Distinct membrane fusion events in the polarized pancreatic acinar cell involve highly specific interactions between distinct sets of SNARE proteins forming exocytotic complexes, whose assembly is modulated by distinct Munc18 proteins. The Munc18 isoform responsible for these exocytotic events in the acinar cell is unknown. Here, I postulate Munc18b to regulate apical exocytosis in the acinar cell. Current dogma for the pathogenesis of acute pancreatitis, including alcoholic pancreatitis, is mis-targeting and deregulated fusion of zymogen granules with lysosomal bodies in the acinar cells. This derangement results in premature activation of proteolytic zymogens and autophagic digestion of cellular contents. I have hypothesized an alternate mechanism, which is pathologic exocytosis occurring at the basolateral plasma membrane, and further propose Munc18c to mediate this process in alcoholic pancreatitis. The aims of this thesis are to demonstrate the roles of Munc18b and Munc18c in regulated apical exocytosis and pathologic basolateral exocytosis underling alcoholic pancreatitis, respectively. In Chapter Three, using both real-time and static imaging techniques and biochemical tools, I demonstrated that Munc18c is dissociated from the acinar basal plasma membrane (BPM) when stimulated with postprandial CCK8 preceding preincubation of acini with postprandial 20-50mM ethanol concentrations. This activated Syntaxin (Syn)-4 and SNAP-23 on the BPM to complex with VAMP proteins on the granule to form the exocytotic SNARE complex that triggered basolateral exocytosis. This molecular mechanism of pathologic basolateral exocytosis was recapitulated in an Ethanol-diet rat model of pancreatitis. In Chapter Four, I determined Munc18b to be in the apical pole of the acinar cell to appropriately bind cognate Syn-2 and Syn-3 in the apical PM and ZGs. Here, I examined the structure-function of Munc18b on amylase secretion by employing Munc18b mutants with distinct affinities to Syn-2 and Syn-3. In Chapter Five, I discovered a novel EF-hand Ca2+-binding protein called Cab45b, which binds Munc18b to regulate its membrane targeting and interactions with Syntaxins in the acinar cell in a manner that influenced Ca2+-induced amylase release. Taken together, these studies clarify our understanding of the role of Munc18 proteins involved in regulated and pathologic membrane fusion events underlying physiologic digestive enzyme secretion and clinical alcoholic pancreatitis.

The inter-relation between inflammation, the immune system and leukocytes is multifaceted, with communication between stroma and immune cells mediated by cytokines, growth factors, chemokines, integrins and other molecules. Proteolysis plays an important role in regulating these molecules. Proteolytic cleavage can not only destroy some molecules but can activate or shed others, converting local juxtacrine signalling proteins into effectors that act at a distance. Shedding can also convert membrane-bound receptors into soluble ligand-binding inhibitors. Finally, cleavage can convert agonist molecules into antagonists. As a wide-ranging inhibitor of metalloproteinases, tissue inhibitor of metalloproteinase 3 (TIMP3) has the potential to down-regulate many of these activities. We explore the role of TIMP3 in the regulation of inflammation, revealing that loss of TIMP3 leads to a more rapid increase of soluble TNF, higher levels of soluble TNF receptors and ultimately to increased TNF signalling in systemic inflammation. We also demonstrate TIMP3 loss impacts local inflammation. In addition we investigate the importance of TIMP3 in the expansion of hematopoietic cells.

Mood and cognition, along with numerous other physiological processes, are under circadian regulation. The synthesis and secretion rhythm of the pineal hormone melatonin is under the direct regulation of the central circadian pacemaker and the secretion rhythm of melatonin can be used to assess circadian alterations. In this thesis, it was demonstrated that low levels of endogenous nocturnal melatonin was associated with subsyndromal depression and alterations in sleep architecture. Studies in individuals with endogenous circadian rhythm disorder, with and without comorbid depressive symptoms, revealed that individuals with depressive symptoms had a greater phase delay in melatonin profiles as compared to individuals without depressive symptoms. Furthermore, in the same study, exogenous melatonin administered to induce phase advances significantly improved depression scores and sleep initiation. In addition to endogenous circadian disruption, circadian rhythms can also be disrupted by repeated atypical alterations in environmental time cues. In mammals, light is the strongest environmental cue that can modulate circadian rhythms. Recent studies suggest that circadian response to photic stimuli is preferentially sensitive to short wavelengths in the range of 450-480 nm. Using an animal model it was demonstrated that filtering a 10 nm bandwidth between 470-480 nm from polychromatic white light prevents nocturnal light exposure induced disruptions in melatonin and corticosterone secretion as well as central and peripheral clock gene expression. These findings were further investigated in humans and revealed that filtering short wavelengths below 480 nm attenuates 12 h nocturnal light exposure induced suppression of melatonin secretion, increased cortisol secretion and disrupted peripheral clock gene expression. Furthermore, attenuation of these changes was associated with improvements in mood, alertness and vigilance at a time close to the endogenous circadian wake drive. However, filtering short wavelengths below 460 nm or reducing the optical transmission by up to 30% below 480 nm did not attenuate the disruptive effects of nocturnal light exposure on physiological and behavioural variables. Overall, the results presented in this thesis support the role of circadian dysfunction in neuropsychometric impairment and presents evidence supporting spectral modulation as a promising approach to attenuate light-mediated chronodisruption.

Zinc levels in pancreatic islets are amongst the highest in the body and reduction in its levels in the pancreas has been associated with diabetes. The link between zinc, diabetes and islet dysfunction has recently been reiterated by genome-wide association studies that identified an islet cell membrane zinc transporter, SLC30A8 (ZnT8), as one of the risk loci for type 2 diabetes. Here we begin to elucidate the molecular mechanisms linking ZnT8 and type 2 diabetes by characterizing global and beta cell specific ZnT8 knockout mice. Our results associate absence of ZnT8 with a reduction in zinc sequestration into insulin vesicles, abnormal insulin granule morphology, down regulation of insulin processing enzymes, abnormal insulin secretion, elevated plasma proinsulin levels and diet-induced obesity and insulin resistance. Furthermore, we observed differential zinc uptake properties by two human ZnT8 variants. We report here that the W325 variant of ZnT8 is more efficient in mediating zinc transport than the at risk variant, R325. Cumulatively, these results suggest that ZnT8 is crucially important for zinc transport and zinc-insulin crystallization in insulin granules of the pancreatic beta cell.

Irx5 is a homeodomain transcription factor that negatively regulates cardiac fast transient outward K+ currents (Ito,f) via the KV4.2 gene and is thereby a major determinant of the transmural repolarization gradient. While Ito,f is invariably reduced in heart disease and changes in Ito,f can modulate both cardiac contractility and hypertrophy, less is known about a functional role of Irx5, and its relationship with Ito,f, in the normal and diseased heart. Here I show that Irx5 plays crucial roles in the regulation of cardiac contractility and proper adaptive hypertrophy. Specifically, Irx5-deficient (Irx5-/-) hearts had reduced cardiac contractility and lacked the normal regional difference in excitation-contraction with decreased action potential duration, Ca2+ transients and myocyte shortening in sub-endocardial, but not sub-epicardial, myocytes. In addition, Irx5-/- mice showed less cardiac hypertrophy, but increased interstitial fibrosis and greater contractility impairment following pressure overload. A defect in hypertrophic responses in Irx5-/- myocardium was confirmed in cultured neonatal mouse ventricular myocytes, exposed to norepinephrine while being restored with Irx5 replacement. Interestingly, studies using mice virtually lacking Ito,f (i.e. KV4.2-deficient) showed that reduced contractility in Irx5-/- mice was completely restored by loss of KV4.2, whereas hypertrophic responses to pressure-overload in hearts remained impaired when both Irx5 and Ito,f were absent. These findings suggest that Irx5 regulates cardiac contractility in an Ito,f-dependent manner while affecting hypertrophy independent of Ito,f. On the other hand, Irx5-ablation attenuated calcineurin (Cn)-induced hypertrophy in hearts and cultured cardiomyocytes, suggesting that the effect of Irx5 on hypertrophy involves the Cn-NFAT signalling cascade. Biochemical assessments further revealed that Irx5 can positively mediate Cn-NFAT activities as well as Nfatc3 and Gata4 expression, and interacts with Nfatc3 and Gata4, suggesting the formation of a transcription complex for hypertrophic gene regulation. Taken together, these studies have identified Irx5 as a vital cardiac transcription factor, important for contractile function of the heart by regulating Ito,f, and compensatory hypertrophic response to biomechanical stress in the heart by affecting the Cn-NFAT (and Gata4) signaling pathway.

Mechanisms to augment the cellular function and mass of beta-cells may be effective means of treating type 2 diabetes. Important in the physiological control of beta-cell function and nutrient disposal are factors released from gut enteroendocrine cells during nutrient digestion. In enteroendocrine L-cells, post-translational processing of proglucagon gives rise to a number of proglucagon-derived peptides. One such peptide, glucagon-like peptide-1 (GLP-1), acts via its own receptor (GLP-1R) to stimulate beta-cell insulin secretion, proliferation and survival. Another, oxyntomodulin (OXM), weakly activates the GLP-1R and inhibits food intake in a GLP-1R-dependent manner in rodents, which led us to hypothesize that OXM modulates GLP-1R-dependent glucoregulation. While OXM did not mimic the inhibitory effect of GLP-1 on gastric emptying in mice, OXM stimulated insulin secretion, beta-cell survival and improved glucose tolerance in a GLP-1R-dependent manner. In a similar manner to GLP-1, glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine K-cells, physiologically stimulates insulin secretion via a distinct GIP receptor (GIPR) in beta-cells. Beyond the beta-cell, GIP and GLP-1 appear to exert divergent actions for the control of glucose homeostasis. Moreover, I illustrate that physiological and pharmacological GLP-1R signalling may be comparatively more important for the preservation of beta-cell mass and glucose homeostasis in murine streptozotocin-induced diabetes. Lastly, studies in rodents and humans have showed that metformin increases circulating levels of GLP-1, leading us to hypothesize that GIP and GLP-1 may be involved in the glucoregulatory effects of metformin. Interestingly, transcripts for the Glp1r and Gipr were significantly increased within islets of metformin-treated mice, and metformin treatment enhanced the sensitivity of cultured beta-cells to GIP and GLP-1. In summary, these studies illustrate mechanisms by which enteroendocrine peptides compare and contrast with respect to beta-cell survival and function and the control of glucose homeostasis.

During pregnancy, 64-96% of women take at least one prescription drug. The placenta is the primary barrier between substrates in maternal and fetal circulation. The blood-brain barrier (BBB) acts as an additional barrier for the fetal brain, which is particularly susceptible to the effects of xenobiotics. Multidrug resistance phosphoglycoprotein (P-gp; encoded by Abcb1 mRNA) and breast cancer resistance protein (Bcrp1; encoded by Abcg2 mRNA) are efflux transporters localized on placental syncytiotrophoblast and capillary endothelial cells of the BBB. Placental Abcb1/P-gp and Abcg2/Bcrp1 limit maternal-fetal transfer of endogenous and exogenous substrates. Similarly, the neuroprotective roles of Abcb1/P-gp and Abcg2/Bcrp1 in the adult BBB have been demonstrated. However, developmental changes in expression and function and regulation of Abcb1/P-gp and Abcg2/Bcrp1 in these tissues are poorly understood. This thesis investigates gestational changes in expression and function of Abcb1/P-gp and Abcg2/Bcrp1 in the placenta and fetal brain, in addition to regulation by steroids, progesterone and glucocorticoids. The effects of glucocorticoids on Abcb1/P-gp and Abcg2/Bcrp1 in the placenta and fetal brain are of importance given that 10% of pregnant women are treated with synthetic glucocorticoids during the management of threatened preterm labour. These studies demonstrate that the decrease in placental Abcb1/P-gp mediated fetal protection near term is compensated by an increase in Abcb1/P-gp and Abcg2/Bcrp1 mediated neuroprotection in the fetal brain; likely in preparation for life ex-utero. The lack of effects of progesterone and the dose-, age- and sex- dependent regulatory effects of synthetic glucocorticoid have highlighted the complexity associated with regulation of these transporters. Further, these studies are the first to report sexually dimorphic glucocorticoid effects on Abcb1/P-gp and Abcg2/Bcrp1 expression and function, with the female fetus being particularly susceptible to glucocorticoid these effects. In this regard, Abcb1/P-gp and Abcg2/Bcrp1 transport capacity may be altered when synthetic glucocorticoid is administered as a co-therapy, and as such, recipient sex should be considered during pharmacotherapy. Understanding the regulation of Abcb1/P-gp and Abcg2/Bcrp1 expression and function in the placenta and fetal brain during normal development and under pathological conditions is critical for fetal health and development, particularly when therapeutic strategies are utilized in pregnancy.

Slow population activities (SPAs) are population activities in the brain with frequencies of less than 5 Hz. SPAs are prominent in many brain structures including the neocortex and the hippocampus. Examples of SPAs include the neocortical EEG δ waves and the hippocampal large amplitude irregular activities during NREM sleep. These in vivo SPAs are believed to play a fundamental role in brain plasticity. However, despite many experimental attempts to understand SPAs, their mechanisms are still not well understood. It is unclear how the individual neurons can sustain low frequency activities on the network as a whole. In this thesis, we demonstrate that a mathematical and computational perspective is indispensable in understanding slow population phenomena and generating testable hypotheses for future experiments. Our focus is on a hippocampal slice preparation exhibiting spontaneous, inhibitory-based SPAs (hippocampal SPAs). We develop a multi-pronged approach consisting of parameter extraction, simulation, and mathematical analysis to elucidate the mechanisms responsible for hippocampal SPAs. Our results suggest that hippocampal SPAs are an emergent phenomenon. In other words, the network “slowness” is not directly represented by any particular individual element within the network. Instead, the low frequency activities on the network are the result of interactions between synaptic and intrinsic characteristics of individual inhibitory interneurons. Our simulations quantify these characteristics which underlie hippocampal SPAs. Specifically, our simulations predict that individual interneurons should 1) be moderately fast-spiking above threshold before the increase in spike frequency slows down with increasing drive, and 2) be well connected with one another for SPAs to occur. We also predict that excitatory noise levels have a larger influence on hippocampal SPAs than mean excitatory drive. Subsequent mathematical analyses show that the synaptic and intrinsic conditions of individual interneurons as predicted by simulations promote network multi-stability. Hippocampal SPAs occur when the network switches from one network firing state to another. Since many of the parameters we use for simulations are extracted from experiments, our simulation model is likely a reasonable representation of actual biological mechanisms in hippocampal networks.

Autosomal-dominant mutations in the amyloid precursor protein (APP) gene increase the production and aggregation of toxic amyloid-β (Aβ) peptides and cause early-onset Alzheimer’s disease (AD). Noradrenergic cell loss is well documented in AD and has been posited to play a role in cognitive symptoms as well as disease progression. We investigated memory and affect, tissue levels of catecholamines, brain-derived neurotrophic factor (BDNF) mRNA and bioenergetic homeostasis in TgCRND8 mice that express a double mutant (K670N/M671L + V717F) human APP695 transgene. We found that TgCRND8 mice develop object memory impairment and behavioural despair, as well as reductions in noradrenaline and BDNF expression in the hippocampus and cortex, before the appearance of Aβ plaques. Animals with more advanced Aβ pathology exhibit disruptions in energetic status, along with diminished complex I+III activity in the electron transport chain. To test whether the AD-like phenotypes of TgCRND8 mice might be due to altered noradrenergic tone, pre-plaque mice were treated with dexefaroxan, an antagonist of presynaptic inhibitory α2-adrenoceptors that are highly expressed on both noradrenergic and cholinergic terminals. Effects of dexefaroxan were compared to those of rivastigmine, a cholinesterase inhibitor. Both dexefaroxan and rivastigmine improved behavioural phenotypes and BDNF expression without affecting tissue Aβ load. Drug treatments also restored complex I+III mitochondrial activity and increased ATP levels. Reductions in noradrenergic tone appear to underlie Aβ-induced functional impairment in TgCRND8 mice, in addition to BDNF deficits and bioenergetic stress. These studies suggest that α2-adrenoceptor targeting may warrant consideration as a therapeutic strategy in AD.

Autosomal-dominant mutations in the amyloid precursor protein (APP) gene increase the production and aggregation of toxic amyloid-β (Aβ) peptides and cause early-onset Alzheimer’s disease (AD). Noradrenergic cell loss is well documented in AD and has been posited to play a role in cognitive symptoms as well as disease progression. We investigated memory and affect, tissue levels of catecholamines, brain-derived neurotrophic factor (BDNF) mRNA and bioenergetic homeostasis in TgCRND8 mice that express a double mutant (K670N/M671L + V717F) human APP695 transgene. We found that TgCRND8 mice develop object memory impairment and behavioural despair, as well as reductions in noradrenaline and BDNF expression in the hippocampus and cortex, before the appearance of Aβ plaques. Animals with more advanced Aβ pathology exhibit disruptions in energetic status, along with diminished complex I+III activity in the electron transport chain. To test whether the AD-like phenotypes of TgCRND8 mice might be due to altered noradrenergic tone, pre-plaque mice were treated with dexefaroxan, an antagonist of presynaptic inhibitory α2-adrenoceptors that are highly expressed on both noradrenergic and cholinergic terminals. Effects of dexefaroxan were compared to those of rivastigmine, a cholinesterase inhibitor. Both dexefaroxan and rivastigmine improved behavioural phenotypes and BDNF expression without affecting tissue Aβ load. Drug treatments also restored complex I+III mitochondrial activity and increased ATP levels. Reductions in noradrenergic tone appear to underlie Aβ-induced functional impairment in TgCRND8 mice, in addition to BDNF deficits and bioenergetic stress. These studies suggest that α2-adrenoceptor targeting may warrant consideration as a therapeutic strategy in AD.