Upon the loss of glucose as the main carbon source cells have developed different mechanisms in order to adapt to this stress and promote survival. In Saccharomyces cerevisiae one such mechanism is acetylation, a post-translational modification performed by a lysine acetyltransferase (KAT) complex, such as NuA4, which has been previously shown to regulate different glucose metabolic pathways. Despite its known role upon glucose starvation, it is not currently understood how NuA4 itself is regulated in response to acute glucose deprivation (GD). I determine here that NuA4 complex protein levels (including catalytic protein Esa1), structure, activity, and localization are not impacted by acute GD. Despite GD showing no impact to NuA4 itself, it does result in the remodelling of both the interactome and acetylome of the complex where 160 proteins were identified to change interaction with Esa1-TAP and 93 acetylation sites were identified. As well GD results in a shift in localization of interacting proteins from nuclear upon standard growth to cytoplasmic. As well the changing interactome shows enrichment for proteins related to regulation of transcription and translation, metabolic pathways like glycolysis and gluconeogenesis, and others related to the cellular stress response. From the interactome three sets of proteins, Pab1, Eaf5/7/3, and Fas1 and Fas2, were studied further to greater characterize their interaction with NuA4 as they change interaction upon GD. Eaf7 protein levels were shown to decrease upon GD and both Fas1 and Fas2 levels were shown to increase in response to NuA4 deletion mutants. Together this work provides a greater understanding of the cellular response to acute GD stress, and how NuA4 plays a role in response to that stress in order to promote cell survival.

Transient Receptor Potential Melastatin 7 (TRPM7) is a ubiquitously expressed divalent cation channel implicated as a key regulator of neuronal cell death in stroke. Our research group has previously shown that TRPM7 dependent cytoskeletal regulation particularly via cofilin mediates neuronal death in oxygen glucose deprivation (in vitro stroke model). LIMK1 phosphorylation was also shown to decrease downstream of TRPM7 activation during anoxia. In the present study we investigated the effects of TRPM7 activation during anoxia, on three regulators of LIMK and cofilin; Rho-associated kinase 2 (ROCK2), P-21 activated kinase 3 (PAK3) and Slingshot family phosphatase 1 (SSH1). Our findings suggest that PAK3 activity is downregulated during OGD through TRPM7 independent mechanisms. However, SSH1 activity appears to be regulated downstream of TRPM7 in a manner that is consistent with LIMK and cofilin regulation. Overall, our work suggests that SSH1 is a new link between anoxia-induced TRPM7activity and cofilin hyperactivation.

Transient Receptor Potential Melastatin 7 (TRPM7) is a ubiquitously expressed divalent cation channel implicated as a key regulator of neuronal cell death in stroke. Our research group has previously shown that TRPM7 dependent cytoskeletal regulation particularly via cofilin mediates neuronal death in oxygen glucose deprivation (in vitro stroke model). LIMK1 phosphorylation was also shown to decrease downstream of TRPM7 activation during anoxia. In the present study we investigated the effects of TRPM7 activation during anoxia, on three regulators of LIMK and cofilin; Rho-associated kinase 2 (ROCK2), P-21 activated kinase 3 (PAK3) and Slingshot family phosphatase 1 (SSH1). Our findings suggest that PAK3 activity is downregulated during OGD through TRPM7 independent mechanisms. However, SSH1 activity appears to be regulated downstream of TRPM7 in a manner that is consistent with LIMK and cofilin regulation. Overall, our work suggests that SSH1 is a new link between anoxia-induced TRPM7activity and cofilin hyperactivation.

There is increasing evidence that inflammatory processes play a pivotal role in the pathophysiology of ischaemic brain injury. Cerebrovascular endothelial cells that form the blood-brain barrier are critical for maintaining brain homeostasis, however, during cerebral ischaemia they contribute to the post-ischaemic inflammatory responses. It is not yet fully understood how different cerebral cells interact during this inflammatory response. This study aimed to test the hypothesis that oxygen-glucose deprivation (OGD) induces the inflammatory activation of the cerebrovascular endothelium and glial cells in vitro and that intercommunication between these cells regulate their responses to OGD. Primary murine brain endothelial cells (MBECs) monocultures, murine mixed-glial monocultures and MBEC-glial co-cultures were exposed to OGD for up to 24 hours (h), then reperfused cultures were returned to normoxia for a further 24 hours. MBECs and glia remained viable over a 24 h OGD exposure and during reperfusion. OGD induced a time-dependent increase in MBEC glucose transporter 1 (GLUT-1) expression but a time-dependent decline in expression and secretion of monocyte chemoattractant protein-1 (MCP-1). A significant increase in keratinocyte-derived chemokine (KC) secretion by MBEC monocultures was observed during reperfusion after prolonged exposure (18-24 h) to OGD whereas, KC secretion by co-cultured MBECs was increased during reperfusion after short exposure (4 h) to OGD. Co-cultured MBECs displayed a significant increase in intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in response to a short or prolonged exposure to OGD with 24 h of reperfusion. Neither OGD nor reperfusion had any effect on permeability of the MBEC monolayer. OGD induced a time-dependent increase in nuclear stabilisation of hypoxia inducible factor-1 alpha (HIF-1α) in glial cells which correlated to vascular endothelial growth factor (VEGF) secretion during OGD and subsequent reperfusion. Nuclear stabilisation of the nuclear factor kappa B (NFκB)p65 subunit by glial cells was dependent upon the duration of OGD. Reperfusion induced a significant increase in KC secretion by co-cultured glial cells after short exposure to OGD. Inflammatory activation of co-cultured MBECs and glia after 4 or 24 h OGD caused a significant increase in neutrophil transendothelial migration which correlated with MBEC expression of ICAM-1 and VCAM-1. A combination of these cell adhesion molecules with neutrophil integrins and soluble glial-derived mediators contributed to neutrophil transendothelial migration. These studies provide evidence that combined hypoxia and glucose withdrawal induces the activation of MBECs and glial cells in vitro. Cross-talk between these two cell types may further regulate their activation. As a result of this inflammatory activation, soluble MBEC and glial-derived mediators may contribute to neutrophil transendothelial migration through the regulation of MBEC cell adhesion molecule expression.

Acute brain cell swelling (cytotoxic edema) can occur in the first minutes of stroke, presumably as a result of brain cells taking up water. In extreme hypo-osmotic situations such as excessive water-loading by patients, uptake by brain cells can expand the brain, causing seizures. But is ischemic brain cell swelling the same as hypo-osmotic swelling? Water can passively diffuse across the plasma membrane. However the presence of water channels termed aquaporins (AQP) facilitates passive water diffusion by 10-100 times. Unlike astrocytes, there is no evidence of water channels on neuronal plasma membrane. However, there is still much debate about which cells (neurons or astrocytes) swell during over-hydration or during stroke and if neurons and astrocytes can volume-regulate during osmotic stress. The purpose of this study was to examine and compare the volume responses of PyNs and magnocellular neuroendocrine cells (MNCs) to acute osmotic challenge and to OGD. We examined MNCs because they are intrinsically osmosensitive to small changes (2-3 mOsm) of plasma osmolality. We also examined if the same neurons behave similarly in brain slices or when dissociated and if they respond differently to acute osmotic stress and stroke-like stress. Our results indicate that during acute osmotic stress (±40 mOsm) half of dissociated PyNs and MNCs tended to show appropriate responses. MNCs in brain slices showed similar responses to when they were dissociated, while brain slice PyNs were less responsive than when dissociated. Exposure to OGD resulted in obvious differences between the two types of in vitro preparations. Dissociated PyNs and MNCs showed no consistency in their volume responses to 10 minutes of OGD. Dissociated neurons swelled, shrunk or were unchanged in about equal numbers. In contrast, brain slice PyNs underwent profound swelling whereas, brain slice MNCs showed minor volume decreases. We conclude that about half of our dissociated neurons were too variable and unpredictable in their osmotic volume responses to be useful for osmotic studies. They also were too resistant to stroke-like stress to be good models for ischemia. Brain slice neurons were similar in their osmotic responses to dissociated neurons but proved to have consistent and predictable responses to stroke-like stress. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2011-02-07 17:55:08.333

Exposure to ethanol (ETOH) during fetal development results in a range of cognitive/behavioral deficits. There are differences in sensitivity to the effects of ETOH that could be explained by other factors, such as hypoxia. Similar mechanisms of damage underlie both ETOH, more specifically ETOH withdrawal, and hypoxia. Based on this overlap, it was hypothesized that sub threshold levels of these insults may interact to produce increased damage in sensitive brain regions. This study used a rodent organotypic hippocampal slice culture model to investigate the interaction of hypoxia and ETOH withdrawal and to determine possible developmental differences in the sensitivity to these insults. The combination of ETOH and hypoxia produced greater damage in the CA1 and CA3 hippocampal regions, as measured by propidium iodide uptake. Differences in outcome were noted between on postnatal (PND) 2 and PND 8 tissue. ETOH alone caused damage as measured by the neuronal marker NeuN, suggesting the ETOH/hypoxia interaction involves different cell types and that caution should be taken when determining appropriate levels of exposure. This data could explain why some offspring appear more sensitive to ETOH and/or hypoxic challenges during early life.

The role of gap junctions in modulating the dynamics of axonal dysfunction in spinal cord white matter injury remains uncertain; hence, I examined the functional role and changes in expression of gap junctions following CNS injury. I hypothesized that inhibition of gap junctions improves axonal conduction during oxygen and glucose deprivation (OGD) in vitro. Carbenoxolone and octanol, gap junction blockers, did not change CAP amplitude in non-injured tissue, yet they significantly reduced the extent of its decline during OGD. No difference in mRNA expression of connexins 32, 36 was found. However, during OGD in the presence of gap junction blockers, expression of connexins 30, 43 was downregulated. Immunohistochemistry confirmed the presence of connexins in spinal cord slices: connexins 30, 43 overlapping with GFAP, connexin 32 with MBP and connexin 36 with CC1. Thus, blocking gap junctions enhances axonal conduction during OGD and promotes dynamic changes in connexin mRNA expression.

The role of gap junctions in modulating the dynamics of axonal dysfunction in spinal cord white matter injury remains uncertain; hence, I examined the functional role and changes in expression of gap junctions following CNS injury. I hypothesized that inhibition of gap junctions improves axonal conduction during oxygen and glucose deprivation (OGD) in vitro. Carbenoxolone and octanol, gap junction blockers, did not change CAP amplitude in non-injured tissue, yet they significantly reduced the extent of its decline during OGD. No difference in mRNA expression of connexins 32, 36 was found. However, during OGD in the presence of gap junction blockers, expression of connexins 30, 43 was downregulated. Immunohistochemistry confirmed the presence of connexins in spinal cord slices: connexins 30, 43 overlapping with GFAP, connexin 32 with MBP and connexin 36 with CC1. Thus, blocking gap junctions enhances axonal conduction during OGD and promotes dynamic changes in connexin mRNA expression.

Studies on the survival of central nervous system of hypoxia-tolerant species under challenges of reduced energy availability have characterised adaptive mechanisms of brain at the cell and tissue level that lead to reduced excitability and protection. However, evidence of hypoxic suppression of retinal activity in these species has not been followed up with mechanistic studies. Microspectrofluorometric monitoring of intracellular free Ca2+ concentration ([Ca2+]i) is useful for identifying cellular mechanisms that may lead to adaptive strategies, as unregulated increases in [Ca2+]i cause toxicity. Horizontal cells (HCs) are second order retinal neurons that receive tonic excitatory input from photoreceptors, and possess voltage-gated Ca2+ conductances and other channels that can facilitate toxic increases in [Ca2+]i under conditions of reduced energy availability (modeled as oxygen-glucose deprivation, OGD). It was demonstrated that isolated HCs of the hypoxia-tolerant goldfish display spontaneous, transient [Ca2+]i activity (SA) which decreased in amplitude and area under the curve following OGD or glucose removal (20 min) without recovery. SA was shown to be dependent on extracellular Ca2+ influx through voltage-gated Ca2+ channels, though mechanisms of SA generation and regulation has yet to be determined. Additionally, glutamate-elicited peak increases in [Ca2+]i were reduced after 20 min of OGD. The removal of O2 during OGD insult seemed to be protective as an increase in baseline [Ca2+]i was seen during and following glucose removal under normoxic conditions. The mechanisms mediating these decreases in spontaneous and elicited [Ca2+]i activity are currently unknown, though candidate pathways are discussed. This thesis contributes a hint towards how HCs may tolerate conditions of low energy availability, which may also inform investigations on their role in situ during these insults.

Les signalisations oncogéniques induisent une consommation accrue de glucose qui n'est que partiellement satisfaite par le microenvironnement. Pour s'adapter et survivre à ce stress métabolique, les cellules malignes mettent en jeu des mécanismes qui restent mal compris. Nos travaux montrent que cette limitation en glucose a pour principale conséquence de déclencher une apoptose via la voie de signalisation PERK-CHOP de la réponse à un stress du réticulum endoplasmique (SRE), nommée Unfolded Protein Response (UPR). Nous avons découvert que le RE est capable de sentir la carence en glucose via la diminution de la disponibilité en UDP Nacétylglucosamine produit par la voie des hexosamines. La délétion du facteur pro-Apoptotique CHOP dans un modèle de cancer spontané du poumon induit par KrasG12V chez la souris augmente l'incidence tumorale, confirmant que le SRE constitue un mécanisme cellulaire de sauvegarde anti-Tumoral. Nous montrons également que le franchissement de cette barrière implique l'atténuation sélective de la voie PERK-CHOP par la protéine chaperon p58IPK, qui permet aux cellules de bénéficier en retour des effets protecteurs des autres voies d'un UPR devenu chronique. Ces résultats révèlent une dualité fonctionnelle pour le stress du RE dans la tumorigenèse contrôlée, au moins pour partie, par la protéine p58IPK / During carcinogenesis, oncogene activation induces high glucose avidity that outstrips the microenvironment supply until angiogenesis occurs. How malignant cells cope with this potentially lethal metabolic stress remains poorly understood. We found that oncogene-Driven glucose shortage triggers apoptosis through the PERK-CHOP pathway of the endoplasmic reticulum (ER) unfolded protein response (UPR). Deletion of the pro-Apoptotic UPR effector CHOP in a mouse model of KrasG12V induced lung cancer increases tumour incidence, strongly supporting the notion that ER stress serves as a barrier to malignancy. Overcoming this barrier requires the selective attenuation of the PERK-CHOP arm of the UPR by the molecular chaperone p58IPK. Furthermore, p58IPK-Mediated adaptive response enables cells to benefit from the protective features of chronic UPR. Altogether, these results show that ER stress activation and p58IPK expression control the fate of malignant cells facing glucose shortage