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Stroke is a pathological condition that causes extensive brain damage. During ischemic stroke, an excess of the excitatory neurotransmitter glutamate exerts many deleterious effects, which leads to cellular damage and cell death, a phenomenon appropriately termed excitotoxicity. Among the events triggered is the activation of the enzyme calpain, a protease whose action is dependent on the intracellular concentration of calcium, which is known to be elevated during excitotoxicity. In this thesis, I hypothesize that neuroprotective drugs can be better accumulated into stroke-affected regions by utilizing the actions of calpain. The extent of calpain activation was first investigated, and it was found to increase over time in both in vitro and in vivo models of stroke. Different amino acid sequences recognized and cleaved by calpain were then incorporated into the neuroprotective Tat-GluR2/3Y peptide. Although in vivo detection of modified Tat-GluR2/3Y peptides was unsuccessful due to technical difficulties, the accumulation of the therapeutic 3Y peptide fragments in neurons under excitotoxic conditions in vitro was found to increase with the CP-3 peptide, a peptide that is a modified version of the Tat-GluR2/3Y, with a sequence cleavable by calpain from the protein Collapsin Response Mediator Protein-3 (CRMP-3). These results suggest that it is possible to concentrate therapeutic agents into stroke-affected neurons, and this may translate into enhanced neuroprotective properties in both in vitro and in vivo animal stroke models.

Stroke is a pathological condition that causes extensive brain damage. During ischemic stroke, an excess of the excitatory neurotransmitter glutamate exerts many deleterious effects, which leads to cellular damage and cell death, a phenomenon appropriately termed excitotoxicity. Among the events triggered is the activation of the enzyme calpain, a protease whose action is dependent on the intracellular concentration of calcium, which is known to be elevated during excitotoxicity. In this thesis, I hypothesize that neuroprotective drugs can be better accumulated into stroke-affected regions by utilizing the actions of calpain. The extent of calpain activation was first investigated, and it was found to increase over time in both in vitro and in vivo models of stroke. Different amino acid sequences recognized and cleaved by calpain were then incorporated into the neuroprotective Tat-GluR2/3Y peptide. Although in vivo detection of modified Tat-GluR2/3Y peptides was unsuccessful due to technical difficulties, the accumulation of the therapeutic 3Y peptide fragments in neurons under excitotoxic conditions in vitro was found to increase with the CP-3 peptide, a peptide that is a modified version of the Tat-GluR2/3Y, with a sequence cleavable by calpain from the protein Collapsin Response Mediator Protein-3 (CRMP-3). These results suggest that it is possible to concentrate therapeutic agents into stroke-affected neurons, and this may translate into enhanced neuroprotective properties in both in vitro and in vivo animal stroke models.

As the major excitatory neurotransmitter, glutamate (Glu) is physiologically important in brain function. Excessive Glu release, however, is a critical underlying pathological mechanism in neurodegenerative disease, especially stroke. Strategies to protect neurons from cell death under these conditions are scarce; in part because of incomplete understanding of inherent neuroprotective mechanisms. The suprachiasmatic nucleus (SCN) is a region of the brain that exhibits endogenous resistance to Glu excitotoxicity. A previous study demonstrated that SCN2.2 cells (an immortalized SCN cell line) were resistant to Glu excitotoxicity as compared to GT1-7 neurons (from the neighboring hypothalamus). This thesis explored the cellular mechanisms underlying this endogenous neuroprotection in SCN2.2 cells. Extracellular regulated kinase (ERK) is expressed in the SCN, activated by Glu, and is anti-apoptotic in other systems. Therefore, this thesis was designed to test the following central hypothesis: SCN2.2 cells are dependent on ERK signaling for survival in the presence of an excitotoxic insult. Glu increased ERK activity in SCN2.2 cells and importantly, resistance to Glu excitotoxicity in SCN2.2 cells was compromised by pre-treatment with an ERK inhibitor (PD98059; PD). ERK inhibition + Glu mediated SCN2.2 cell death in an N-methyl-D-aspartate receptor (NMDAR)-dependent manner; specifically via the NMDAR 2B (NR2B) subunit. Glu treatment increased expression of NR2B, phosphorylated NR2B and NR1 proteins and decreased NR2A and NR2D mRNA in the GT1-7 cells. Glu-treated SCN2.2 cells showed decreased NR2B, phosphorylated NR2B, increased NR2C proteins and increased NR2A and NR2D mRNA levels. These data are consistent with varied NMDAR responses to Glu in GT1-7 vs. SCN2.2 cells, which might underlie the different physiological responses to Glu in the two cell types. Further experiments investigated the role of several signaling kinases, e.g. protein kinase A (PKA), protein kinase C (PKC), calcium/calmodulin-dependent kinase II (CaMK-II) and c-Jun N-terminal kinase-II (JNK-II) in regulation of ERK activation and on SCN2.2 cell fate. PKA and PKC inhibition together, CaMK-II inhibition and JNK-II inhibition resulted in SCN2.2 cell death in the presence of Glu. PKA + PKC inhibition and CaMK-II inhibition resulted in a corresponding decrease in Glu-induced ERK phosphorylation. Combined inhibition of ERK, CaMK-II and JNK-II resulted in exacerbation of cell death as compared to when the inhibitors were used individually. These results suggest that ERK activity is regulated by a number of different kinases. Glu treatment resulted in a persistent increase in ERK phosphorylation (activation) for up to 48 h in the SCN2.2 cells whereas the pro-apoptotic p38 was phosphorylated (activated) in the GT1-7 cells exposed to Glu. JNK-II was transiently phosphorylated (activated) in the SCN2.2 cells. This suggests an activation of a short-term stress response which can result in activation of a long-term neuroprotective response in these cells. Pro-apoptotic Bid mRNA and cleaved Bid protein levels were increased in the Glu-treated GT1-7 cells. The effect of Glu treatment on the expression of several downstream effector molecules of ERK activation was also explored. Neuritin mRNA was increased with Glu treatment in the SCN2.2, but not in the GT1-7 cells. However, there was no change in the neuritin protein levels in either cell type with Glu treatment. Bcl2 levels remained unchanged in the Glu-treated GT1-7 cells. Although there was no change in the Bcl2 mRNA levels in the SCN2.2 cells, Bcl2 protein was significantly increased with Glu treatment, thus suggesting a post-translational mechanism of neuroprotection involving Bcl2. Taken together, these results are consistent with activation of an apoptotic mechanism in the GT1-7 cells exposed to Glu as opposed to a pro-survival effect in similarly treated SCN2.2 cells. Future studies should be able to take advantage of these mechanisms in developing therapeutic strategies in the treatment of neurodegenerative disorders.

Stroke is a pathological condition that causes extensive brain damage. During ischemic stroke, an excess of the excitatory neurotransmitter glutamate exerts many deleterious effects, which leads to cellular damage and cell death, a phenomenon appropriately termed excitotoxicity. Among the events triggered is the activation of the enzyme calpain, a protease whose action is dependent on the intracellular concentration of calcium, which is known to be elevated during excitotoxicity. In this thesis, I hypothesize that neuroprotective drugs can be better accumulated into stroke-affected regions by utilizing the actions of calpain. The extent of calpain activation was first investigated, and it was found to increase over time in both in vitro and in vivo models of stroke. Different amino acid sequences recognized and cleaved by calpain were then incorporated into the neuroprotective Tat-GluR2/3Y peptide. Although in vivo detection of modified Tat-GluR2/3Y peptides was unsuccessful due to technical difficulties, the accumulation of the therapeutic 3Y peptide fragments in neurons under excitotoxic conditions in vitro was found to increase with the CP-3 peptide, a peptide that is a modified version of the Tat-GluR2/3Y, with a sequence cleavable by calpain from the protein Collapsin Response Mediator Protein-3 (CRMP-3). These results suggest that it is possible to concentrate therapeutic agents into stroke-affected neurons, and this may translate into enhanced neuroprotective properties in both in vitro and in vivo animal stroke models. / Medicine, Faculty of / Graduate

The transcriptional coactivator PPARgamma coactivator 1alpha (PGC-1α) is a regulator of mitochondrial biogenesis and function and is decreased in the striatum of patients with Huntington’s Disease (HD). HD is an autosomal dominant neurological disorder caused by a polyglutamine repeat in the huntingtin protein which leads to degeneration of striatal and cortical tissues. PGC-1α undergoes targeted downregulation by mutant huntingtin protein (mtHtt) and PGC-1α knockout mice have striatal lesions similar to HD transgenic mice. Exogenous PGC-1α partially reverses the toxic effects of mutant huntingtin in cultured striatal neurons while in vivo administration of PGC-1α to the striatum in a mouse model of HD reduces neuronal volume loss. Synaptic N-methyl-D-aspartate receptor (NMDAR)- activity can drive the expression of PGC-1α which is neuroprotective against oxidative and excitotoxic stress in vitro whereas extrasynaptic NMDAR expression is increased in HD. Excessive NMDAR activity, specifically through extrasynaptic rather than synaptic NMDARs, leads to excitotoxic death in neurons and its regulation has been targeted in the search for therapeutic interventions for multiple neurological disorders. The data presented in this thesis show that the repression of PGC-1α by mtHtt may be significant in the dysregulation of NMDARs in HD. Both PGC-1α knockdown and mutant huntingtin are found to increase extrasynaptic NMDAR activity and excitotoxicity in a non-additive way, suggesting common regulatory mechanisms. Furthermore exogenous PGC- 1α expression is sufficient to reverse this increase in extrasynaptic NMDAR currents and excitotoxicity by mtHtt. This thesis adds mechanistic insight into previous understanding of the synergistic roles of mtHtt, NMDAR activity and PGC-1α in HD. Finally, we show that chronic knockout of PGC-1α in the PGC-1α(-/-) mouse causes distinct alterations in glutamatergic signaling that do not mimic the observation of acute knockdown of PGC-1α. We propose that the loss of PGC-1α in a number of neurological disorders contributes to concurrent increases in aberrant glutamate signaling and excitotoxicity in these diseases.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system, playing critical roles in basal synaptic transmission and the molecular correlates of learning and memory, long-term potentiation and long-term depression. However, glutamate is also neurotoxic during prolonged exposure and the dysfunction of the glutamatergic system has been implicated in most neurological disorders, including stroke and epilepsy, and in certain neurodegenerative diseases, including Alzheimer’s disease. In these conditions, an increased concentration of extracellular glutamate causes an over-activation of local ionotropic glutamate receptors that trigger neuronal cell death (excitotoxicity). In this study, we have used dissociated hippocampal neurons cultured on coverslips and within novel microfluidic devices to study neuronal responses, both functional and morphological, to prolonged exposure to glutamate. We find that high glutamate concentrations evoke a rapid retraction of dendritic spines, the collapse of microtubules, the formation of dendritic beads and the inhibition of basal neurotransmitter release. These responses have been identified in many neurological disorders where excitotoxicity is reported, suggesting they may be a sign of imminent cell death. However, the development of dendritic beads and the inhibition of network activity also occurs at subtoxic concentrations of glutamate and neuronal morphological changes recover rapidly post-insult. We therefore hypothesised that beading and the inhibition of neurotransmitter release may be a protective mechanism and render neurons resistant to subsequent glutamatergic insults. However, a subtoxic stimulation is not protective against a subsequent excitotoxic insult delivered immediately afterwards. However, given that neurotransmitter release can confer protection to neurons, it is possible that protection is realised, not on the neurons exposed to the subtoxic insult, but on those neurons with which they communicate, as a ‘warning’ signal. To assess the impact of a localised insult to a wider neuronal network, hippocampal neurons were cultured in novel microfluidic devices, to environmentally isolate neuronal populations, whilst preserving synaptic contacts between them. We observe that bystander naïve neurons downstream of a localised excitotoxic insult succumb to a secondary, activity-dependent, spreading toxicity. In addition, we reveal a novel mechanism by which neuronal networks also transmit a rapid and robust (albeit transient) protection from excitotoxicity. The protective phenotype acquired by neurons during this protective process requires neuronal inhibitory activity to quench overexcitation, along with the retraction of dendritic spines and/or dendritic beading. Therefore, we highlight a dichotomous role that dendritic beading plays following a direct glutamatergic insult (large beads) and as a result of GABAergic recruitment in downstream neurons (small beads). We determine that a network neuroprotective capacity exists that limits spreading toxicity, which may be recruited from a distal site even after an excitotoxic insult has occurred. Together, we may have identified a new therapeutic opportunity to limit on-going brain damage in conditions of acute neuronal injury.

Mitochondrial Ca2+ buffering is an important physiological modulator of neuronal signaling and bioenergetics, but this propensity toward Ca2+ regulation proves pathological during excitotoxic insult. Specifically, excessive mitochondrial Ca2+ uptake is a key component of glutamate toxicity within the penumbra surrounding the ischemic core following stroke. This mitochondrial toxicity and Ca2+ dyshomeostasis may be visualized in real time as delayed calcium deregulation (DCD). DCD is a predictor of neuronal, excitoxic death, and is composed of three phases: 1) an initial response; 2) a latent period of elevated, but stable cytosolic Ca2+; and 3) failure of mitochondrial Ca2+ retention, termed deregulation. The duration of the latent period is an index of neuronal resistance. Mitochondria are dynamic organelles that rapidly and reversibly undergo fission and fusion (MFF). MFF is tightly regulated by the phosphoregulation of fission inducing Drp1 at serine 656. Drp1-S656 phosphorelation is mediated by PKA/AKAP1, and it is dephosphorylated by PP2A/Bβ2. Phosphorylation of Drp1-S656 inactivates this contractile GTPase resulting in inhibition of mitochondrial fission and a shift toward elongated mitochondria. This PKA/AKAP1 dependent Drp1-S656 phosphorylation has proven to be neuroprotective. Likewise, attenuation of PP2A/Bβ2 signaling enhances neuronal survival during ischemia and excitotoxic insult. Based on the mitochondrial buffering role in excitotoxicity and MFF modulation of neuronal survival, we began investigating the role of Ca2+ buffering as a function of MFF during glutamate toxicity. Noted above, resistance to excitoticity is visualized by the duration of the DCD latent period. Overexpression of AKAP1 in cultured hippocampal neurons greatly prolonged DCD latency in a PKA dependent manner, while Bβ2 ablation prolonged DCD latency by hours. Pharmacological modulation of PKA required PDE4 inhibition to reproduce the AKAP1 observations. Preliminary experiments studying the effect of Bβ2 overexpression on matrix Ca2+ load suggests possible mechanism of MFF regulated of matrix Ca2+ accumulation. Using mtPericam DRG neurons as a model system for individual mitochondrial Ca2+ recording, we discovered impaired extrusion kinetics in mitochondria fragmented by both Drp1 and Bβ2 overexpression. Ca2+ uptake was comparable to that of control. Extreme elongation of mitochondria via dominant negative Drp1-K38A enhanced recovery. Understanding these observations, however, requires knowledge of the mitochondrial Ca2+ buffering mechanism. Mitochondrial uptake candidates include MCU and ccdc109b. Our neuronal characterization of MCU confirms a role in mitochondrial Ca2+ buffering, but not a requirement; other components must be involved. Ccdc109b remains an inconclusive candidate, but may be an important regulator of MCU. Mitochondrial efflux transporters include Letm1 and NCLX. Though Letm1 observations are hindered by control artifact, preliminary evidence supports a role in extrusion. The role of NCLX is complicated by possible tissue specificity. Functional expression experiments utilizing Na+ free Li+ external solution suggests absence of NCLX in hippocampal neurons; DRG neurons were capable of Li+ exchange. The above observations confirm the significance of mitochondrial Ca2+ extrusion in neuronal survival. Understanding the mechanisms and regulation of mitochondrial Ca2+ transport has the potential to provide novel therapeutic targets in pathologies of excitotoxic etiology.

There is evidence that the excitotoxin quinolinic acid (QUIN) synthesized through the kynurenine pathway (KP) by activated microglia may play a role in the pathogenesis of several major neuroinflammatory diseases and more particularly in Alzheimer??s disease (AD). The hypothesis of this project is QUIN affects the function and morphology of astrocytes. In this study I used human foetal astrocytes stimulated with AD associated cytokines including IFN-gamma, TNF-alpha, TGF-alpha and different concentrations of QUIN ranging from low physiological to high excitotoxic concentrations. I found that QUIN induces IL-1beta expression in human astrocytes and subsequently, contribute to the inflammatory cascade that is present in AD pathology. Glial fibrillary acid protein (GFAP) and vimentin protein expression were complementary in expression to each other after 24 hr stimulation with different QUIN doses. However, there were marked increases in GFAP levels and reduction in vimentin levels compared to controls with QUIN treatment indicating that QUIN can trigger astrogliosis in human astrocytes. Glutamine synthetase (GS) activity was used as a functional metabolic test for astrocytes and I found a dose-dependent inhibition of GS activity by QUIN. This inhibition was inversely correlated with iNOS expression whereby reduced GS activity is accompanied with an increase expression of iNOS in human astrocytes. These results suggest that reduction in GS activity can lead to accumulation of extracellular glutamate then leading to exacerbated excitotoxicity via NMDA receptor over-activation and ultimately neuronal death. PCR array results showed that at least four different pathways were activated with pathological concentration of QUIN including p38 MAPK that is associated with pro-inflammatory cytokine production, ERK/MAPK growth and differentiation that can modulate structural proteins, mitochondrial-induced apoptotic cascade and cell cycle control pathway. QUIN-induced astrogliosis and excitotoxicity could lead to glial scar formation and prevention of axonal growth thus exacerbation of neurodegeneration via synaptosomal NMDA receptor over-activation. All together, this study showed that, in the context of AD, QUIN is an important factor for astroglial activation, dysregulation and death, which can be mediated by the previously mentioned pathways.