouse model with endothelial specific gain of TRPC3 function. Compared with non-transgenic control mice, atherosclerotic lesions of transgenic mice had significantly increased plaque size and macrophage content, and notably increased endothelial cell apoptosis. These studies show for the first time, a role of TRPC3 in endothelial apoptosis in the context of atherosclerosis and suggest that this pro-apoptotic role contributes to the pro-atherogenic effect of endothelial TRPC3.

Alzheimer’s disease (AD) is characterized by extensive cell death and accumulation of misfolded proteins in the brain, which consist of amyloid beta (Aβ) plaques and hyperphosphorylated tau neurofibrillary tangles (NFTs). We sought to determine the effects of endoplasmic reticulum (ER) stress on classical AD characteristics in neurons and neural stem cells (NSCs). We used isogenic human induced pluripotent stem cell (hiPSC)-derived neurons and NSCs that were genetically modified with the presenilin 1 exon 9 deletion (PS1-ΔE9) mutation. We found that after two days, tunicamycin induced cell death in the PS1-ΔE9 neurons, but not wild type neurons; treatment for five days with thapsigargin, however, induced more cell death in wild type neurons than PS1-∆E9 neurons.While previous studies have suggested connections between ER stress, Aβ, and phosphorylated tau (P-tau), results have remained controversial. To measure the effects of ER stress on Aβ42/Aβ40 and P-tau/T-tau (total tau) ratios, we treated neurons with tunicamycin or thapsigargin for two or five days. We observed no change in Aβ42/Aβ40 ratio with tunicamycin or thapsigargin, but observed a dose- and time-dependent reduction in overall Aβ42 and Aβ40 levels. P-tau/T-tau ratio was decreased in PS1-∆E9, but not wild type neurons in response to both tunicamycin and thapsigargin at 2 days. However, after 5 days of treatment we observed reduced P-tau/T-tau ratio in response to tunicamycin only in wild type and PS1-∆E9 neurons. The results presented in this study show that the PS1-∆E9 mutation sensitizes and confers resistance to neurons in response to different ER stressors, and suggest that ER stress may rescue AD phenotypes.

The tumor microenvironment is harbor to a variety of insults that privilege tumor cells to co-opt host immunity. Many of these insults, including nutrient deprivation and hypoxia, create endoplasmic reticulum (ER) stress for resident tumor cells. Emerging reports also suggest that ER stress in infiltrating myeloid cells leads to the immune dysfunction, rendering these inept in the eliminating the tumor and instead facilitative of tumor growth. We previously reported that this polarization may be mediated by factors derived from tumor cells undergoing ER stress that target immune infiltrating cells to then similarly undergo ER stress, termed transmissible ER stress (TERS).This dissertation advances the findings of TERS through three central aims. In Chapter 2, I probe the role of TERS on cancer cells using a prostate cancer cell model. My findings reveal that TERS experienced cancer cells gain cytoprotection over tumor microenvironmental challenges as well as chemotherapies, which was centrally mediated by the PERK arm of the UPR. Chapter 3 reports that myeloid cells infiltrating the tumor microenvironment undergo a polarization consistent with those affected by TERS. Chemical inhibition of IRE1α during TERS led to dramatic reduction in TERS driven inflammation, immune-suppression, and angiogenesis. Finally, Chapter 4 elucidates the identity of the molecules responsible for TERS. I found TERS activity is assignable to lipid soluble molecules. Lipidomic analysis identified TERS contained enriched eicosanoids, which could not cause ER stress but drive immune suppressive features. The ER stress inducing factor behind TERS is a novel mono-unsaturated fatty acid that caused increases in ER stress, pro-inflammation, and angiogenesis in myeloid cells as well as drove the surface expression of CD86 and PD-L1. These lines provide the elucidation of the novel factors tumor cells undergoing ER stress secrete to promote immune evasion. These results identify potential novel biomarkers of the tumor microenvironment as well as emphasize that targeting the UPR, and specifically the IRE1α axis, may serve an important target to bolster anti-tumor immunity.

Mammalian UNC-51–like kinases 1 and 2 (ULK1 and ULK2), Caenorhabditis elegans UNC-51 and Drosophila melanogaster Atg1 are redundant serine/threonine kinases that regulate flux through the autophagy pathway in response to various types of cellular stress. C. elegans UNC-51 and D. melanogaster Atg1 also promote axonal growth and defasciculation, and disruption of these genes results in defects in axon guidance in invertebrates. Germline Ulk1/2-deficient mice die perinatally. Therefore, we used a conditional-knockout approach to investigate the roles of ULK1/2 in the brain. Mice lacking Ulk1 and Ulk2 in their central nervous systems (CNS) showed defects in axonal pathfinding and defasciculation affecting the corpus callosum (CC), anterior commissure (AC), corticothalamic axons (CTAs) and thalamocortical axons (TCAs) and mossy fibers. These defects led to impaired midline crossing of callosal axons, anterior commissure hypoplasia and disorganization of the somatosensory cortex. The axon guidance defects observed in Ulk1/2 double knockout (dko) and in CNS-specific (Nestin-Cre) Ulk1/2 conditional double knockout (cdko) mice were not recapitulated in mice lacking other autophagy genes (i.e. Atg7 or Fip200), and was associated with abnormal localization of the axon guidance molecule, transient axonal glycoprotein-1 (TAG-1) in the distal CTAs. Approximately 40% of the Ulk1/2 cdko animals died shortly after birth; the remaining animals survived up to 4 months. Although the mice showed neuronal degeneration, specifically in the hippocampal CA1 region, the neurons showed no accumulation of P62+/ubiquitin+ inclusions or abnormal membranous structures, which are observed in mice lacking other autophagy genes, such as Atg7, and Fip200. Rather, neuronal death was associated with activation of the unfolded protein response (UPR) pathway. An unbiased proteomics approach identified SEC16A as a novel ULK1/2-interacting partner. ULK-mediated phosphorylation of SEC16A regulated the assembly of endoplasmic reticulum (ER) exit sites and ER-to-Golgi trafficking of specific cargo such as, the serotonin transporter SERT, and did not require other autophagy proteins (e.g. ATG13). The defect in ER-to-Golgi trafficking activated the UPR pathway in ULK-deficient cells; both processes were reversed upon expression of SEC16A with a phosphomimetic substitution. Thus, the regulation of ER-to-Golgi trafficking by ULK1/2 is essential for cellular homeostasis. Moreover, the defect in SERT trafficking may also contribute to the disrupted formation of the barrel cortex in the Ulk1/2 cdko mice. Together, these data highlight the autophagy-independent role of ULK1 and ULK2 in maintaining cellular homeostasis and regulating axon guidance in the mammalian brain.

The endoplasmic reticulum is an organelle that responsible for the folding and processing of newly synthesized proteins. When unfolded or misfolded proteins accumulate in the ER and disrupt proper function this leads to a phenomenon known as ER stress. When this occurs, the UPR is activated in order to reduce ER load and restore the ER to its normal function. ATF6, IRE1, and PERK are the three pathways of the UPR. Here, I studied ER stress and the UPR in relation to retinitis pigmentosa, achromatopsia, and retinal degeneration in a tauopathy mouse model. I found that P23H rhodopsin, which is one of the most common mutated proteins involved with the development of retinitis pigmentosa, is highly ubiquitinated by several E3 ubiquitin ligases in order to be targeted for degradation. In addition, knocking out ATF6 seems to increase activation of IRE1 and preserve rhodopsin levels in the case of Rho+/P23H mice. In the achromatopsia study, Class 2 ATF6 mutants experience constitutive transcriptional activation and Class 3 ATF6 mutants are transcriptionally inactive, which can help explain causation of disease development. In the preliminary studies of the PS19 mouse model, I found that there is retinal degeneration and loss of visual function occurring in these mice compared to their wildtype littermates.

Diets rich in saturated fatty acids (SFAs) produce a form of tissue inflammation driven by “metabolically activated” macrophages, which contributes to the development of obesity-associated metabolic diseases. To better understand this form of macrophage activation, we analyzed the transcriptomes of mouse macrophages treated with either SFAs or the classical inflammatory stimulus lipopolysaccharide (LPS). SFA treatment induced a transcriptional signature distinct from that of LPS and strongly enriched by endoplasmic reticulum (ER) stress markers. In particular, SFA treatment increased the expression of the ER stress sensor IRE1α and many of its target genes comprising the adaptive unfolded protein response. SFAs also activate the NLRP3 inflammasome in macrophages, resulting in secretion of the pro-inflammatory cytokine IL-1β. We found that IRE1α mediates SFA-induced IL-1β secretion by macrophages and that its activation by SFAs does not rely on unfolded protein sensing. Instead, the ability of SFAs to stimulate either IRE1α activation or IL-1β secretion can be specifically reduced by preventing their flux into the phosphatidylcholine (PC) class of membrane lipids or by increasing unsaturated PC levels. Thus, IRE1α is an unrecognized intracellular PC sensor critical to the process by which SFAs stimulate macrophages to secrete IL-1β, a driver of diet-induced tissue inflammation and metabolic dysfunction.

Abstract: Phosphatidylethanolamine N-methyltransferase (PEMT), a liver enriched enzyme, is responsible for approximately one third of hepatic phosphatidylcholine (PC) biosynthesis via three sequential methylations of phosphatidylethanolamine (PE). PEMT is also expressed in white adipose tissue (WAT). When fed a high-fat diet (HF), Pemt-/-mice are protected from diet-induced obesity (DIO), but develop steatohepatitis. This thesis is endeavored to seek the underlying mechanisms for the resistance to DIO and the development of steatohepatitis in HF-fed Pemt-/- mice, mainly focusing on the liver, WAT and brown adipose tissue (BAT). The vagus nerve relays signals between the liver and brain, regulating energy metabolism. Thus, a possible role of the hepatic branch of the vagus nerve in the resistance to DIO and the development of steatohepatitis in Pemt-/- mice was first explored. Pemt-/- and Pemt+/+ mice were subjected to hepatic vagotomy (HV), compared with sham operation; or subjected to capsaicin treatment, which selectively disrupts the afferent nerve, versus vehicle-treated mice. After surgery, mice were fed the HF for 10 weeks. HV abolished the protection against DIO and prevented the development of steatohepatitis in Pemt-/- mice. However, disruption of the hepatic afferent vagus nerve by capsaicin failed to reverse either the protection against DIO or the development of HF-induced steatohepatitis in Pemt-/- mice. Thus, neuronal signals, relayed particularly via the efferent vagus nerve, contribute to the development of steatohepatitis and protection against obesity in HF-fed Pemt-/- mice. Endoplasmic reticulum (ER) stress is associated with the development and progression of steatohepatitis. PEMT is located on the ER and mitochondrial-associated membranes. Thus, we proposed that PEMT deficiency might cause aberrant composition of PC and PE in the ER and consequently ER stress, which sensitized mice to HF-induced steatohepatitis. Indeed, chow-fed Pemt-/- mice had reduced PC and increased PE in hepatic ER fractions. Chow-fed Pemt-/- mice presented ER stress in the livers with higher expression of CHOP and BIP compared to Pemt+/+ mice, without activating the unfolded protein response (UPR). HF led to more severe ER stress and activated UPR including all three branches PERK-eIF2α, IRE1α-XBP1s and ATF6, in the livers from Pemt-/- mice than Pemt+/+ mice. Similarly, McArdle cells without PEMT activity exhibited ER stress and activated UPR compared with McArdle cells expressing PEMT. Furthermore, the chemical chaperone 4-phenyl butyric acid (PBA) reversed the activation of UPR and ER stress caused by PEMT deficiency in McArdle cells. PBA had a minor impact on hepatic ER stress or the development of steatohepatisis in HF-fed Pemt-/- mice. However, PBA decreased triglyceride accumulation and alleviated apoptotic signaling in the livers from these mice. Together, PEMT deficiency leads to hepatic ER stress and sensitizes mice to HF-induced steatohepatitis. The contribution of WAT and BAT to the protection against DIO in Pemt-/- mice has also been studied. PEMT deficiency had no effect on adipocyte differentiation or lipolysis in WAT, but resulted in a decreased lipogenesis in WAT, which at least partially contributes to the lower weight gain in HF-fed Pemt-/- mice. Contrary to WAT, BAT is a highly oxidative tissue, responsible for non-shivering thermogenesis. In BAT, the protein and enzyme activity of PEMT was not detectable, compared to that in the liver or WAT. The capability for thermogenesis in mice was evaluated by cold exposure at 4˚C. When fed standard chow diet, both Pemt+/+ and Pemt-/- mice were able to maintain their body temperature above 33˚C for up to 4 h upon cold exposure. However, after fed the HF diet for 2 weeks, Pemt-/- mice developed hypothermia, whereas Pemt+/+ mice maintained their body temperature throughout the 4 h cold challenge. However, thermogenic capacity in BAT remained intact in HF-fed Pemt-/- mice. Dietary choline supplementation prevented the cold-induced hypothermia in Pemt-/- mice, coinciding with restoration of lower plasma glucose and increased expression of hepatic gluconeogenesis-related genes. Thus, cold-induced hypothermia in HF-fed Pemt-/- mice is likely due to an insufficient glucose supply caused by impaired hepatic gluconeogenesis, highlighting the importance of glucose as a substrate for thermogenesis.

Inflammation is a complex biological response to stress triggered by microbial infections. Although inflammation drives microbial clearance, if uncontrolled it leads to cellular damage and cell death. Previous studies have demonstrated that endoplasmic reticulum (ER) and mitochondrial stress programs can contribute to immunity by mediating production of inflammatory cytokines; however, the regulatory mechanisms are unclear. By utilizing Brucella abortus cattle vaccine strain RB51, we elucidated how ER and mitochondrial stress mediate inflammatory responses and the eventual fate of infected cells. Our initial findings revealed that RB51-induced programmed cell death (PCD), mediated by caspase-2-induced mitochondrial damage, led to activation of proapoptotic caspase-3 and -8. Notably, this caspase-2 mediated PCD was accompanied by the production of proinflammatory cytokines, TNFα and IL-1β, a phenomenon associated with pyroptosis, not PCD. These observations led us to further investigate how caspase-2 could regulate inflammation and proinflammatory cell death. As early as 2 hours post infection, RB51 triggered activation of ER stress sensor, IRE1, leading to increased ROS levels in the mitochondrial network. This sharp increase in mitochondrial ROS elicited the recruitment of inflammasome component NLRP3 and caspase-2 to the mitochondrial network, which triggered mitochondrial damage. Damage promoted release of mitochondrial danger signals into the cytosol and the activation of the NLRP3-ASC-caspase-1 inflammasome, which processes the inflammatory cytokine, IL-1β. Damaged mitochondria are usually tagged for removal by a mitochondria-specific form of autophagy called mitophagy. We observed that killed RB51 activated a second ER stress sensor, PERK, which suppressed IL-1β production. Upon PERK activation, the downstream transcription factor ATF4 initiated transcription of Parkin, a key regulator of mitophagy, which decreased mitochondrial damage signals and activation of the inflammasome. We have discovered that the partnership between the ER and mitochondria is essential for mediating inflammatory responses and determining cell fate. By fine-tuning the immune response via selective activation of ER stress sensors, we have laid the groundwork for therapeutic targeting of these regulators in inflammatory diseases associated with cellular stress.

Type 2 diabetes is an epidemic health crisis in concert with the worldwide rise in obesity. Dietary lipid oversupply and members of the lipid activated protein kinase C (PKC) family have been associated in the development of insulin resistance, a hallmark of diabetes. Deletion of either the PKCδ or PKCε isoform improves insulin action in fat-fed mice, but surprisingly leads to either a loss or an accumulation of hepatic triglyceride respectively. This thesis aims to examine the molecular mechanisms by which PKCδ and PKCε regulate insulin action and hepatic lipid metabolism. Using an in vivo spike-in SILAC proteomics approach, this work provided comprehensive and quantitative protein profiles of livers from fat-fed mice deficient in either PKC isoform. The study is the first to show that enzymes of retinol metabolism are affected by the absence of either PKCδ or PKCε. Pathway analysis indicated that monosaccharide metabolism was affected only upon PKCδ deletion, while isoprenoid biosynthesis was affected in a PKCε-specific manner. Two novel candidates, flavin-containing monooxygenase 3 (FMO3) and HIV-tat-interacting protein 2 (Htatip2), were further studied as they were significantly regulated by both PKC isoforms, in a similar or reciprocal fashion respectively. Overexpression of Htatip2 in hepatocytes affected triglyceride accumulation and oxidation, which phenocopied the effect of PKCε deletion on lipid storage in fat-fed mice. This is the first report to demonstrate that Htatip2 is involved in lipid metabolism as a potential downstream mediator of PKC isoforms. In FMO3-overexpressing cells, palmitate-induced ER stress, apoptosis and the expression of gluconeogenic enzymes were reduced. Thus this thesis offers an alternative mechanism in which PKC can induce production of glucose: by inhibiting FMO3 expression which causes an upregulation of ER stress, rather through direct effects on insulin signalling as per the current dogma. Overall, this thesis provides a detailed comparison of the effects of fat feeding and PKC deletion at the protein level. The study further supports the idea that PKC isoforms are not merely effectors of lipid second messengers, but can modulate lipid metabolism itself, and also emphasize that the link between hepatic lipid accumulation and defective insulin action is not necessarily causative.

Phospholipids (PLs) are important structural components of biological membranes and precursors of numerous signaling molecules. The fatty acyl composition of PLs determines the biophysical characteristics of membranes. Multiple lines of evidence demonstrated that changes in fatty acyl composition could potentially affect the properties of proteins associated with membranes and influence the biological processes that occur on membranes. However, there is little understanding of how regulatory pathways control PL fatty acyl composition in vivo or how such regulation dictates physiological responses. In this work, we investigated the regulation of membrane fatty acyl composition by the Liver X Receptor (LXR)-Lysophosphatidylcholine Acyltranferase 3 (Lpcat3) pathway and its physiological or pathological relevance in lipid homeostasis and metabolic diseases.In chapter 2, we define a nuclear receptor pathway for the dynamic modulation of membrane composition in response to changes in cellular lipid metabolism. Ligand activation of LXR preferentially drives the incorporation of polyunsaturated fatty acids into PLs through induction of the remodeling enzyme Lpcat3. Promotion of Lpcat3 activity decreases endoplasmic reticulum (ER) stress induced by saturated free fatty acids in vitro or by obesity and hepatic lipid accumulation in vivo. Conversely, Lpcat3 knockdown in liver exacerbates ER stress and inflammation. Mechanistically, Lpcat3 modulates inflammation both by regulating c-Src (Proto-oncogene tyrosine-protein kinase Src) and JNK (c-Jun N-terminal kinases) activation through changes in membrane composition and by affecting substrate availability for inflammatory mediator production. These results outline an endogenous mechanism for the preservation of membrane homeostasis during lipid stress and identify Lpcat3 as an important mediator of LXRs effects on metabolism.In chapter 3, we show that Lpcat3 is a critical determinant of triglyceride secretion due to its unique ability to catalyze the incorporation of arachidonate into membranes. Mice lacking Lpcat3 in the intestine fail to thrive during weaning and exhibit massive enterocyte lipid accumulation and reduced plasma triglycerides. Mice lacking Lpcat3 in the liver show reduced plasma triglycerides, hepatosteatosis, and secrete lipid-poor very low density lipoprotein (VLDL) lacking arachidonoyl PLs. Mechanistic studies indicate that Lpcat3 activity controls membrane lipid mobility in living cells, suggesting a biophysical basis for the requirement of arachidonoyl PLs in lipidating lipoprotein particles. These data identify Lpcat3 as a key factor in lipoprotein production and illustrate how manipulation of membrane composition can be used as a regulatory mechanism to control metabolic pathways.

Abstract: Biopsies and post-mortem tissue of patients with multiple sclerosis (MS) as well as inflammatory demyelinating animal models show that endoplasmic reticulum (ER) stress is a hallmark of the progression of these pathologies. Moreover, MS biopsies and animal models of neuroinflammatory diseases have detected axonal damage associated with mitochondria fragmentation and impaired distribution as an early event in absence of demyelination. It is thought that a combination of these phenomena makes cells more susceptible to inflammatory–mediated neurodegeneration and subsequent progression of the disease. Recent studies have demonstrated that Rab32, a small GTPase in the Ras protein family, plays a role in regulating mitochondrial mobility and ER stress induced apoptosis. Liang et al. showed that Rab32 expression sharply increases in response to acute brain inflammation, but subsequently drops. Based on the finding that activation of Rab32 induces ER stress related apoptosis and facilitates mitochondrial fragmentation via activation of dynamin-related protein 1 (Drp1), we hypothesize that Rab32 could play a role in altering the axonal mitochondrial distribution and inducing neurodegeneration in MS. In this study, we probed and measured the levels of Rab32 protein and functional related proteins Rab38 and Rab7L1, ER stress and apoptosis related proteins in acute as well as chronic lesions and normal-appearing white matter (NAWM) of inflamed MS brain tissues by Western blot and immunohistochemistry. Indeed, we found that high levels of Rab32 coincide with ER stress-associated apoptosis in acute lesions and its activation leads to shorter neurites with fragmented mitochondria in human neurons. Moreover, abnormal expression and activity of Rab32 accelerates apoptosis of human neurons, suggesting a role for Rab32 in neurodegenerative progression of MS.

A long-standing goal of the Eckhert Lab has been to understand the biology of boron and its role in human health at the molecular level. The argument for boron's essentiality in humans is weakened by the fact that no molecular mechanism has been identified. Boron is an essential nutrient for plants, and the human consumption of boron from dietary plants equals an intake of approximately 1 mg/day. Boron from plants is nearly completely absorbed as boric acid (BA) and circulates unchanged with a half-life of about 23 hours. This work focuses on three unsolved problems in the area of human boron biology: 1) characterization of the molecular target of BA and how it interacts with protein targets, 2) the signal transduction consequences of BA induced Ca2+ signaling dysregulation, and 3) a molecular explanation of how BA can inhibit cell proliferation without resulting in apoptosis. Collectively, the work presented herein defines a mechanism for BA in human cells.The Eckhert Lab has previously reported that the molecular target of BA is cyclic ADP-ribose (cADPR), the endogenous agonist of the ryanodine receptor (RyR), an endoplasmic reticulum (ER) Ca2+ channel. There were two questions remaining. First, it was unknown if cADPR binds to RyR or one of its accessory proteins. Second, it was unclear how BA interacted with the RyR complex to inhibit cADPR induced Ca2+ release from the ER. In the first chapter, I provide evidence that demonstrates cADPR binds directly to the accessory protein FKPB12. In this chapter, I also provide evidence that demonstrates that in the presence of BA, the binding of cADPR to FKBP12 is inhibited.The Eckhert Lab has also reported that BA induces mild ER stress, a consequence of ER Ca2+ dysregulation. Building on the work of Kimberly Henderson and Sarah Kobylewski, I describe in chapter 2 the molecular pathways that are activated by BA treatment of prostate cancer cells at physiological concentrations. I describe evidence that BA activates the eIF2alpha/ATF4 and ATF6 pathways. These results raise two additional questions. EIF2alpha; phosphorylation can occur through one of four kinases, each sensitive to different environmental stresses, but it was not known which kinase is activated by BA. In addition, model activators of the eIF2alpha; pathway usually activate CHOP, an apoptotic protein. However, BA does not cause apoptosis or increase levels of CHOP protein or mRNA and we did not have a molecular explanation for this observation.The question of which kinase was responsible for BA induction of ph- eIF2alpha; needed to be addressed. In the third chapter, I identify PERK as the kinase sensitive to BA. PERK has two known substrates, eIF2alpha; and Nrf2. I also provide evidence that demonstrates that BA treatment activates Nrf2, which is a transcription factor for the antioxidant response element and induces transcription of several antioxidant genes. This data indicates that BA treatment can increase levels of antioxidant proteins in the cell and provides evidence for why BA does not induce CHOP .The work described in these three chapters make a large contribution to the field of boron biology. Future studies should explore the link between the molecular mechanisms outlined herein with the specific effects observed with boron supplementation in animal models.

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 heightened sensitiviy to ER stress at early larval stages, and identified XBP1s as an early marker of stress. Unfortunaltey, over time we were not able to demonstrate that XBP1s was a valid marker for early ER stress. We were also able to show that AMPA recepotor subunits were changed in mutant larvae, but we could not tie these changes to higher intracellular Ca2+ levels in the motoneuron. This work contributes to increasing the knowledge of an ALS pathway and this knowledge may contribute to future therapies and cures.