<p> Diabetes mellitus is a major independent risk factor for cardiovascular disease (CVD) and stroke, however the cellular mechanisms by which diabetes contributes to vascular dysfunction are not fully understood. In recent decades, multiple molecular mechanisms have been implicated in hyperglycemia-associated vascular damage and CVD [1]. It is well established that hyperglycemia promotes intracellular glucose flux through the hexosamine pathway where the rate-limiting enzyme, glutamine:fructose-6-phosphate amidotransferase (GFAT) produces glucosamine-6-phosphate [2,3]. We have shown that elevated levels of intracellular glucosamine cause ER stress and activation of the UPR in multiple cell types [4]. Additionally, we have previously shown that ER stress is associated with lipid accumulation, activation of inflammatory pathways, and is associated with atherosclerotic plaque formation in hyperglycemic mice [ 4,5]. We hypothesize that the accumulation of intracellular glucosamine, observed in conditions of hyperglycemia, promotes atherogenesis via a mechanism that involves the hexosamine pathway, protein glycosylation and ER stress.</p> <p> Using in vitro over-expression studies, we investigated the role of GFAT in hyperglycemia-associated ER stress. We developed methods to increase GFAT expression in both HepG2 cells and HASMC. However, we found that GFAT over-expression is insufficient to induce an ER stress response. Further investigation of this system suggests that the over-expressed GFAT does not increase intracellular glucosamine levels to sufficiently promote ER stress.</p> <p> We have also investigated the role of protein glycosylation in glucosamine-induced ER stress. We have shown that O-linked glycosylation plays a role in ER stress induction. We have also shown that N-linked protein glycosylation is affected by elevated cellular glucosamine levels. Thus, dysregulated glycosylation of newly synthesized proteins may contribute to the accumulation of unfolded protein in the ER and lead to the activation of the UPR.</p> / Thesis / Master of Science (MSc)

Endoplasmic reticulum (ER) stress, which results from the aggregation of misfolded proteins in the ER, has been implicated in many forms of kidney injury, including hypertensive nephrosclerosis. ER stress induction increases levels of active TGFβ1, a pro-fibrotic cytokine, which can lead to epithelial-to-mesenchymal transition (EMT) in renal proximal tubular cells. EMT occurs when epithelial cells undergo phenotypic changes, which can be prevented by inhibiting ER stress. Further, the ER stress protein TDAG51 is essential for the development of TGFβ1-mediated fibrosis. The low molecular weight chemical chaperone 4-phenylbutyrate (4-PBA) can protect against ER stress-mediated kidney injury. It acts directly on the kidney, and can prevent ER stress, renal tubular damage, and acute tubular necrosis. In a tunicamycin-mediated model of kidney injury, this damage is prevented primarily through repression of the pro-apoptotic ER stress protein CHOP. Along with providing renoprotective effects, 4-PBA can inhibit endothelial dysfunction and elevated blood pressure in a rat model of essential hypertension. In addition to lowering blood pressure, 4-PBA reduces contractility, augments endothelial-dependent vasodilation, and normalizes media-to-lumen ratio in mesenteric arteries from spontaneously hypertensive rats. Further, ER stress leads to reactive oxygen species generation, which is reduced with 4-PBA. Dahl salt-sensitive rats given 4-PBA are protected from hypertension, proteinuria, albuminuria, and renal pathology. Rats provided with vasodilatory medications demonstrate that lowering blood pressure alone is not renoprotective. In fact, endothelial dysfunction, as demonstrated by an impaired myogenic response, is culpable in the breakdown of the glomerular filtration barrier and subsequent renal damage. As such, alleviating ER stress using 4-PBA serves as a viable therapeutic strategy to preserve renal function and prevent ER stress-mediated endothelial dysfunction, renal fibrosis, glomerular filtration barrier destruction, and progression of hypertensive nephrosclerosis. / Thesis / Doctor of Philosophy (PhD) / Chronic kidney disease is characterized by progressive loss of kidney function, and is a major public health problem. Kidney cells make proteins that help the kidney function properly. However, if the proteins are made improperly, the kidney does not function as well. This can lead to poor filtration and protein in the urine, damage to important kidney structures, and kidney scarring. High blood pressure, a risk factor for kidney disease, is often accused of causing kidney damage. This thesis shows that malfunctioning blood vessels can cause kidney injury, and lowering blood pressure may not prevent this. However, there are pharmacological molecules that can protect the kidney from damage. These molecules help the cells make proteins properly, preventing blood vessel malfunction and kidney damage. Our findings suggest that helping blood vessels and kidney cells create properly functioning proteins is more protective for the kidney than lowering blood pressure alone.

In response to starvation, mis-folded proteins accumulate in the endoplasmic reticulum (E.R.) causing E.R. stress. This triggers a series of signaling pathways known as the unfolded protein response (UPR). The response helps to both enhance protein folding capacity and initiate mis-folded protein degradation, reducing E.R. stress. Alternatively, misfolded proteins are degraded and nutrients are recycled through autophagy. Thus, E.R. homeostasis depends on both UPR and autophagy. However, if E.R. stress is not resolved, UPR and autophagy can also cause apoptosis by mechanisms that are not fully understood. In chicken embryo fibroblasts, gamma-aminobutyric acid receptor-associated protein or GABARAP (a protein involved in autophagy) can promote apoptosis in conditions of prolonged starvation (Maynard et al. 2015). In these conditions, the down-regulation of GABARAP by shRNA/RNA interference reduces the expression of the pro-apoptotic CHOP (CAAT-enhancer-binding protein homologous protein) transcription factor (a marker of E.R. stress) and enhances cell survival. This suggests that elevated levels of autophagy compromises E.R. homeostasis and promotes the expression of CHOP in UPR lethal pathways. While GABARAP induction and processing/activation has been linked to the expression of CHOP upon prolonged starvation (Maynard et al. 2015), nothing is known about the pathway mediating CHOP expression and the relationship with other pathways of the UPR in cells with GABARAP mis-expression. Understanding these pathways will allow us to determine if GABARAP is a general determinant of E.R. stress or acts specifically on the expression of CHOP to control cell survival. Elucidating mechanisms which are involved in E.R. stress and the cellular transition between pro-survival to pro-apoptotic roles can allow understanding of processes associated with several pathological conditions like cancer and neuro-degenerative diseases. Additionally, establishing a role for GABARAP tumor suppressor in the control of the UPR and cell fate is also important. / Thesis / Bachelor of Science (BSc) / In response to starvation, mis-folded proteins accumulate in the endoplasmic reticulum (E.R.) causing E.R. stress. This activates both the Unfolded Protein Response (UPR) and Autophagy as both processes help to reduce E.R. stress. GABARAP, a protein involved in autophagy, has been shown to be involved in the promotion of apoptosis in conditions of prolonged starvation as its downregulation reduces apoptosis and CHOP expression (Maynard et al. 2015). However, how GABARAP regulates apoptosis remains unknown. Here, we investigate if GABARAP mis-expression affects multiple pathways in the UPR relieving global E.R. stress or if its specifically involved in blocking CHOP expression.

Several studies have shown that the loss in tension during fatigue can be virtually reversed by exposure of the muscle to agents which evoke Ca²⁺ release from the SR. The purpose of this study was to determine whether the SR Ca²⁺ release mechanism of fatigued muscle is less sensitive to caffeine than that of rested muscle. Following a fatigue bout of electrically evoked tetanic pulses, the functioning of the SR of chemically skinned muscle fibers was determined by the sensitivity of the SR to increasing concentrations of caffeine. Measurements of tension and rate of tension development were made at the maximal Ca²⁺ activated contracture(pCa4.5), the maximal caffeine(25mM) activated contracture and at the caffeine threshold for contraction. All tension and rate values were normalized per cross sectional area and expressed as percents of the maximal calcium activated values. Results of the maximal Ca²⁺ and caffeine data suggest that the both control and fatigue fibers are similar in maximal tension and Ca²⁺ loading characteristics. While no differences were found between rested or fatigued maximal Ca²⁺ or caffeine contractures, significant difference was found at the caffeine threshold (p<.05) with the fatigued muscle tending to contract at a higher caffeine concentration. This suggests that fatigued muscle is less sensitive to the caffeine stimulus for Ca²⁺ release from the SR. / Master of Science

No / Catecholamines that accompany acute physiological stress are also involved in mediating the development of hypertrophy and failure. However, the cellular mechanisms involved in catecholamine-induced cardiac hypertrophy, particularly Ca2+ handling, are largely unknown. We therefore investigated the effects of cardiac hypertrophy, produced by isoprenaline, on INa/Ca and sarcoplasmic reticulum (SR) function in isolated myocytes. Methods: INa/Ca was studied in myocytes from Wistar rats, using descending (+80 to ¿110 mV) voltage ramps under steady state conditions. Myocytes were also loaded with fura-2 and either field stimulated or voltage clamped to assess [Ca2+]i and SR Ca2+ content. Results: Ca2+-dependent, steady state INa/Ca density was increased in hypertrophied myocytes (P<0.05). Ca2+ release from the SR was also increased, whereas resting [Ca2+]i and the rate of decline of [Ca2+]i to control levels were unchanged. SR Ca2+ content, estimated by using 10.0 mmol/l caffeine, was also significantly increased in hypertrophied myocytes, but only when myocytes were held and stimulated from their normal resting potential (¿80 mV) but not from ¿40 mV. However, the rate of decline of caffeine-induced Ca2+ transients or INa/Ca was not significantly different between control and hypertrophied myocytes. Ca2+-dependence of INa/Ca, examined by comparing the slope of the descending phase of the hysteresis plots of INa/Ca vs. [Ca2+]i, was also similar in the two groups of cells. Conclusion: Data show that SR Ca2+ release and SR Ca2+ content were increased in hypertrophied myocytes, despite an increase in the steady state INa/Ca density. The observation that increased SR function occurred only when myocytes were stimulated from ¿80 mV suggests that Na+ influx may play a role in altering Ca2+ homeostasis in hypertrophied cardiac muscle, possibly through increased reverse Na+/Ca2+ exchange, particularly at low stimulation frequencies.

Most seed storage proteins accumulate in protein bodies which are derived from the vacuole. Zeins, the major corn storage proteins, however, are retained in the endoplasmic reticulum (ER) and their protein bodies are derived from the ER. There are circumstantial and preliminary data indicating that 27K zein, the proline-rich zein, may span the ER membrane. This potential transmembrane feature is considered very significant to understand the mechanism for zeins' ER retention. The transmembrane feature may retain the 27K zein in the ER where it could serve as an anchor for other classes of zein through specific protein interactions. In this study, a heterologous system (<i>Xenopus laevis</i> oocytes) was used to investigate the potential transmembrane domain of 27K zein. This study utilized physical assays of proteolytic digestion (proteinase K) and chemical modification (biotinylation) on isolated protein vesicles from <i>Xenopus</i> oocytes injected with <i>in vitro</i> transcribed 27K zein mRNA. In addition, the transmembrane features were analyzed by monitoring the protein's mobility in the lumen of the ER by pulse-chase experiments. The results showed that the possibility of 27K zein as a transmembrane protein was consistently refuted in this study. The 27K zein protein was not affected by the proteinase K treatment or biotinylation. Moreover, 27K zein and total zeins moved freely in the lumen of the ER similar to a secretory protein (ovalbumin), totally different from an ER membrane protein (a mutant transmembrane hemagglutinin envelope protein). The free movement, within the ER lumen, of total zeins under conditions where zein aggregates should form necessitates a reevaluation of the mechanisms responsible for zein polypeptides' ER retention and protein body formation. This study, therefore, concludes that 27K zein is not a protein body nucleating factor by virtue of an ER transmerrlbrane feature or association with the ER membrane and that the significance of zein solubility should be reconsidered to explain the zeins' ER retention leading to protein body formation in the ER. / Ph. D.

La signalisation calcique joue un rôle crucial dans la réponse des cellules aux signaux extracellulaires. Ces dernières années, les progrès des techniques de microscopie ont permis de montrer que l’organisation spatio-temporelle du signal calcique était un élément fondamental de la réponse des cellules. L'organisation temporelle du signal calcium se caractérise ainsi par l'observation d'oscillations de calcium dont la fréquence varie en fonction du stimulus. Pour de nombreux types cellulaires, et notamment pour les hépatocytes, modèle cellulaire de notre étude, l’amplitude et la fréquence des oscillations de calcium sont très régulières et finement régulées. Les nombreux facteurs qui interviennent pour assurer cette régulation rendent l'étude de ce processus difficile. Ceci explique que l’étude des oscillations de calcium est un des domaines dans lequel l’aspect expérimental est communément complété avec la modélisation mathématique.Parmi les nombreux éléments qui participent à la régulation du signal calcique, les mitochondries qui ont longtemps été considérées comme ayant un rôle passif, pourraient jouer un rôle important. En effet il a été établi que les mitochondries étaient capables d’accumuler de façon active le calcium libéré par le reticulum endoplasmique. Notre objectif a donc été d’étudier l’importance des mitochondries dans la régulation des oscillations de calcium dans les hépatocytes, et grâce à nos résultats expérimentaux, de mettre au point un modèle mathématique. Pour cela nous avons choisi de modifier la prise de calcium par les mitochondries en altérant le moins possible leur fonctionnement normal. Nous avons utilisé une protéine altérant directement la prise de calcium par les mitochondries (HINT 2) et une autre protéine altérant l’interaction entre le reticulum endoplasmique et les mitochondries (R-1). Nos résultats expérimentaux et computationnels démontrent que la protéine HINT 2 augmente l’activité de la chaine respiratoire, ce qui a pour effet d’accélérer l’accumulation de calcium par les mitochondries et d’augmenter la fréquence des oscillations de calcium cytosolique dans les hépatocytes. En effet le modèle élaboré permet à la fois de reproduire les observations expérimentales mais également de prédire avec succès que l’absence de la protéine Hint 2 rend les mitochondries plus sensibles à l’ouverture du pore de transition mitochondrial (mPTP). Nous avons également démontré que le R-1 n’affecte pas significativement la prise de calcium par les mitochondries mais que sa surexpression aboutit à une élévation de la concentration calcique de base des cellules hépatocytaires. A la lumière de nos résultats, cette élévation de la concentration de calcium basale semble être due à son interaction physique avec la pompe ATPase SERCA 2. Nos résultats nous ont permis de mettre en évidence à la fois la contribution des mitochondries dans la régulation des oscillations de calcium dans les hépatocytes grâce à la protéine Hint 2, mais aussi le lien direct entre la surexpression du R-1 et l’élévation de la concentration en calcium de base dans les cellules HepG2. / Calcium signaling plays a crucial role in cell response to external stimuli. These last years, the progress of the microsocopy techniques allowed demonstrate that spatio-temporal organisation of the calcium signal is fundamental for cell physiology. This spatio-temporal organization is characterized by the observation of calcium oscillations whose frequency varies according to the stimulus. For many cell types in particular hepatocytes, calcium oscillations amplitude and frequency are regular and finely regulated. The large range of actors involved in this regulation explains the complexity of studying this process. This explains why the calcium oscillations studies are one of the area in which the experimental approach is commonly associated with computational approaches.Among all the elements participating in the regulation of calcium signaling, mitochondria that have been thought for many years to play a passive role may have a greater role. Indeed it has been shown that mitochondria are able to accumulate actively calcium from the endoplasmic reticulum.Our goal was to study the importance of mitochondria in calcium oscillations regulation in hepatocytes and to elaborate a model based on these experimental results. For that purpose we choose to change the calcium uptake by mitochondria using techniques that didn’t kill them. We used a protein that alters directly mitochondrial calcium uptake (Hint 2), and a protein involved in the interaction between endoplasmic reticulum and mitochondria (R-1).Our experimental and computational results show that HINT 2 enhances the electron transport chain activity, which lead to faster mitochondrial calcium uptake and faster calcium oscillations in the cytosol of hepatocytes. The model allows us not only to reproduce experimental data but also to successfully predict that the loss of HINT 2 lead the mitochondria to be more sensitive to the mitochondrial transition pore opening (mPTP).We also demonstrate that R-1 doesn’t change mitochondrial calcium uptake, but its over expression lead to a higher calcium concentration in resting cells. In the light of our results, this higher calcium concentration seems to be the result of R-1 interaction with ATPase pump SERCA 2.Our results allowed us to show not only the role of mitochondria in the regulation of hepatocytes calcium oscillations by HINT 2, but also the link between R-1 over expression and a higher calcium concentration in the cytosol of HepG2 cells.

La maladie d’Alzheimer à forme tardive, exempte de mutations, représente près de 99% des 850 000 cas répertoriés en France. Hormis l’âge, des facteurs génétiques comme BIN1 apparaissent déterminant dans l’établissement de l’amyloïdopathie et de la tauopathie, marqueurs constitutifs de cette maladie. Le travail de thèse est basé sur l’étude d’une surexpression du gène humain de BIN1 et de son impact dans un contexte murin de tauopathie. La surexpression seule de BIN1 entraine des défauts mnésiques à court terme associés à des anomalies cellulaires et moléculaires au niveau de la voie temporo-hippocampique. Ces altérations sont exacerbées par la combinaison de la souris TgBIN1 avec le modèle de tauopathie, à la fois chez les mâles et les femelles. Pour autant il apparait que la surexpression de BIN1 préserve la mémoire spatiale dépendamment de l’âge et du sexe. L’hippocampe apparait en grande partie préservé des inclusions intracellulaires de Tau et la myéline des fibres axonales est retrouvé intacte. Ces éléments mettent en évidence que BIN1 est un acteur important dans l’établissement de la tauopathie et que son activité neuro-protectrice peut être médiée par un complexe moléculaire direct impliquant à la fois Tau et RNT4-A/Nogo-A. / Late Onset Alzheimer Disease represents more than 99% of total Alzheimer cases and it is not caused by genetic mutations. Among risk factors such as age, genetic compounds as BIN1 appear to be determinant for the pathological process establishment. This study aims to determine the BIN1 overexpression effect in mice and in a tauopathy context. In this study, BIN1 overexpression alone caused short term memory impairments linked with the cellular and molecular abnormalities. These disorders are exacerbated by a combination of TgBIN1 mice with a tauopathy model, both in males and females. Surprisingly, BIN1 overexpression rescued long term and spatial memory regarding the age and sex. Hippocampus appeared to be preserved from intracellular Tau inclusions. Moreover, fornix myelin is found intact. These elements highlighted BIN1 which is a key gene in tauopathie establishment. BIN1 neuroprotective activity is mediated by direct molecular interactions both with Tau and RTN4-A/Nogo-A.

How organelles acquire their unique shapes is a fundamental question of cell biology. The peripheral endoplasmic reticulum (ER) consists of a vast network of membrane sheets and tubules, the formation of which requires homotypic membrane fusion. Previous studies suggest that the dynamin-like GTPase, atlastin (ATL), mediates ER fusion, but the mechanism by which this occurs is unclear. In this study, I investigate 1) the role of dimerization and conformational changes in the N-terminal domain of ATL, 2) how the C-terminal amphipathic helix and the transmembrane domain of ATL cooperate with the N-terminal domain, and 3) the formation of cis and trans ATL dimers in the fusion mechanism. ATL has a cytosolic N-terminal domain, consisting of a GTPase domain and three-helix bundle (3HB), followed by two transmembrane segments (TMs) and a cytosolic C-terminal tail (CT). Crystal structures of ATL and biochemical experiments suggest that nucleotide-dependent dimerization between ATL molecules sitting in different membranes can tether the membranes together. A subsequent conformational change triggered by GTP hydrolysis could pull the membranes toward one another for fusion. This mechanism is supported by in vitro membrane tethering and fusion assays using vesicles containing full-length Drosophila ATL. The CT and TMs of ATL are also required for efficient membrane fusion. A synthetic peptide corresponding to a conserved amphipathic helix in the CT can act in trans to restore the fusion activity of a tailless ATL mutant. We characterize CT mutants to show that the C-terminal helix promotes fusion by perturbing the lipid bilayer. The TMs of ATL also mediate nucleotide-independent oligomerization, which may allow ATL molecules in the same membrane to synchronously undergo the conformational change leading to fusion. Lastly, we show that continuous GTP hydrolysis is required for membrane tethering, occasionally resulting in fusion. The N-terminal cytosolic domain mediates trans dimer formation between ATL molecules on different membranes. GTP binding induces dimerization through the GTPase domains and 3HBs. We propose that GTP hydrolysis and phosphate release are required not just to drive fusion, but also to dissociate cis dimers that form on the same membrane, thus allowing ATL molecules to form trans dimers.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.