No description available.

Infusion of the "mononuclear fraction" of human cord blood cells (HUCBC), which is composed of immature blood cells and hematopoietic progenitors, is known to reduce neurobehavioral deficits in rats subject to middle cerebral artery occlusion (MCAO). When MCAO rats are infused with 106 cells 24 hours after the induction of the stroke, their motor function improves. To extend these findings, we first examined the behavioral recovery of MCAO rats in the presence of increasing doses of HUCBC. The recovery in behavioral performance seen with measurements of spontaneous activity and motor deficits, depended on the amount of cells delivered, with 106 HUCBC being the threshold for significant behavioral recovery. Measurements of the ischemic volume revealed an inverse relationship between HUCBC dose and damage volume, which reached significance at the higher HUCBC doses (107 and 3-5x107 cells). Moreover, investigation of the distribution of the intravenously injected cells showed that HUCBC were localized to the injured brain hemisphere and the spleen. Given these findings, we hypothesized that there may be a role of HUCBC in the modulation of the peripheral or brain-localized immune response that is normally evoked after stroke. Results on the effect of HUCBC infusion on splenocytes indicated that HUCBC treatment prevented the alterations in splenocyte type (CD8+ depletion) and function (T-cell suppression) induced by stroke. In particular, examination of cytokine production from splenocyte cultures of HUCBC-treated MCAO rats revealed increased production of IL-10 and decreased production of IFNgamma relative to MCAO rats. Microglia (immunostained with a CD11b antibody) and B cells (identified with the B220 cell marker) that were increased after MCAO were dramatically decreased after HUCBC treatment. Proinflammatory cytokines such as TNF-alpha, IL-1beta and IL-2 were upregulated after MCAO surgery and their expression was abrogated after HUCBC infusion. All these findings indicate that the action of HUCBC may be specifically related to the modulation of the stroke-induced inflammatory response, providing a possible mechanism by which cord blood cells have been repeatedly reported to promote functional recovery from ischemic injury.

Autophagy is a physiological process by which the cell eliminates damaged organelles, toxic agents, and long-lived proteins by degradation through lysosomal system. Mitophagy, the specific autophagic elimination of mitochondria, regulates mitochondrial number to match metabolic demand and is a core machinery of quality control to remove damaged mitochondria. A neuroprotective role of physiological autophagy/mitophagy has been discovered. However, recent studies suggested that highly accelerated autophagy/mitophagy might contribute to neuronal death in various pathological situations including cerebral ischemia. In this study, we aimed to investigate the activation of excessive autophagy, particularly, the more specific mitophagy, in neuronal tissues and its contribution to ischemia/hypoxia (I/H)-induced delayed neuronal death. I/H injury was induced by oxygen and glucose deprivation (OGD) followed by reperfusion (RP) on primary cortical neurons in vitro. Cerebral ischemia was induced by unilateral common carotid artery occlusion and hypoxia in neonatal mice in vivo. In order to determine the extent to which autophagy contributes to neuronal death in cerebral ischemia, we performed multiple methods and found that in both primary cortical neurons and SH-SY5Y cells exposed to OGD for 6 h and RP for 24, 48, and 72 h, respectively, an increase of autophagy was observed as determined by the increased ratio of LC3-II to LC3-I and Beclin 1 expression. Using Fluoro-Jade C and monodansylcadaverine double-staining, and electron microscopy we found the increment in autophagy after OGD/RP was accompanied by increased autophagic cell death, and this increased cell death was inhibited by the specific autophagy inhibitor, 3-methyladenine. The presence of large autolysosomes and numerous autophagosomes in cortical neurons were confirmed by electron microscopy. Autophagy activities were increased dramatically in the ischemic brains 3-7 days postinjury from a rat model of neonatal cerebral I/H as shown by increased punctate LC3 staining and Beclin-1 expression. We thus obtained the conclusion that excessive activation of autophagy contributes to neuronal death in cerebral ischemia. BNIP3 (Bcl-2/adenovirus E19 kD interacting protein 3), a member of a unique subfamily of death-inducing mitochondrial proteins, is highly associated with mitochondrial dysfunction and delayed neuronal death in stroke. It is known that BNIP3-induced neuronal death is caspase-independent and characterized by early mitochondrial damage. Recent evidence suggested that the BNIP3 family of proteins might be important regulators of mitophagy. Here, using both stroke models, we found that homodimer (60 kD) of BNIP3/NIX (BNIP3L) were highly expressed in a ‘delayed’ manner. Particularly, significant mitophagic activation was confirmed by electron microscopy. In contrast, both neonatal mitophagy and apoptosis were significantly inhibited in the BNIP3 knockout (KO) mice after I/H, which was also accompanied by a significantly increased autophagic response. In addition, the infarct volume in the BNIP3 KO mice was significantly reduced as compared to wild-type (WT) mice after 7 or 28 days recovery, showing a prominent neuroprotection of BNIP3 gene silencing. A protein-to-protein interaction of mitochondria-localized BNIP3 (60 kD) with the autophagosome marker, LC3, was confirmed by co-ip, immunocytochemistry and further quantified by ELISA, indicating BNIP3 was an effective LC3-binding target on damaged mitochondria. These data demonstrated a novel role of BNIP3 in regulating neuronal mitophagy and cell death during ischemic stroke.

Humanin (HN) is an anti-apoptotic peptide that suppresses neuronal cell death induced by Alzheimer's disease, prion protein fragments, and serum deprivation. Recently, we demonstrated that Gly14-HN (HNG), a variant of HN in which the 14th amino acid serine is replaced with glycine, can decrease apoptotic neuronal death and reduce infarct volume in a focal cerebral ischemia/reperfusion mouse model. In this study, we postulate that the mechanism of HNG's neuroprotective effect is mediated by the PI3K/Akt pathway. Oxygen-glucose deprivation (OGD) was performed in cultured mouse primary cortical neurons for 60 min. The effect of HNG and PI3K/Akt inhibitors on OGD-induced cell death was examined at 24 h after reperfusion. HNG increased cell viability after OGD in primary cortical neurons, whereas the PI3K/Akt inhibitors wortmannin and Akti-1/2 attenuated the protective effect of HNG. HNG rapidly increased Akt phosphorylation, an effect that was inhibited by wortmannin and Akti-1/2. Mouse brains were injected intraventricularly with HNG before being subjected to middle cerebral artery occlusion (MCAO). HNG treatment significantly elevated p-Akt levels after cerebral I/R injury and decreased infarct volume. The protective effect of HNG on infarct size was attenuated by wortmannin and Akti-1/2. Taken as a whole, these results suggest that PI3K/Akt activation mediates HNG's protective effect against hypoxia/ischemia reperfusion injury.

L’accident vasculaire cérébral (AVC) déclenche une cascade de changements qui conduisent à la mort cellulaire mais aussi des processus endogènes de protection en réponse à l'ischémie. La compréhension de ces processus est très importante pour le développement d’agents neuro-protecteurs potentiels qui peuvent être seulement des amplificateurs des processus endogènes. Le traitement par la progestérone est neuro-protecteur dans des modèles expérimentaux d’occlusion de l'artère cérébrale moyenne (MCAO). Cependant, des niveaux endogènes significatifs de progestérone sont mesurables dans le cerveau de souris mâles et femelles.Le but de notre travail était d'étudier: 1) les effets de l’ischémie sur les niveaux endogènes des stéroïdes et le rôle des récepteurs de la progestérone (PR) à la phase aiguë après ischémie chez les souris jeunes adultes et âgées des deux sexes; 2) la base cellulaire de la neuroprotection par la progestérone après ischémie et le rôle de PR neural. Nous avons utilisé un modèle d’ischémie expérimentale (MCAO); une lignée transgénique de souris (PRNesCre) dans laquelle l’expression de PR a été sélectivement invalidée dans les cellules neurales; la chromatographie en phase gazeuse-spectrométrie de masse en tandem (GC-MS/MS); et des analyses histologiques, comportementales, et d’immunofluorescence.Dans la première partie, nous avons montré que dans le cerveau de souris mâles la progestérone est principalement convertie en 5a-dihydroprogestérone (5a-DHP), qui est un agoniste naturel de PR. Après MCAO, les niveaux de progestérone et de 5a-DHP cérébrales augment chez les mâles, mais pas chez les femelles. En revanche, les femelles peuvent utiliser l’inter-conversion de la 20a-dihydroprogestérone et de la progestérone pour réguler la disponibilité des pregnanes actifs au niveau de PR. De plus, les souris PRNesCre, mâles et femelles jeunes adultes et âgées, ont des infarctus plus grands et des déficits sensori-moteurs plus importants ainsi qu’une diminution de la densité des neurones et de la microglie activée comparativement aux souris témoins PRloxP/loxP. En outre, nos résultats ont révélé des différences liées au sexe chez les souris jeunes, mais pas chez les souris âgées. Dans la deuxième partie de l'étude, nous avons montré que, chez les souris mâles PRloxP/loxP, le traitement par la progestérone améliore la récupération fonctionnelle et réduit le volume de l'infarctus. Dans le péri-infarctus, la progestérone augmente la densité des neurones, des oligodendrocytes et de leurs précurseurs, et diminue la densité des astrocytes et de la microglie activée, et l'expression de l’aquaporine 4. Ces effets de la progestérone n’ont pas été observés chez les souris PRNesCre.Nos résultats montrent 1) l'importance des pregnanes endogènes et du PR neural pour la protection cérébrale à la phase aiguë précoce après une ischémie; 2) que le traitement par la progestérone chez les souris mâles après ischémie a des effets neuro-protecteurs, pro-myélinisants et anti-inflammatoires et que PR neural est requis pour la médiation de ces effets. Ces données suggèrent fortement que les ligands de PR ou des agents ciblant leur signalisation en aval pourraient être développés pour la neuro-protection après un AVC. / Ischemic stroke initiates a cascade of changes that lead to cell death and also coordinates endogenous processes that counteract the nocuous consequences of ischemia. Understanding these processes is very important for the development of potential neuroprotectants which can be just boosters of endogenous processes. Treatment with exogenous progesterone is neuroprotective after middle cerebral artery occlusion (MCAO). However, the male and female brains contain significant amounts of endogenous progesterone.The aim of our work was to study: 1) the effects of MCAO on the endogenous levels of steroids and the role of neural progesterone receptors (PR) at the acute phase after stoke in young and aging mice of both sexes; 2) the cellular basis of the neuroprotection by progesterone following stroke and the role of neural PR. We used an in vivo model of MCAO; a transgenic mice line (PRNesCre) selectively lacking the expression of PR in neural cells; gas chromatography-tandem mass spectrometry (GC-MS/MS); and histological, behavioral, and immunofluorescence analyses.In the first part of the study, we showed that in the male mouse brain, progesterone is mainly converted to 5a-dihydroprogesterone (5a-DHP), which is a natural PR agonist ligand. After MCAO, brain levels of progesterone and 5a-DHP are rapidly upregulated in males but not in females. In contrast, females may use the interconversion of 20a-dihydroprogesterone and progesterone for regulating the availability of PR-active pregnanes. Moreover, young and aging male and female PRNesCre mice exhibited increased infarcts, severe sensorimotor deficits, and decreased densities of neurons and microglia comparatively to age-matched control mice PRloxP/loxP. In addition, our results revealed sex differences in stroke outcomes in young but not in aging mice. In the second part of the study, we showed that, in male PRloxP/loxP mice, progesterone improved sensorimotor outcomes and reduced infarct volumes. In the peri-infarct, progesterone increased the densities of neurons, oligodendrocytes and their precursors, decreased the densities of activated astrocytes and microglia, and the expression of the aquaporin 4. These beneficial effects of progesterone were not observed in PRNesCre mice.Our findings 1) uncover the importance of endogenous pregnanes and neural PR for the cerebroprotection at the early acute phase after stroke; 2) show that progesterone treatment in male mice has neuro-protectant, pro-myelinating and anti-inflammatory effects after MCAO and that neural PR is required for the mediation of these effects. These data strongly suggest that ligands of PR or agents targeting their downstream signaling could be developed for neuroprotection after stroke.

No description available.

No description available.

An increasing body of evidence points to a key role of endoplasmic reticulum (ER) stress in chronic and acute neurodegenerative diseases. Indeed, markers of ER stress are common features of neurons destined to die in these conditions. In the present study we demonstrate that PUMA, a BH3-only member of the Bcl-2 family is essential for ER stress-induced cell death. PUMA is known to be a key transcriptional target of p53, however we have found that ER stress triggers PUMA induction and cell death through a p53-independent mechanism involving instead the ER stress inducible transcription factor ATF4. Specifically, we demonstrate that ectopic expression of ATF4 sensitizes neurons to ER stress induced apoptosis, and that ATF4-deficient neurons exhibit markedly reduced levels of PUMA expression and cell death. However, chromatin immunoprecipitation experiments suggest that ATF4 does not directly regulate the PUMA promoter. Rather, we found that ATF4 induces expression of the transcription factor CHOP, and that CHOP in turn directly activates PUMA induction. Specifically, we demonstrate that CHOP binds to the PUMA promoter during ER stress and that CHOP knockdown attenuates PUMA induction and neuronal apoptosis. In summary, we have identified a key signaling pathway in ER stress induced neuronal death involving ATF4-CHOP mediated transactivation of the pro-apoptotic Bcl-2 family member PUMA. Protein aggregates and markers of ER stress response have also been observed in dying neurons in several animal models of cerebral ischemia. Therefore, to decipher the significance of the ER stress apoptotic response, we investigate the role of ATF4-CHOP signaling pathway in ischemic neuronal injury. Ischemic stroke results from a transient or permanent reduction in cerebral blood flow in the brain. In spite of much research in trying to develop therapeutic strategies, most clinical trials have failed. These failures demonstrate that effective treatments require a more complete understanding of molecular signals that lead to neuronal death. However, stroke is a complex scenario since distinct mechanisms may involve in rapid and/or delayed neuronal death. The signaling pathways regulating these mechanisms however are not fully defined. Previous studies had suggested that ER stress playing a pivotal role in post-ischemic neuronal death. Yet, the relevance of ER stress signals was not fully known in ischemic neuronal injury. Accordingly, this thesis research attempts to explore the functional role of ER stress -inducible pathway, ATF4-CHOP axis, in different models of neuronal death (delayed and excitotoxic cell death) evoked by ischemia. The data indicates that ATF4 is essential in delayed type of death in vitro. In focal ischemia model (tMCAO) ATF4 also plays a role as a mediator of death signal in vivo. However, CHOP function looks more complex, and our data did not support the role of CHOP in ischemic neuronal death.

High resolution imaging of wide fields has been a prerogative of space telescopes for decades. Multi-conjugate adaptive optics (MCAO) is a key technology for the future of ground-based astronomy, especially as we approach the era of ELTs, where the large apertures will provide diffraction limits that will significantly surpass even the James Webb Space Telescope. NFIRAOS will be the first light MCAO system for the Thirty Meter Telescope and to support its development I have worked on HeNOS, its test bench integrated in Victoria at NRC Herzberg. I have aligned the optics, tested the electronic hardware, calibrated the subsystems (cameras, deformable mirrors, light sources, etc.) and characterized the system parameters. Development and support for future MCAO instruments also involves data analysis, a critical process in delivering the expected performance of any scientific instrument. To develop a strategy for optimal stellar photometry with MCAO, I have observed the Galactic globular cluster NGC 1851 with GeMS, the MCAO system on the 8-meter Gemini South telescope. From near-infrared images of this target in two bands, I have found the optimal parameters to employ in the profile-fitting photometry and calibration. As testimony to the precision of the results, I have obtained the deepest near-infrared photometry of a crowded field from the ground and used it to determine the age of the cluster with a method recently proposed that exploits the bend in the lower main sequence. The precise color-magnitude diagram also allows us to clearly observe the double subgiant branch for the first time from the ground, caused by the multiple stellar populations in the cluster. As the only facility MCAO system, GeMS is an important instrument that serves to illuminate the challenges of obtaining accurate photometry using such a system. By coupling the knowledge acquired from an instrument already on-sky with experiments in the lab on a prototype of a future system, I have addressed new challenges in photometry and astrometry, like the promising technique of point spread function reconstruction. This thesis informs the development of appropriate data processing techniques and observing strategies to ensure the ELTs deliver their full scientific promise over extended fields of view. / Graduate

An increasing body of evidence points to a key role of endoplasmic reticulum (ER) stress in chronic and acute neurodegenerative diseases. Indeed, markers of ER stress are common features of neurons destined to die in these conditions. In the present study we demonstrate that PUMA, a BH3-only member of the Bcl-2 family is essential for ER stress-induced cell death. PUMA is known to be a key transcriptional target of p53, however we have found that ER stress triggers PUMA induction and cell death through a p53-independent mechanism involving instead the ER stress inducible transcription factor ATF4. Specifically, we demonstrate that ectopic expression of ATF4 sensitizes neurons to ER stress induced apoptosis, and that ATF4-deficient neurons exhibit markedly reduced levels of PUMA expression and cell death. However, chromatin immunoprecipitation experiments suggest that ATF4 does not directly regulate the PUMA promoter. Rather, we found that ATF4 induces expression of the transcription factor CHOP, and that CHOP in turn directly activates PUMA induction. Specifically, we demonstrate that CHOP binds to the PUMA promoter during ER stress and that CHOP knockdown attenuates PUMA induction and neuronal apoptosis. In summary, we have identified a key signaling pathway in ER stress induced neuronal death involving ATF4-CHOP mediated transactivation of the pro-apoptotic Bcl-2 family member PUMA. Protein aggregates and markers of ER stress response have also been observed in dying neurons in several animal models of cerebral ischemia. Therefore, to decipher the significance of the ER stress apoptotic response, we investigate the role of ATF4-CHOP signaling pathway in ischemic neuronal injury. Ischemic stroke results from a transient or permanent reduction in cerebral blood flow in the brain. In spite of much research in trying to develop therapeutic strategies, most clinical trials have failed. These failures demonstrate that effective treatments require a more complete understanding of molecular signals that lead to neuronal death. However, stroke is a complex scenario since distinct mechanisms may involve in rapid and/or delayed neuronal death. The signaling pathways regulating these mechanisms however are not fully defined. Previous studies had suggested that ER stress playing a pivotal role in post-ischemic neuronal death. Yet, the relevance of ER stress signals was not fully known in ischemic neuronal injury. Accordingly, this thesis research attempts to explore the functional role of ER stress -inducible pathway, ATF4-CHOP axis, in different models of neuronal death (delayed and excitotoxic cell death) evoked by ischemia. The data indicates that ATF4 is essential in delayed type of death in vitro. In focal ischemia model (tMCAO) ATF4 also plays a role as a mediator of death signal in vivo. However, CHOP function looks more complex, and our data did not support the role of CHOP in ischemic neuronal death.