published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Cigarette smoking is a major risk factor in the development of age-related chronic obstructive pulmonary disease (COPD) with chronic airway inflammation as a key feature. Currently, no effective treatment can reduce the protracted inflammation in the lung of COPD. Further research on the inflammatory mechanisms would therefore be important in determining new potential therapeutic targets in COPD. Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter that plays an important role in pulmonary functions and inflammatory responses. The serotoninergic system including serotonin transporter (SERT), serotonin receptors (5-HTR) and its metabolic enzyme monoamine oxidase (MAO) have been reported to associate with cigarette smoking and/or COPD. Blockade of serotonin receptor 2A (5-HTR2A) with its selective antagonist ketanserin has been shown to improve lung function in COPD patients. In this study, we hypothesize that the serotoninergic system is involved in cigarette smoke-induced oxidative stress, inflammation and COPD. Exposure to cigarette smoke medium (CSM) caused the elevation of interleukin (IL)-8 levels in primary normal human bronchial epithelial (NHBE) cells and a human bronchial epithelial cell line (BEAS-2B) in vitro via activation of p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathway. Besides, CSM was found to disrupt the glutathione (GSH) system, resulting in the translocation of nuclear factor-erythroid 2 related factor 2 (Nrf2) to the nucleus. Knock-down of Nrf2 by small interference RNA (siRNA) blocked CSM-induced IL-8 release. Pretreatment with ketanserin was found to attenuate CSM-induced IL-8 release by inhibiting the p38, ERK1/2, and Nrf2 signaling pathways, and by partially restoring the GSH system. On the other hand, CSM reduced MAO activity in BEAS-2B, indicating a reduced catabolism of 5-HT. Furthermore, 5-HT was found to share the common p38 and ERK1/2 signaling pathway with CSM in IL-8 release. In the cigarette smoke-exposed rat model, the GSH system in the lung was found to be disrupted compared to the sham-air control, supporting our in vitro findings. Interestingly, we found an increased MAO-A activity in the lung of cigarette smoke-exposed rats in comparison to sham air-exposed rats. The increased MAO-A activity in the lung was associated with the reduction of 5-HT levels in bronchoalveolar lavage (BAL) and lung homogenates, while the increased metabolism of 5-HT may be involved in cigarette smoke-induced superoxide anion levels. On the other hand, serum, but not plasma level of 5-HT was elevated in cigarette smoke-exposed group, which may be due to platelet activation caused by cigarette smoke. In the clinical study, the elevated plasma 5-HT levels were found to be associated with an increased odds ratio for COPD and positively correlated with age in COPD patients. Furthermore, plasma 5-HT was also demonstrated to be a significant mediator on the relation between cigarette smoking and COPD. In summary, our study supports the hypothesis that the serotoninergic system contributes to cigarette smoke-induced oxidative stress, inflammation and COPD. The serotoninergic system (e.g. 5-HTR2A) may constitute potential therapeutic targets for the treatment of COPD, which is worthy for further investigation. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide expansion in the SCA7 gene resulting in progressive ataxia and retinal dystrophy. SCA7 belongs to a group of neurodegenerative disorders called polyglutamine (polyQ) diseases, that share the common feature of glutamine tract expansions within otherwise unrelated proteins. Common suggested mechanisms by which polyQ expanded proteins induce toxicity include aggregation and induction of oxidative stress.  In this work we examined the connection between oxidative stress, aggregation and toxicity in SCA7 disease. We show that expression of the SCA7 disease protein, ataxin-7 (ATXN7), results in elevated levels of ROS and oxidative stress which in turn lead to toxicity. Our results also revealed that the oxidative stress further contributes to mutant ATXN7 aggregation. Moreover, we show, for the first time, that the major source of the elevated ROS in mutant ATXN7 cells is the increased activation of NOX1 enzymes. Interestingly, our results further revealed that the increased level of NOX1 activity together with altered p53 function leads to a metabolic shift in mutant ATXN7 expressing cells. Treatments with antioxidants, a NOX1 specific inhibitor or NOX1 knock-down, all decreased the ROS level, restored the metabolic shift and ameliorated the mutant ATXN7 induced toxicity. Taken together, we conclude that mutant ATXN7 activate NOX1 enzymes which results in oxidative stress, increased mutant ATXN7 aggregation, metabolic dysfunction and toxicity. NOX1 specific inhibition could thus be a potential therapeutic strategy for SCA7. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

The blood-brain barrier (BBB) is located at the level of the cerebral microvasculature and is critical to maintain central nervous system (CNS) homeostasis. The tight junction (TJ) protein complexes between endothelial cells at the BBB are primarily responsible for limiting paracellular diffusion of substances from the blood to the CNS. The BBB’s functional integrity is compromised in a number of disease states which affect the CNS, suggesting BBB dysfunction causes or contributes to many diseases of the CNS. A common component of most of these diseases is oxidative stres. Oxidative stress is associated with hypoxia-reoxygenation (HR) and peripheral inflammatory pain (PIP). Both HR and PIP have been shown to compromise BBB functional integrity. Using in vivo rat models of HR and PIP, we examined the role of ROS on BBB permeability as well as the TJ protein occludin using the free radical scavenger tempol. First, we subjected rats to HR with or without pre-treatment with tempol (200 mg/kg). We showed that tempol prevents up-regulation of the cellular stress marker heat shock protein 70 at the BBB during HR. Next we showed tempol reverses HR-mediated BBB permeability increase to ¹⁴C-sucrose, a marker of BBB paracellular permeability. Tempol also attenuated changes in the structure and localization of occludin, suggesting ROS produced during HR alter occludin and lead to disruption of BBB. We then investigated whether ROS production have similar effects on occludin and BBB permeability during PIP by administering 3% λ-carrageenan into the hind paw of rats. We found tempol attenuated carrageenan-induced increase in paw edema and thermal hyperalgesia. Tempol also attenuated up-regulation of the cellular stress marker NF-κB in cerebral microvessels. Tempol significantly decreased BBB permeability to ¹⁴C sucrose during PIP. We found PIP reduces disulfide bonds in occludin oligomeric assemblies thought to be important in maintaining the structural integrity of the BBB. Tempol significantly inhibited disulfide bond reduction, suggesting ROS mediate BBB disruption during inflammatory pain by reducing occludin disulfide bonding. Taken together, these findings show the involvement of ROS during HR and PIP contributes to BBB dysfunction by altering the structure of high molecular weight occludin oligomeric assemblies.

Non-Hodgkin lymphoma is commonly associated with chronic infection and inflammation. Such conditions are characterized by chronic oxidative stress. Because apoptosis signaling is often mediated by reactive oxygen species, lymphoma arising in the context of oxidative stress may become resistant to these apoptosis signals. Resistance to oxidative stress could contribute to tumorigenesis and limit response to chemotherapy, as apoptosis induced by many drugs involves reactive oxygen species. We used a cell culture model to understand how changes in the ability to handle oxidative stress contribute to apoptosis resistance. WEHI7.2 murine thymic lymphoma cells transfected with catalase or selected for resistance to hydrogen peroxide acquire a concomitant resistance to apoptosis induced by glucocorticoids. Cytochrome c release is delayed in these variants, demonstrating that apoptosis resistance lies upstream, in the signaling phase, or in the mitochondria themselves. By comparing the apoptosis-sensitive WEHI7.2 parental cells with the oxidative stress- and apoptosis-resistant variant cells, we investigated the contribution of cytosolic and mitochondrial changes to glucocorticoid-induced apoptosis. We showed that neither JNK kinase signaling, nor GSTπ, a redox sensor protein which regulates JNK, is activated during glucocorticoid-induced apoptosis. Our work using isolated mitochondria and recombinant tBid protein in cell-free apoptosis assays showed that the apoptosisresistant variants are intrinsically resistant to the release of cytochrome c and other intermembrane space proteins. The resistance was mediated upstream and within the mitochondria, and occurred at both steps controlling cytochrome c release. Given that the resistant variants demonstrated alterations in mitochondrial apoptotic function, we investigated mitochondrial protein changes that could explain these differences. An increased expression of cytochrome c was observed in the resistant variants, but selective reduction of cytochrome c expression showed that this change alone was not sufficient to affect sensitivity. The balance of pro- and anti-apoptotic Bcl-2 family members in untreated cells also did not explain intrinsic resistance. Alterations in Bcl-2 protein levels following treatment could contribute to glucocorticoid resistance, but additional work to test Bcl-2 family interactions will be required. We have identified points of resistance that are important in glucocorticoid-induced apoptosis and may also contribute to resistance to novel mitochondrial-targeting drugs.

Genetic perturbations and foreign chemicals can result in a multitude of changes across a wide range of biochemical processes in a biological system. These perturbations may affect the metabolome, the small molecule metabolites in an organism. Recently, liquid-chromatography coupled to mass spectrometry (LC-MS) technology has been used to quantify large proportions of the metabolome, however standardized protocols are not yet available for use with Drosophila melanogaster. Here, I developed an ion-pairing LC-MS protocol for the metabolomic characterization of D. melanogaster and demonstrated its implementation in establishing the metabolomic profile of flies under oxidative stress and in the metabolic profiles of four different Drosophila species. I demonstrated that this new method allows for the detection of otherwise difficult metabolites and that it is repeatable and sensitive with acceptable levels of ionsuppression, matrix effects, limits of detection and quantification. I then used this method to determine and quantify the metabolomic fingerprints of loss of Superoxide dismutase activity and paraquat-induced stress. Comparing and contrasting the effects of these two sources of oxidative stress, I document both similarities and stressor-specific effects.

Oxidative stress resulting from an imbalance between free radicals and antioxidants is considered to be an important etiological factor in the development and maintenance of hypertension. Angiotensin II (Ang II) has been shown to be an important regulator of blood pressure and acts to elevate blood pressure by its pressor effects. The pressor effects of Ang II are well documented but recent evidence has suggested another possible role of Ang II in elevating blood pressure, whereby it acts via an independent mechanism that is directly linked with oxidative stress. The spontaneously hypertensive rat (SHR) is a widely used model in the investigation of the pathophysiological mechanisms involved in hypertension. This study was therefore undertaken to determine whether Ang II acts as a causative factor via oxidative stress in the development and maintenance of hypertension in the SHR. This was elucidated by evaluating the role of both the endogenous in vivo levels of Ang II as well as an exogenous sub-pressor dose of Ang II, on oxidative stress and its associated parameters. The parameters evaluated included, the antioxidant status of the model on the basis of the levels of the major antioxidant enzymes viz. SOD, GPx and catalase; the free radical generating capacity, by assessing the activity of the membrane bound enzyme NADPH oxidase and the levels of H2O2. The study also evaluated the levels of the endogenous vasodilator nitric oxide (NO), remodelling of the vasculature and the level of tissue oxidative stress in the kidney. The results show that the SHR has an elevated plasma Ang II level and an elevated level of oxidative stress, thus showing that in this model there is an intimate link between oxidative stress and Ang II. The SHR also shows depleted levels of NO and thus a decreased vasodilatory capacity and increased remodelling of the vasculature. The kidney showed an increased level of lipid peroxidation, which was due to the elevated levels of oxidative stress. All of these pathophysiological changes contribute to the elevation in blood pressure in this model. The long term infusion of the sub-pressor dose of Ang II affected the SHR to a greater extent than the Wistar. Although the dose of Ang II elevated the blood pressure in both models, the degree of the pathophysiological changes associated with the elevation in blood pressure was greater in the SHR. The Ang II infusion in both these models demonstrated that in the SHR which is genetically predisposed to hypertension, adjustments are made to the antioxidant system, that result in an elevated level of protection against oxidative stress. These results show that Ang II acts as a causative factor in the pathogenesis of hypertension in the SHR via its well documented pressor effects, as well as via a multitude of independent mechanisms that are linked to oxidative stress. This is substantiated by the significant decrease in NO that is caused by the elevated oxidative stress, as well as the previously described pathophysiological changes. This study has therefore shown that Ang II has an intimate causative link with oxidative stress that results in parallel mechanisms that work concomitantly with each other in hypertension in this model. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

Type 2 diabetes is now recognized as a worldwide epidemic. Pancreatic beta-cell decompensation in the presence of insulin resistance is a major mechanism for the development of type 2 diabetes and may be triggered by mitochondrial dysfunction. Alcoholism is a known risk factor for type 2 diabetes. Excessive or chronic alcohol consumption leads to increased oxidative stress and mitochondrial dysfunction in beta-cells. Prohibitin is a multifunctional protein that also regulates mitochondrial biogenesis and function. Although it has anti-oxidant effects in some cell types, its role in pancreatic beta-cells is not known. This study has investigated the effects of prohibitin in ethanol treated pancreatic beta-cells using RINm5F and INS-1E cell lines. Prohibitin was found to be expressed in pancreatic beta-cells with localization to the nucleus and the perinuclear area. Ethanol increased the expression of prohibitin and induced its translocation from the nucleus to the mitochondria. Ethanol, through its metabolism by alcohol dehydrogenase (ADH), increased oxidative stress and altered mitochondrial membrane potential, decreased the activity of mitochondrial respiratory complexes I and IV, and uncoupled energy production with resulting reduction in ATP production. This was associated with activation of the proinflammatory enzyme c-Jun N-terminal kinase and proapoptotic proteins Bax and caspase-3, leading to beta-cell apoptosis. Ethanol also reduced glucose induced insulin secretion without alteration of the beta-cell transcription factors PDX-1 and MafA. Treatment with exogenous prohibitin or cellular overexpression of endogenous prohibitin attenuated ADH activity, prevented the deleterious effects of ethanol on mitochondrial function and reduced apoptosis, whereas prohibitin knockdown enhanced ethanol-induced apoptosis. In addition, prohibitin per se increased PDX-1 and MafA levels. Through the above mechanisms, prohibitin restored glucose induced insulin secretion in ethanol exposed beta-cells. In brief, ethanol causes mitochondrial dysfunction and induces apoptosis in beta-cells, which result in a reduction of insulin secretion; whereas prohibitin prevents mitochondrial dysfunction, apoptosis, and -cell failure by stabilizing mitochondrial complexes I and IV and inhibiting ADH activity during ethanol metabolism. In addition, prohibitin in itself increases the levels of beta-cell transcription factors. As a consequence, prohibitin maintains normal pancreatic beta-cell function and could be useful in diabetes prevention and treatment.

Converging evidence indicates that SOD1 aggregation is a common feature of mutant SOD1 (mSOD1)-linked FALS, and seems to be directly related to the gain-of-function toxic property. However, the mechanisms of protein aggregation are not fully understood. To study the contribution of modification on cysteine residues in SOD1 aggregation, we systematically examined the redox state of SOD1 cysteine residues in the G37R transgenic mouse at different stages of ALS and under oxidative stress induced by H2O2. Our data showed that under normal circumstances, cysteine 111 in SOD1 is free. Under oxidative stress, it is prone to oxidative modification by providing the thiolate anion (S-). With the progression of ALS, increased levels of oxidative insults facilitated the oxidation of thiol groups of cysteine residues. Human mutant SOD1 could generate an upper shifted band in SDS-PAGE, which turned out to be a Cys111-peroxidized SOD1 species. We also found that at different stages of ALS, accumulated oxidative stress facilitated the aggregates formation, which were not mediated by disulfide bond. The oxidative modification of cysteine 111 may promote the formation of disulfide bond-independent SOD1 aggregates. In addition, we investigated the correlation between nitrosative stress and S-nitrosylation of protein disulfide isomerase (PDI) in the mechanism of aggregates formation. Our data showed that up-regulated inducible nitric oxide synthase (iNOS) generated high levels of nitric oxide (NO), which induced S-nitrosylation of PDI with the progression of ALS in the spinal cords of mSOD1 transgenic mice. This correlation was confirmed by treating SH-SY5Y cells with NO donor SNOC to trigger the formation of S-nitrosylated PDI (SNO-PDI). When mSOD1 was overexpressed in SH-SY5Y cells, iNOS expression was up-regulated, NO generation was increased consequently. Furthermore, both SNO-PDI and mSOD1 aggregates were detected in these cells. Blocking NO generation with NOS inhibitor N-nitro-L-arginine (NNA) attenuated the S-nitrosylation of PDI; the formation of mSOD1 aggregates was inhibited as well. We conclude that NO-mediated S-nitrosylation of PDI is highly linked to the accumulation of mSOD1 aggregates in ALS.

No description available.