No description available.

Pancreatic beta cells sense fluctuations in circulating nutrients and adjust the rate of insulin secretion to maintain glucose homeostasis. Mitochondria integrate changes in nutrient flux to the generation of signals that modulate insulin secretion via oxidative phosphorylation. Type 2 Diabetes (T2D) is characterized by beta cell mitochondrial dysfunction and impairment of insulin secretion. Early stage progression of this disease in obese and pre-diabetic subjects is characterized by basal hypersecretion of insulin and increased insulin resistance in peripheral tissues including muscle, liver and adipose tissue. Whether basal hypersecretion of insulin or insulin resistance is the primary defect in T2D progression is still debated. The molecular mechanism underlying basal insulin hypersecretion and how it may lead to beta cell failure are not understood. Herein, we optimize a model of glucolipotoxicity that results in increased basal and reduced stimulated insulin secretion response. Furthermore, we show that pancreatic islets exposed to excess nutrients in vitro or isolated from high fat diet fed animals, have a decreased bioenergetic efficiency, which is characterized by increased mitochondrial proton leak. Leak represents the fraction of oxygen consumed that is not coupled to ATP production. We show that leak is sufficient to induce insulin secretion at basal glucose levels and that nutrient-induced insulin secretion at basal glucose is leak-dependent. Finally, we identify the mitochondrial permeability transition pore (PTP) as the source of the leak. Our findings suggest the PTP may be a potential therapeutic target to prevent/delay the onset of hyperinsulinemia in pre-diabetic subjects.

No description available.

In the hours and days following acute CNS injury, a secondary wave of events is initiated that exacerbate spinal tissue damage and neuronal cell death. A potential mechanism driving these secondary events is opening of the mitochondrial permeability transition pore (mPTP) and subsequent release of several cell death proteins. Previous studies have shown that inhibition of cyclophilin D(CypD), the key regulating component in mPTP opening, was protective against insults that induce necrotic cell death. We therefore hypothesized that CypD-null mice would show improved functional and pathological outcomes following spinal cord injury (SCI) and traumatic brain injury (TBI). Moderate and severe spinal contusion was produced in wild-type (WT) and CypD-null mice at the T-10 level using the Infinite Horizon impactor. Changes in locomotor function were evaluated using the Basso Mouse Scale (BMS) at 3 days post-injury followed by weekly testing for 4 weeks. Histological assessment of tissue sparing and lesion volume was performed 4 weeks post SCI. Calpain activity, measured by calpain-mediated spectrin degradation, was assessed in moderate injury only by western blot 24 hours post SCI. Results showed that following moderate SCI, CypD-null mice had no significant improvement in locomotor recovery or tissue sparing compared to wild-type mice. Following severe SCI, CypD-null mice showed significantly lower locomotor recovery and decreased tissue sparing compared to WT mice. Calpain-mediated spectrin degradation was not significantly reduced in CypD-null mice compared to WT mice 24h post moderate SCI. The lack of protective effects in CypD-null mice suggests that more dominant mechanisms are involved in the pathology of SCI. In addition, CypD may have a pro survival role that is dependent on the severity of the spinal cord injury.

A subset of cyclophilins have been implicated in mechanisms of neuroinflammation and neurodegeneration that contributes to the pathogenesis of Multiple Sclerosis. Mitochondrial dysfunction leading to mitochondrial permeability transition plays a pivotal role in axonal damage and disease progression in Multiple Sclerosis. Cyclophilin D (CypD) is a crucial regulatory component of the mitochondrial permeability transition pore and it was demonstrated that the cyclophilin D knockout animals showed reduced experimental allergic encephalomyelitis (EAE) clinical disease severity and axonal injury. We investigated the effect of N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive and non-selective cyclophilin inhibitor, on the course and severity of EAE. EAE mice treated with NIM811 showed a significant reduction in clinical disease severity compared to vehicle treated mice. FACS analysis performed with the dissociated thoracolumbar spine showed that NIM811 treatment was associated with a reduction in CNS macrophages but does not alter T-helper lineage frequencies. In addition, we demonstrated NIM811’s effect on crude mitochondrial fraction obtained from brain and liver homogenates of both wild type and CypD knockout mice in order to determine drug specificity. Benefits observed from the pharmacological inhibition of cyclophilin may lead to a novel MS therapy but NIM811’s exact mechanism of action has yet to be elucidated.

With an estimated incidence of 1.5 million each year, traumatic brain injury (TBI) is a major cause of mortality and morbidity in the United States. Opening of the mitochondrial permeability transition pore (mPTP) is a key event contributing to TBI pathology. Cyclophilin D (CypD), a matrix peptidyl-prolyl cis-trans isomerase, is believed to be the regulating component of the mPTP. Cyclosporin A, an immunosuppressant drug, inhibits CypD and blocks mPTP formation and has been shown to be neuroprotective following TBI. However, it is unclear if CsA’s neuroprotective mechanism is due to inhibition of CypD and/or immuno-suppression. Therefore to directly assess the contribution of CypD to TBI pathology, CypD knockout mice were subjected to a controlled cortical impact model of TBI. CypD ablation resulted in increased tissue sparing, hippocampal protection, and improved mitochondrial complex I driven respiration. Next a dose-response study of the Cyclophilin D inhibitor, NIM811, was performed. NIM811 administration following TBI resulted in improved cognition, increased tissue sparing, and improved mitochondrial function. These results suggest a major role for CypD in TBI pathology and validate CypD as a potential therapeutic target for TBI. TBI has been proposed to be the signature injury of the current Middle Eastern conflicts with an estimated prevalence of 15-60 % among combat soldiers. Although the brain does appear to be vulnerable to blast, the exact mechanisms underlying the injury remain unclear. Adult male Sprague-Dawley rats were exposed to a moderate level of blast overpressure. Following blast, blood brain barrier disruption was evident at 3 and 24 h post-exposure, oxidative damage increased at 3 h post-exposure, and microglia were activated in the hippocampus at 5 and 10 days post-exposure. This may widen future neuroprotective avenues for blast since blast brain injury appears to share similar mechanisms of injury with other TBI models.

Les phénomènes d’ischémie-reperfusion sont rencontrés dans plusieurs situations physiopathologiques. Le seul traitement de l’ischémie repose sur une restauration précoce du flux sanguin. Paradoxalement, la reperfusion génère des lésions supplémentaires, appelées « lésions de reperfusion », dont la mitochondrie est un acteur majeur via l’ouverture du pore de transition de perméabilité mitochondrial (mPTP). L’ouverture du mPTP est principalement modulée par la cyclophiline D (CypD), une protéine de la matrice mitochondriale, dont l’inhibition pharmacologique par la cyclosporine A (CsA) permet de limiter l’ouverture du pore. Cette inhibition, obtenue in vitro et in vivo, permet de réduire les lésions de reperfusion. Néanmoins, de récents essais cliniques n’ont pas permis de confirmer ce bénéfice dans le cadre de l’infarctus du myocarde, soulignant la nécessité de développer de nouveaux inhibiteurs du mPTP. Dans ce travail, nous avons étudié l’effet de nouveaux ligands de la CypD sur l’ouverture du mPTP. Ces petites molécules innovantes, de structure radicalement différente de la CsA inhibent l’ouverture du mPTP de mitochondries isolées et le dérivé le plus actif, le C31, permet une inhibition plus efficace du mPTP que la CsA. Le C31 inhibe également le mPTP au niveau cellulaire, dans des hépatocytes primaires et dans des cardiomyocytes isolés. In vivo, le C31 atteint les mitochondries hépatiques et protège le foie dans un modèle d’ischémie-reperfusion hépatique. Cependant, la stabilité métabolique du C31 ne lui permet pas d’atteindre le cœur. La poursuite du développement de ces inhibiteurs pourrait aboutir à de nouveaux candidats pour protéger les organes des lésions de reperfusion. / Ischemia-reperfusion can occur in various pathophysiological situations such as myocardial infarction or organ transplantation. The only available treatment of ischemia relies on a timely reperfusion which paradoxically causes additional damage, so-called « reperfusion injury ». Mitochondria play a central role in this phenomenon through the opening of the mitochondrial permeability transition pore (mPTP) which extends cell death. mPTP opening is modulated by the matrix protein cyclophilin D (CypD). CypD inhibition by cyclosporin A (CsA), the most described CypD inhibitor, limits reperfusion injury in vivo. Nevertheless, recent clinical trials failed to recapitulate such protection in the context of myocardial infarction, emphasizing the urge to develop new mPTP inhibitors. Here, we investigated the effects of new CypD ligands on mPTP opening. We demonstrated that these small molecules unrelated to CsA are potent mPTP inhibitors and that the most active compound, C31, exhibited stronger mPTP-inhibiting properties as compared to CsA. C31 also inhibited mPTP opening in primary hepatocytes and isolated cardiomyocytes. In vivo, C31 reaches liver mitochondria and protects mitochondrial function in a hepatic ischemia-reperfusion model. Nevertheless, C31 metabolic stability hampers cardiac uptake of the compound. Further development of these new inhibitors might lead to interesting candidates to protect organs against ischemia-reperfusion injury.

No description available.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

L’arrêt cardiaque (AC) est un problème de santé publique dont la mortalité reste très élevée malgré les progrès de la réanimation. La majorité des décès à distance de la réanimation cardio-pulmonaire (RCP) initiale est la conséquence du syndrome post‑AC provoqué par les lésions cellulaires d'ischémie-reperfusion (I/R) qui touchent principalement le cœur et le cerveau. L’ouverture du pore de transition de perméabilité mitochondrial (PTP), en lien avec la cyclophiline D (CypD), est un déterminant majeur des lésions d'I/R. L’hypothermie thérapeutique est le seul traitement adjuvant de la RCP qui a démontré un bénéfice sur la survie des patients en limitant le syndrome post-AC. Ses mécanismes d’action complexes ne sont que partiellement compris. Dans ce travail de thèse, nous avons, dans un premier temps, utilisé notre modèle d'AC chez le lapin pour étudier les mécanismes mitochondriaux de l’hypothermie thérapeutique. Notre modèle a d'abord permis de mettre en évidence le rôle important joué par la température dans la gravité du syndrome post‑AC et les effets protecteurs de l’hypothermie sur le cœur et le cerveau. Notre travail montre également que l'hypothermie inhibe l’ouverture du PTP par un mécanisme dépendant de la CypD et agit sur une voie de signalisation cellulaire. Nous avons, dans un second temps, mis au point un nouveau modèle d'AC chez la souris déficiente en CypD montrant que l'inhibition complète du mécanisme dépendant de la CypD d'ouverture du PTP améliore le succès de la RCP ainsi que le pronostic à long terme des animaux traités par hypothermie thérapeutique. Ainsi, nos travaux contribuent à identifier les mécanismes d'action mitochondriaux de l’hypothermie thérapeutique ainsi que des pistes pour améliorer le pronostic des patients après un AC réanimé / Despite advances in resuscitation science, mortality after cardiac arrest (CA) remains very high. A substantial proportion of cardiac arrest deaths occur in patients following successful resuscitation and can be attributed to the development of post-CA syndrome caused by cellular ischemia-reperfusion (I/R) injury that primarily affect heart and brain cells. The opening of the mitochondrial permeability transition pore (PTP), promoted by cyclophilin D (CypD), is a major determinant of I/R injuries. Therapeutic hypothermia is the only adjuvant therapy to cardiopulmonary resuscitation (CPR) known to improve survival by limiting the post-AC syndrome. The mechanisms of action of therapeutic hypothermia are still poorly understood. In this present work, we used our model of CA in rabbit to study the role of the PTP in the mechanisms of action of therapeutic hypothermia. Our model first highlighted the important role played by temperature in the severity of post-AC syndrome and the protective effects of hypothermia on heart and brain injuries. Our work also determined that hypothermia inhibited PTP opening by a CypD-dependent mechanism and acted on a pro-survival signaling pathway. We then developed a new CA model using CypD-deficient mice showing that complete inhibition of the CypD-dependent mechanism of PTP opening improved success of CPR and long-term survival of hypothermia-treated animals. Thus, our work contributes to identifying the mitochondrial mechanisms of action of therapeutic hypothermia and ways to improve the prognosis of patients after CA