No description available.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitous membrane protein that exchanges one intracellular H+ for an extracellular Na+, thereby regulating cell pH and volume. NHE1 catalytic activity is mediated by a transmembrane (TM) domain with 12 transmembrane segments. We performed cysteine scanning mutagenesis on TMVI (Asn227–Ile249) of NHE1. Each residue of TMVI was mutated into a cysteine in the background of a cysteineless NHE1 protein. MTSET and MTSES are sulfhydryl reactive membrane impermeable compounds able to react with accessible cysteines. Asp238Cys, Pro239Cys, and Glu247Cys expressed inactive NHE1. Asn227Cys, Ile233Cys, and Leu243Cys were strongly inhibited by MTSET, suggesting their pore lining properties. More mutations were introduced to characterize critical residues in TMVI. The Glu248Gln and Leu243Ala mutants were more susceptible to limited proteolytic attack by trypsin suggesting an altered conformation. The results suggest that Glu248 and Leu243 are important in protein structure, stability, and folding.

The ubiquitously expressed plasma membrane Na⁺/H⁺ exchanger isoform 1 (NHE1) plays an important role in directed cell migration in non-neuronal cells, an effect which requires both the ion translocation and actin cytoskeleton anchoring functions of the protein. In the present study, an analogous role for NHE1 as a modulator of neurite outgrowth was evaluated in vitro utilizing NGF-differentiated PC12 cells as well as mouse neocortical neurons in primary culture. Examined at 3 d.i.v., endogenous NHE1 was found to be expressed in growth cones, where it gave rise to an elevated intracellular pH in actively-extending neurites. Application of the NHE inhibitor cariporide at an NHE1-selective concentration (1 μM) resulted in reductions in neurite extension and elaboration while application of 100 μM cariporide, to inhibit all known plasmalemmal NHE isoforms, failed to exert additional inhibitory effects, suggesting a dominant role for the NHE1 isoform in modulating neurite outgrowth. In addition, whereas transient overexpression of full-length NHE1 enhanced neurite outgrowth in a cariporide-sensitive manner in both NGF-differentiated PC12 cells and WT neocortical neurons, neurite outgrowth was reduced in NGF-differentiated PC12 cells overexpressing NHE1 mutants deficient in either ion translocation activity or actin cytoskeleton anchoring, suggesting that both functional domains of NHE1 are important for modulating neurite elaboration. A role for NHE1 in modulating neurite outgrowth was confirmed in neocortical neurons obtained from NHE1-/- mice which displayed reduced neurite outgrowth when compared to neurons obtained from their NHE1⁺/⁺ littermates. Further, neurite outgrowth in NHE1-/- neurons was rescued by transient overexpression of full-length NHE1 but not with mutant NHE1 constructs again suggesting that both functional domains of NHE1 are important for modulating neurite outgrowth. Finally, the growth promoting effects of netrin-1 but not BDNF or IGF-1 were abolished by cariporide in WT neocortical neurons and while both BDNF and IGF-1 were able to promote neurite outgrowth in NHE1-/- neurons, netrin-1 was unable to elicit this effect. Taken together, these results indicate that NHE1 is a permissive regulator of early neurite morphogenesis and also plays a novel role in netrin-1-stimulated neurite outgrowth.

The ubiquitously expressed plasma membrane Na⁺/H⁺ exchanger isoform 1 (NHE1) plays an important role in directed cell migration in non-neuronal cells, an effect which requires both the ion translocation and actin cytoskeleton anchoring functions of the protein. In the present study, an analogous role for NHE1 as a modulator of neurite outgrowth was evaluated in vitro utilizing NGF-differentiated PC12 cells as well as mouse neocortical neurons in primary culture. Examined at 3 d.i.v., endogenous NHE1 was found to be expressed in growth cones, where it gave rise to an elevated intracellular pH in actively-extending neurites. Application of the NHE inhibitor cariporide at an NHE1-selective concentration (1 μM) resulted in reductions in neurite extension and elaboration while application of 100 μM cariporide, to inhibit all known plasmalemmal NHE isoforms, failed to exert additional inhibitory effects, suggesting a dominant role for the NHE1 isoform in modulating neurite outgrowth. In addition, whereas transient overexpression of full-length NHE1 enhanced neurite outgrowth in a cariporide-sensitive manner in both NGF-differentiated PC12 cells and WT neocortical neurons, neurite outgrowth was reduced in NGF-differentiated PC12 cells overexpressing NHE1 mutants deficient in either ion translocation activity or actin cytoskeleton anchoring, suggesting that both functional domains of NHE1 are important for modulating neurite elaboration. A role for NHE1 in modulating neurite outgrowth was confirmed in neocortical neurons obtained from NHE1-/- mice which displayed reduced neurite outgrowth when compared to neurons obtained from their NHE1⁺/⁺ littermates. Further, neurite outgrowth in NHE1-/- neurons was rescued by transient overexpression of full-length NHE1 but not with mutant NHE1 constructs again suggesting that both functional domains of NHE1 are important for modulating neurite outgrowth. Finally, the growth promoting effects of netrin-1 but not BDNF or IGF-1 were abolished by cariporide in WT neocortical neurons and while both BDNF and IGF-1 were able to promote neurite outgrowth in NHE1-/- neurons, netrin-1 was unable to elicit this effect. Taken together, these results indicate that NHE1 is a permissive regulator of early neurite morphogenesis and also plays a novel role in netrin-1-stimulated neurite outgrowth.

The ubiquitously expressed plasma membrane Na⁺/H⁺ exchanger isoform 1 (NHE1) plays an important role in directed cell migration in non-neuronal cells, an effect which requires both the ion translocation and actin cytoskeleton anchoring functions of the protein. In the present study, an analogous role for NHE1 as a modulator of neurite outgrowth was evaluated in vitro utilizing NGF-differentiated PC12 cells as well as mouse neocortical neurons in primary culture. Examined at 3 d.i.v., endogenous NHE1 was found to be expressed in growth cones, where it gave rise to an elevated intracellular pH in actively-extending neurites. Application of the NHE inhibitor cariporide at an NHE1-selective concentration (1 μM) resulted in reductions in neurite extension and elaboration while application of 100 μM cariporide, to inhibit all known plasmalemmal NHE isoforms, failed to exert additional inhibitory effects, suggesting a dominant role for the NHE1 isoform in modulating neurite outgrowth. In addition, whereas transient overexpression of full-length NHE1 enhanced neurite outgrowth in a cariporide-sensitive manner in both NGF-differentiated PC12 cells and WT neocortical neurons, neurite outgrowth was reduced in NGF-differentiated PC12 cells overexpressing NHE1 mutants deficient in either ion translocation activity or actin cytoskeleton anchoring, suggesting that both functional domains of NHE1 are important for modulating neurite elaboration. A role for NHE1 in modulating neurite outgrowth was confirmed in neocortical neurons obtained from NHE1-/- mice which displayed reduced neurite outgrowth when compared to neurons obtained from their NHE1⁺/⁺ littermates. Further, neurite outgrowth in NHE1-/- neurons was rescued by transient overexpression of full-length NHE1 but not with mutant NHE1 constructs again suggesting that both functional domains of NHE1 are important for modulating neurite outgrowth. Finally, the growth promoting effects of netrin-1 but not BDNF or IGF-1 were abolished by cariporide in WT neocortical neurons and while both BDNF and IGF-1 were able to promote neurite outgrowth in NHE1-/- neurons, netrin-1 was unable to elicit this effect. Taken together, these results indicate that NHE1 is a permissive regulator of early neurite morphogenesis and also plays a novel role in netrin-1-stimulated neurite outgrowth. / Medicine, Faculty of / Graduate

Sodium-Glucose Linked Transporter 2 (SGLT2) inhibitors block renal glucose reabsorption and have shown marked cardiac protection in type 2 diabetics, and surprisingly, also in non-diabetics. However, the mechanism by which these drugs improve cardiovascular outcomes is unknown. Metabolic heart disease, which is characterized by cardiac hypertrophy and diastolic dysfunction, is associated with obesity and insulin resistance and leads to adverse cardiovascular outcomes including heart failure with a preserved ejection fraction. A high fat, high sucrose “Western” diet can induce metabolic syndrome, an aggregate of obesity-driven clinical phenotypes including insulin resistance, elevated triglycerides, hypertension, and abnormal cholesterol. Using a mouse model of metabolic syndrome and adult rat ventricular myocytes (ARVMs) in vitro, we aim to determine if the SGLT2 inhibitor Ertugliflozin (ERTU) can prevent metabolic syndrome-induced cardiac pathophysiology and whether ERTU can exert a direct action on cardiomyocytes, a cell type lacking SGLT2. SGLT2 inhibitors have been proposed to act directly on the Sodium-Hydrogen Exchanger 1 (NHE1) and thus could have direct action on cardiomyocytes that may mediate cardioprotective effects. Mice were fed either a control diet (CD) or a high fat high sucrose (HFHS) diet ± ERTU for 16 weeks. Echocardiography was performed and heart weights were obtained. ARVMs were used to assess ERTU’s effect on insulin sensitivity in vitro in a high-palmitate, insulin resistance model, and to test the efficacy of the known NHE1 inhibitor Cariporide (NHEi). A NHE1 activity ammonium chloride pulse assay was performed in HEK293 cells over-expressing either wild-type (WT) or a known NHEi-insensitive point mutant NHE1 ± NHEi or ERTU. In HFHS-fed mice, ERTU attenuated weight gain and restored blood glucose, insulin, hemoglobin A1c, and HOMA-IR to CD levels. HFHS-induced cardiac hypertrophy and diastolic dysfunction were prevented with ERTU. In vitro, high palmitate media decreased insulin stimulated AKT signaling compared to low palmitate media and was rescued by either ERTU or NHEi treatment. ERTU inhibited WT NHE1 activity in HEK293 cells by 67%, whereas activity of the NHEi-insensitive mutant NHE1 was unaffected by ERTU treatment. ERTU prevented the hallmarks of diet-induced metabolic heart disease (cardiac hypertrophy and diastolic dysfunction) in mice. These benefits exceed the expected consequences of glucose control alone. The actions of ERTU on ARVMs in vitro suggest the favorable effects on cardiac structure and function may be due, at least in part, to the direct action of the drug on cardiomyocytes. Furthermore, mutational overexpression studies show that ERTU can directly affect NHE1 in cardiac myocytes. Taken together, this thesis provides evidence that the direct cardioprotective effects of ERTU could be via inhibition of NHE1, a critical modulator of intracellular pH and sodium in the cardiomyocyte, with known implications in the pathophysiology of diabetes and heart failure. / 2022-06-04T00:00:00Z

O presente trabalho visa contribuir para o esclarecimento da seqüência de eventos intracelulares produzidos pelas PKCs a e e, na modulação do pHi, via NHE1. Os estudos foram realizados em células MDCK e as medidas de pHi efetuadas por microscopia de fluorescência. A expressão das PKCs a e e, bem como do NHE1 foi investigada por western blot, utilizando anticorpos específicos para cada proteína. Os estudos foram realizados na situação controle ou na vigência de PMA ou ANG II, AVP e /ou inibidores específicos para cada receptor hormonal ou isoforma de PKC. Nossos resultados indicam que PMA (10-7 M) estimula a recuperação do pHi, por modular a atividade das PKCs a e e. ANG II e AVP em concentrações fisiológicas estimulam a recuperação do pHi após sobrecarga ácida, concomitante com o aumento da fosforilação da PKC a. Em concentração elevada, ambos hormônios não alteram estes parâmetros. O efeito de ANG II ou de AVP depende da interação de cada hormônio com receptores específicos para modular as vias de sinalização celular envolvidas com o aumento dos níveis de diacilglicerol, cálcio citosólico e AMPc. / The purpose of this work was to investigate the signaling events of PKCs a e e on the NHE1 activity. The effect of phorbol 12-myristate 13-acetate (PMA), angiotensin II (ANG II) or arginine vasopressin (AVP) on the intracellular pH (pHi) was investigated in MDCK cells by using the fluorescence microscopy and fluorescent probe BCECF/AM. The NHE1 or PKCs a e e expression was examined by western blot and specific antibodies. Our results indicate that PMA (10-7 M) or low concentration of ANG II and AVP induced a significant increase of pH recovery rate and PKC a expression, after intracellular acidification with NH4Cl pulse. ANG II or AVP did not change the PKC a expression. However, in right concentration, both hormones did not change these parameter. In conclusion, the effect of ANG II and AVP on NHE1 activity, depend of specifics membrane receptors and cellular signaling of intracellular calcium, DAG and PKCs a and e.

O presente trabalho visa contribuir para o esclarecimento da seqüência de eventos intracelulares produzidos pelas PKCs a e e, na modulação do pHi, via NHE1. Os estudos foram realizados em células MDCK e as medidas de pHi efetuadas por microscopia de fluorescência. A expressão das PKCs a e e, bem como do NHE1 foi investigada por western blot, utilizando anticorpos específicos para cada proteína. Os estudos foram realizados na situação controle ou na vigência de PMA ou ANG II, AVP e /ou inibidores específicos para cada receptor hormonal ou isoforma de PKC. Nossos resultados indicam que PMA (10-7 M) estimula a recuperação do pHi, por modular a atividade das PKCs a e e. ANG II e AVP em concentrações fisiológicas estimulam a recuperação do pHi após sobrecarga ácida, concomitante com o aumento da fosforilação da PKC a. Em concentração elevada, ambos hormônios não alteram estes parâmetros. O efeito de ANG II ou de AVP depende da interação de cada hormônio com receptores específicos para modular as vias de sinalização celular envolvidas com o aumento dos níveis de diacilglicerol, cálcio citosólico e AMPc. / The purpose of this work was to investigate the signaling events of PKCs a e e on the NHE1 activity. The effect of phorbol 12-myristate 13-acetate (PMA), angiotensin II (ANG II) or arginine vasopressin (AVP) on the intracellular pH (pHi) was investigated in MDCK cells by using the fluorescence microscopy and fluorescent probe BCECF/AM. The NHE1 or PKCs a e e expression was examined by western blot and specific antibodies. Our results indicate that PMA (10-7 M) or low concentration of ANG II and AVP induced a significant increase of pH recovery rate and PKC a expression, after intracellular acidification with NH4Cl pulse. ANG II or AVP did not change the PKC a expression. However, in right concentration, both hormones did not change these parameter. In conclusion, the effect of ANG II and AVP on NHE1 activity, depend of specifics membrane receptors and cellular signaling of intracellular calcium, DAG and PKCs a and e.

Différentes études ont montré que les facteurs liés au mode de vie, de même que le vieillissement ou l’amélioration des tests de diagnostic et de screening, ne peuvent à eux seuls expliquer l’incidence croissante des cancers dans les pays dits industrialisés. Bien que des changements de comportements aient conduit à une diminution du nombre de cancers en France, l’incidence des carcinomes hépatocellulaires est toujours en augmentation. D’autres facteurs oncogéniques, tels que l’exposition à des cancérogènes de l’environnement pourraient intervenir. Parmi ceux-ci, les hydrocarbures aromatiques polycycliques (HAP), dont le benzo[a]pyrène (B[a]P) est le chef de file, et qui sont retrouvés notamment dans la fumée de cigarette, les gaz d’échappements ou les aliments grillés, constituent une priorité en termes de santé publique du fait de leurs effets cancérogènes. Une caractéristique-clé commune à tous les cancers a trait à leur métabolisme énergétique particulier, basé sur la glycolyse. Cependant, rien n’est connu quant à une possible reprogrammation métabolique due aux HAP. L’objectif global de mon projet de thèse a donc été d’étudier l’impact in vitro du B[a]P, utilisé à faible concentration, sur le métabolisme énergétique, de préciser le rôle de la reprogrammation métabolique dans le contrôle de la balance survie/apoptose par le B[a]P et de caractériser les mécanismes cellulaires et moléculaires impliqués. Nous avons d’abord identifié une production de monoxyde d’azote (NO) résultant de l’activation de la iNOS par le B[a]P, et agissant comme un signal de survie, possiblement via une hyperpolarisation mitochondriale. Nous avons ensuite démontré que le B[a]P induit une reprogrammation métabolique des cellules en favorisant un métabolisme glycolytique au détriment de la phosphorylation oxydative. Enfin, nous avons identifié IF1, l’inhibiteur physiologique de la F0F1ATPase, comme une nouvelle cible des HAP, participant à la reprogrammation métabolique et capable de promouvoir la survie sous l’effet du B[a]P. Au total, nous montrons que ces altérations du métabolisme mitochondrial promues par le B[a]P, sont à l’origine de signaux de survie dans notre modèle de cellules épithéliales hépatiques F258. Ces mécanismes pourraient ainsi contribuer à la progression tumorale sous l’effet des HAP. / Various studies have shown that factors related to lifestyle (smoking, diet, etc.), as well as aging or even the improved efficiency of diagnostic and screening tests, cannot explain by themselves the rising incidence of cancers in the industrialized countries. Although evolution of behaviors has helped reducing the number of cancers in France, the incidence of hepatocellular carcinomas is still increasing. This alarming result could be related to other oncogenic factors such as chronic exposure to environmental carcinogens. Among these compounds, polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (B[a]P), the prototype carcinogen of this family, especially found in cigarette smoke, exhaust fumes or grilled food, are a priority in terms of public health due to their high carcinogenic potential. A key feature of cancer cells is related to their predominant glycolytic metabolism. However, nothing is known yet about a possible metabolic reprogramming upon PAH exposure. My PhD project has aimed at characterizing the impact of a low B[a]P concentration on energy metabolism, at clarifying the role of such a metabolic reprogramming in cell fate determination, and at elucidating the cellular and molecular basis of this phenomenon. We first identify a production of nitric oxide (NO), involving the activation of iNOS by B[a]P, and acting as a survival signal. We then demonstrate that B[a]P induces a metabolic reprogramming, thus promoting a glycolytic metabolism at the expense of oxidative phosphorylation. Finally, we identify IF1, the physiological inhibitor of the F0F1ATPase as a new target of PAHs, which participates in the B[a]P-elicited metabolic reprogramming and survival. To sum up, we identify new alterations of mitochondrial metabolism, acting as survival signals in B[a]P-treated rat hepatic epithelial F258 cells. These mechanism could therefore contribute to tumor progression.

Age-related neurologic disease is a significant and growing burden on our society. Although the largest share of research effort has typically been devoted to the common neurodegenerative illnesses (such as Alzheimer’s disease, or AD), the reality is that nearly all cases of neurodegenerative disease possess elements of mixed pathology. Vascular contributions to cognitive impairment and dementia (VCID) is a complex form of dementia, combining aspects of vascular disease and other forms of dementia, such as Alzheimer’s disease. This pathology is heterogeneous and can include cerebral amyloid angiopathy (CAA), hemorrhages, white matter infarcts, and changes to the neurovascular unit. Given the heterogeneous nature of VCID, we hypothesized that we could further elucidate mechanisms that drive dementia in VCID by examining pathology in mouse models and use this data to guide the study of human autopsy cases. Using a mouse model of VCID, we identified NHE1, a sodium hydrogen exchanger that was upregulated in these mice, as a possible candidate for a factor involved in cerebrovascular disease in humans. We saw a significant age effect of NHE1 in cases with Down syndrome (DS), leading us to further examine cerebrovascular pathology in individuals with DS. People with DS are at a high risk of developing cognitive impairment and dementia after the age of 50. In fact, virtually all adults with DS develop the neuropathology for an AD (beta-amyloid (Aß) senile plaques and tau neurofibrillary tangles) diagnosis by the age of 40 due to a triplication of chromosome 21. We found that these individuals develop CAA and microhemorrhages as a function of age, and that these rates are as severe as sporadic AD, despite an age difference of ~30 years. We also found that individuals with DS have different microglial morphologies than controls or individuals with AD. This data indicates that people with DS develop significant cerebrovascular and AD pathology, indicative of VCID. Overall, we found that mixed pathologies, specifically VCID, is an important contributor to the development of dementia and should be studied further to better understand how this pathology drives cognitive impairment. Further, it is clear that mouse models map imperfectly onto complex human diseases, and that significant work remains to be done towards achieving an adequate model of VCID.