Sandhoff disease (SD), a fatal lysosomal storage disease, results from a deficiency of the β-subunit of the β-hexosaminidase A and B enzymes. This deficiency leads to severe accumulation of GM2 gangliosides in lysosomes within the central nervous system (CNS) resulting in mass neuronal apoptosis. The mouse model of SD shows progressive neurodegeneration that closely resembles Sandhoff and Tay Sachs disease (TSD) in humans. SD and TSD consist of infantile, juvenile, and late-onset forms. These diseases can present with a multiplicity of symptoms including cognitive and speech impairments, ataxia, and lower motor neuron disease. Late-onset SD and TSD show motor neuron disease in over 40% of patients. In this study, we explore the role of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in the spinal cord during the development and progression of disease in Sandhoff mice. Using immunocytochemistry and western blotting, we analyzed the expression level and localization of several ER stress and cellular apoptosis markers within the cervical, thoracic, and lumbar regions of the spinal cord of Sandhoff mice. Our results revealed significant upregulation of several ER stress markers in motor neurons that appeared to coincide with significant lysosomal accumulations. In addition, we observed sequential and age-dependent expression changes in ATF6 and CHOP and their prominent nuclear localization within anterior horn motor neurons. Markers of apoptosis, caspases and PARP also appeared to be activated in the spinal cords of Sandhoff mice starting as early as 60 days. Interestingly, we noted more than 50% reduction in neuronal numbers in all regions of the spinal cord of Sandhoff mice between ages 80 and 120 days. Overall, this study provides strong evidence for the role of chronic ER stress and UPR activation in the spine pathophysiology of SD. / Thesis / Master of Science (MSc) / Lysosomal storage diseases are a rare group of inherited neurological disorders that are often fatal at a young age. Two diseases that fall within this category, Sandhoff and Tay Sachs disease, are similar in their cause and symptoms. Current research lacks a complete understanding of the mechanism behind these disorders making the development of new therapeutics challenging. This research highlights a group of cells in the spine that are vulnerable in these diseases. These cells show physical and functional changes in their structure as the diseases progress. We provide evidence of a new stress pathway which appears to be strongly implicated in the development and progression of these diseases. We also show an association between this pathway and the death of these vulnerable cells leading to the symptoms exhibited by patients. These findings expand our current knowledge of these disorders and open new avenues for therapeutic interventions.

Diabetes mellitus dramatically increases the risk for atherosclerotic cardiovascular disease. It has been established that chronic hyperglycemia promotes an increase in glucose flux through the hexosamine biosynthesis pathway (HBP). Central to this pathway is glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme controlling the conversion of glucose to glucosamine. We have shown that glucosamine is a potent inducer of endoplasmic reticulum (ER) stress, which is characterized by the accumulation of misfolded proteins in the ER. Chronic ER stress can initiate a multifaceted response that results in lipid accumulation, inflammation and apoptosis: the hallmark features of atherosclerosis. We hypothesized that conditions of chronic hyperglycemia, associated with diabetes mellitus, can accelerate the development of atherosclerosis by a mechanism that involves increased HBP flux resulting in glucosamine-induced ER stress and the subsequent activation of pro-atherogenic pathways. In support of the hypothesis we found that glucosamine-supplemented apoE-/- mice had elevated levels of ER stress and atherosclerosis. Mechanistically, our data showed that glucosamine induced ER stress by interfering with the lipid-linked oligosaccharide biosynthesis pathway and protein N-glycosylation. These findings support a model by which conditions of hyperglycemia promote vascular complications through a glucosamine-intermediate. / Thesis / Doctor of Philosophy (PhD) / Diabetes mellitus dramatically increases the risk for heart attacks and strokes. High blood glucose is utilized in cells through its conversion into metabolites, such as glucosamine. We hypothesized that conditions of high blood glucose can led to an increase in intracellular glucosamine which can initiate pathways involved in accelerating atherosclerosis. Our results show that this is possible in both human cells and mice.

Essential hypertension is the leading contributor to premature death worldwide. Endoplasmic reticulum (ER) stress has recently been implicated in diseased blood vessels and hypertension. It is unclear whether ER stress is a cause or a consequence of hypertension. We hypothesized that ER stress inhibition would prevent the development of hypertension in the young spontaneously hypertensive rat (SHR) by improving vascular structure and function. The SHR was used as a genetic model of human essential hypertension, and the Wistar Kyoto (WKY) rat as its normotensive control. The first study conducted involved assessing the levels of ER stress in young SHRs, before they developed hypertension. The second study conducted involved treating rats with 1g/kg/day of the sodium salt of 4-phenylbutyric acid (4-PBA) orally for 8 weeks from 5 weeks of age. Blood pressure was measured weekly, noninvasively via radiotelemetry. Mesenteric arteries were collected at sacrifice. Finally, the third study conducted involved treating rats with 1g/kg/day 4-PBA orally for eight weeks from five weeks of age, and then withdrawing the drug for four weeks to determine if drug treatment created a sustained lowering of blood pressure. In the first study, ER stress markers were observed to be significantly increased in the young SHR when compared to the WKY. In the second study, blood pressure was observed to be significantly lower in the 4-PBA-treated SHR groups than in the untreated SHRs. In addition, mesenteric arteries from the 4-PBA treated SHRs had a significant decrease in media/lumen ratio, ER stress marker expressions, as well as improved vasodilatory response to carbachol and reduced contractile responses to phenylephrine. In the third study, 4-PBA was able to keep the blood pressure low for one week after withdrawal, however, blood pressure returned to similar levels as untreated SHRs by the end of three weeks. Overall, ER stress inhibition, via 4-PBA, blunted the development of hypertension in the SHR. / Thesis / Master of Science (MSc)

The proprotein convertase subtilisin/kexin type 9 (PCSK9) was first characterized in 2003 by Seidah and colleagues and marked the beginning of what is now considered by many as the greatest advancement in the field of cardiovascular disease (CVD) research since the discovery of the LDLR nearly half of a century ago. Since its discovery, PCSK9 was shown to enhance the degradation of cell-surface low-density lipoprotein (LDL) receptor (LDLR) and gain-of-function (GOF) mutations were shown to correlate with CVD risk. In contrast, patients carrying loss-of-function (LOF) mutations in PCSK9 highlighted a novel therapeutic approach for LDL lowering as they exhibit a life-long state of hypocholesterolemia and reduced CVD risk. A decade after the cloning of the PCSK9 gene, pharmaceutical companies have now developed a variety of PCSK9 inhibitors, ranging from monoclonal antibodies (mAbs) to small interfering RNA (siRNA) and vaccines, which have been shown to markedly reduce LDL cholesterol levels in pre-clinical models, as well as in patients at high risk of CVD. Despite these advances, there remained several unanswered questions regarding the mechanisms by which PCSK9 expression and secretion is regulated in the liver; the tissue from which the circulating pool of PCSK9 almost exclusively originates. The thought that further development of our understanding of PCSK9 biology may lead to the discovery of a signaling cascade that could be targeted by small molecules, the only class of inhibitor that has not yet been developed, has now merited additional research attention. The focal point of my doctoral studies represents the axis between a cellular process known as endoplasmic reticulum (ER) stress and PCSK9 expression/biosynthesis. ER stress is a deleterious cellular process that is known to occur in secretory cell types, such as liver hepatocytes, and is a well-established causative driver of an array of human diseases ranging from CVD to neurodegenerative diseases. ER stress is prevalent in the livers of patients with metabolic disease and is also known to activate the transcription factor capable of regulating PCSK9 levels, the sterol regulatory element-binding protein 2 (SREBP2). Based on this information, the first aim during the course of my PhD studies was to determine whether ER stress affected the expression and secretory status of PCSK9. In the past several years, I demonstrated that ER stress caused by ER Ca2+ depletion led to a marked increase in PCSK9 protein expression, but blocked its secretion as a result of its retention in the ER. Such a result was also associated with heightened hepatic LDLR expression and reduced LDL cholesterol levels in mice. Additional studies also characterized a variety of agents, including caffeine, as potent inhibitors of PCSK9 expression via increasing ER Ca2+ levels, which antagonized SREBP2 activity. As our initial studies revealed ER PCSK9 retention as a viable strategy for PCSK9 inhibition and LDL lowering, follow-up studies were also carried out to determine the outcome of such a strategy on liver function and injury. Given that heritable mutations in proteins that transit the ER can accumulate in this compartment and cause ER storage disease (ERSD), it was critical to further evaluate whether ER PCSK9 retention would lead to a similar outcome. In a series of experiments with rather surprising outcomes, we observed that the retention of the LOF Q152H PCSK9 mutant in the ER failed to cause ER stress; even in mice overexpressing the protein. Interestingly, tissue culture and mouse models demonstrated that the retention of PCSK9 in this cellular compartment increased the cellular abundance of ER stress response chaperones, such as the glucose-regulated proteins of 78- and 94-kDa (GRP78 and GRP94, respectively), but did not activate transducers of the ER stress signaling cascade. Strikingly, mice expressing the ER-retained PCSK9 Q152H mutant were protected against ER stress, suggesting a novel co-chaperone-like role of intracellular PCSK9. Collectively, the ER environment including secondary messengers like Ca2+ as well as its chaperones, plays a critical regulatory role on PCSK9 expression and secretion. Agents that increase ER Ca2+ levels can be utilized to block PCSK9 expression at the mRNA level to increase hepatic LDL clearance, and ER PCSK9 retention may also represent a safe avenue with a similar LDL lowering outcome. Beyond LDL lowering, hepatic ER PCSK9 retention may also serve as a novel strategy to enhance ER function and protect against ER stress-driven diseases of the liver. / Thesis / Doctor of Philosophy (Medical Science)

No description available.

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.

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.

Scrub typhus, caused by the obligate intracellular bacterium Orientia tsutsugamushi, afflicts one million people annually. Despite being a global health threat, little is known about O. tsutsugamushi pathogenesis. Here, we demonstrate that O. tsutsugamushi modulates the ER and ER-associated processes as mechanisms of nutritional virulence and immune evasion. To obtain amino acids to fuel replication, O. tsutsugamushi simultaneously induces ER stress and the unfolded protein response (UPR) while inhibiting ER-associated degradation (ERAD) during early infection time points. During exponential growth, the bacterium releases the ER bottleneck, resulting in generation of ERAD-derived amino acids that it parasitized for replication. The O. tsutsugamushi effector, Ank4, is linked to this process, as it impedes ERAD when ectopically expressed. O. tsutsugamushi expression of ank4 peaks during the ERAD inhibition window, but is absent when the pathway is restored. These data reveal a novel mechanism of nutritional virulence, whereby an obligate intracellular pathogen coordinates the modulation of multiple ER-associated processes. Like other intracellular pathogens, O. tsutsugamushi inhibits expression of MHC-I, but it does so in a novel manner by degrading the master regulator of MHC-I, NLRC5. This impedes production of the MHC-I components, human leukocyte antigen A and Beta-2 microglobulin. The NLRC5-reduction mechanism recapitulates across diverse cell types, but the degree and duration of inhibition is cell type-specific. NLRC5 modulation and MHC-I inhibition are linked to another O. tsutsugamushi Ank, Ank5. NLRC5 is a putative interacting partner of Ank5. Moreover, NLRC5 and MHC-I levels are reduced in cells ectopically expressing Ank5. To our knowledge, these are the first examples of a pathogen modulating NLRC5 to negatively regulate MHC-I expression and of a bacterial effector interacting with NLRC5. As we learn more about the bacterium’s ability to regulate its host cell, a unifying theme has emerged: modulation of the ER and ER-associated pathways. These projects reveal two novel mechanisms of O. tsutsugamushi pathogenesis, strategies to acquire the amino acids needed for replication and to decrease MHC-I antigen presentation by the host cell. These insights help in understanding how O. tsutsugamushi and potentially other related pathogens co-opt host cell processes to cause disease.

Classic Galactosemia (CG) is an autosomal recessive disorder caused by deleterious mutations of galactose-1-phosphate uridyltransferase (E.C. 2.7.7.12) (GALT) gene, which results in the inability to metabolize galactose and the accumulation of galactose-1-phosphate (gal-1-p) in patients' cells. Newborn screening has resulted in presymptomatic diagnosis and treatment. Although a galactose-restricted diet prevents the neonatal lethality of this disorder, many well-treated patients continue to develop debilitating complications such as premature ovarian insufficiency (POI), dyspraxic speech, ataxia and other neurological defects. The causes of these unsatisfactory outcomes remain unclear, but accumulation of gal-1-p is regarded as the major factor responsible for these chronic complications. In previous studies we found that gal-1-p was a competitive inhibitor of the UTP dependent, glucose-1-phosphate pyrophosphorylase (EC 2.7.7.9) and inositol monophosphatase (EC 3.1.3.25). As a result there were deficiencies in cellular UDP-glucose and UDP-galactose as well as impaired G-protein-stimulated inositol responses of Ca++ release, respectively. In this study, we found that when we challenged GALT-deficient yeast and GALT-deficient human diploid fibroblasts with galactose, these cells exhibited environmental stress and endoplasmic reticulum (ER) stress, which was characterized by the up-regulation of the gene encoding the master regulator of ER stress, GRP78/BiP. In separate studies using GALT-deficient diploid fibroblasts and comparative expression arrays, we found that the expression level of a tumor suppressor gene called aplysia ras homolog I (ARHI) was significantly higher in patient cells under galactose challenge. This ARHI gene was lost in rodents during evolution and GALT knockout mice did not express the human phenotype for galactosemia whereas over-expression of this gene in transgenic mice resulted in phenotypes characteristic of those seen in patients with galactosemia. We therefore propose here that ARHI could be an important target of galactose toxicity in Classic Galactosemia, and also explain the absence of patient phenotypes in GALT knock-out mice. In order to prevent accumulation of gal-1-p caused by GALT-deficiency, we experimentally screened over 300,000 chemical compounds against human galactokinase (GALK) in vitro. To date, we obtained from two high-throughput screenings (HTS), 200 GALK inhibitors with IC50s ranging from 700nM to 35μM. We subsequently established selectivity and toxicity profiles of 34 selected GALK inhibitors. Based on these results, we selected four compounds for further characterization, which included kinetic studies, site-directed mutagenesis and molecular docking experiments. From these experiments, we learned how these compounds interacted with the GALK enzyme and built detailed binding models for each of them. We demonstrated that three of the in vitro inhibitors of GALK could lower intracellular gal-1-p accumulation in GALT-deficient cells. Of considerable interest to us was that one of the compounds, cluster 25-1, not only reduced gal-1-p accumulation, but also corrected the level of GRP78/BiP back to background in the galactose-challenged GALT-deficient cells. These results were the first to demonstrate a direct link between GALT-deficiency and ER stress and provided proof of concept that we could prevent both gal-1-p accumulation and ER stress with GALK inhibitors in GALT deficient human cells. Lastly, we developed a new, virtual method of identifying novel GALK inhibitors by combining software-based, high-throughput virtual screening (HTVS) and fragment-based linkage using docking software. The initial HTVS validation experiments with compounds from the "ZINC" database identified four active GALK inhibitors with IC50s ranging from 70μM to 400μM. We then used HTVS to identify chemical fragments which bind to the active site of the human GALK enzyme. Using fragment-linking software, we identified chemical fragments which could potentially result in high-affinity inhibitors when chemically joined.

Androgen receptor (AR) is a member of the steroid hormone receptor family of molecules, and expansion of a CAG repeat encoding polyglutamine (poly-Q) in AR gene are associated with a progressive neuromuscular disease known as spinal bulbar muscular atrophy (SBMA) or Kennedy disease. The hallmark of SBMA diseases is formation of juxtanuclear AR inclusions that have been termed ¡¥AR aggregates¡¦.Previous studies showed that transgenic mice overexpressing wild-type AR exclusively in the skeletal muscle fibers display similar abnormalities to those observed in models of SBMA disease. To elucidate the mechanisms underlying toxicity conferred by wild-type protein aggregation within normal cells, a mouse embryonic stem cell (ESC) model with non-genetic modified settings in AR overexpression was used to display the common features of polyglutamine disease in this experiment. It was found that wild-type AR proteins are highly expressed and form nuclear aggregate inclusions in response to androgen treatment in ES cells, the formation of AR aggregates inhibit the differentiation of embryonic bodys and enhanced caspase-3 activity in androgens -induced apoptosis. In addition, it was also investigated that relation between chaperones¡BAR and the endoplasmic reticulum (ER) stress-induced pathways in ES cells in this study, and it was found that chaperones could colocalize with AR aggregates, these findings may help us to better understand the roles of the chaperones on AR aggregates.