Neuronal regeneration in the mammalian central nervous system (CNS) is severely compromised due to the presence of extrinsic inhibitory signals and a reduced intrinsic regenerative capacity. Understanding the cellular and molecular processes underlying injury and regeneration in the CNS is necessary for the development of effective therapeutic strategies. Lymnaea stagnalis, a freshwater pond snail, has proven to be a powerful model for studying the fundamental mechanisms underlying neurite outgrowth and regeneration. In this study I designed the first custom L. stagnalis microarray gene chip and carried out microarray analysis to profile gene expression changes following CNS injury. From a pool of significantly regulated genes, I provided the first evidence that C/EBP, a transcription factor, plays an integral role in regeneration by maintaining the viability of the distal neurite. We also proposed a novel signaling network and demonstrated that BCL 7 regulates neurite regeneration, an effect that may be mediated through Ca2+-dependent growth cone formation.

Sirtuins are a family of nicotinamide adenine dinucleotide - dependent enzymes, which have gained recent interest due to their ability to directly or indirectly regulate cell metabolism, oxidative response mechanisms and cellular senescence. A mitochondrial sirtuin SIRT3, although still relatively under-investigated, regulates mitochondrial processes through deacetylation of metabolic enzymes and components of electron transport chain. We hypothesized that SIRT3 is a mitochondrial cytoprotective factor that exerts its function by decreasing reactive oxygen species levels, and protecting cells from oxidative stress. HEK-293 cells over-expressing SIRT3 exhibit reduced mitochondrial membrane potential and reactive oxygen species levels under basal conditions. In addition, cells over-expressing SIRT3 are less sensitive to hydrogen peroxide and glucose deprivation/glucose reperfusion induced-cell death. Since SIRT3 expression in the brain has not yet been investigated, its expression pattern in the rodent brain was characterized. Our results showed that SIRT3 mRNA and protein levels are robustly expressed in different regions of the adult rodent brain and their expression increases with age. Furthermore, SIRT3 is expressed predominantly in astrocytes in cultures derived from rat primary E18 cortical cells. These results suggest that SIRT3 possesses cytoprotective potential, and that its actions in the brain regulate astrocyte physiology.

Previous studies have shown that NACHT, leucine rich repeat and PYD domain containing 5 (Nlrp5) deficient embryos fail to develop beyond the two-cell stage. Despite this strong phenotype, little is known of the function of NALP5 and the pathways affected by its deficiency. We showed that Nlrp5 deficient oocytes and embryos exhibit a decrease in caspase activity. In addition, the kinetics of NF-κB-p65 nuclear translocation is altered, which leads to negative downstream effects. Autophagy is known to be regulated downstream of NF-κB and is a key event during the oocyte-to-embryo transition. We observed defective execution of autophagy in Nlrp5 deficient two-cells evidenced by absence of autophagosome formation and abnormal lysosomal maturation. We found that inactivating autophagy leads to an accumulation of lipid droplets and embryos lacking Nlrp5 exhibit this accumulation. Thus, NALP5 may be an integral component responsible for autophagy mediated lipid metabolism, which when compromised causes developmental arrest.

Neuronal regeneration in the mammalian central nervous system (CNS) is severely compromised due to the presence of extrinsic inhibitory signals and a reduced intrinsic regenerative capacity. Understanding the cellular and molecular processes underlying injury and regeneration in the CNS is necessary for the development of effective therapeutic strategies. Lymnaea stagnalis, a freshwater pond snail, has proven to be a powerful model for studying the fundamental mechanisms underlying neurite outgrowth and regeneration. In this study I designed the first custom L. stagnalis microarray gene chip and carried out microarray analysis to profile gene expression changes following CNS injury. From a pool of significantly regulated genes, I provided the first evidence that C/EBP, a transcription factor, plays an integral role in regeneration by maintaining the viability of the distal neurite. We also proposed a novel signaling network and demonstrated that BCL 7 regulates neurite regeneration, an effect that may be mediated through Ca2+-dependent growth cone formation.

Sirtuins are a family of nicotinamide adenine dinucleotide - dependent enzymes, which have gained recent interest due to their ability to directly or indirectly regulate cell metabolism, oxidative response mechanisms and cellular senescence. A mitochondrial sirtuin SIRT3, although still relatively under-investigated, regulates mitochondrial processes through deacetylation of metabolic enzymes and components of electron transport chain. We hypothesized that SIRT3 is a mitochondrial cytoprotective factor that exerts its function by decreasing reactive oxygen species levels, and protecting cells from oxidative stress. HEK-293 cells over-expressing SIRT3 exhibit reduced mitochondrial membrane potential and reactive oxygen species levels under basal conditions. In addition, cells over-expressing SIRT3 are less sensitive to hydrogen peroxide and glucose deprivation/glucose reperfusion induced-cell death. Since SIRT3 expression in the brain has not yet been investigated, its expression pattern in the rodent brain was characterized. Our results showed that SIRT3 mRNA and protein levels are robustly expressed in different regions of the adult rodent brain and their expression increases with age. Furthermore, SIRT3 is expressed predominantly in astrocytes in cultures derived from rat primary E18 cortical cells. These results suggest that SIRT3 possesses cytoprotective potential, and that its actions in the brain regulate astrocyte physiology.

Previous studies have shown that NACHT, leucine rich repeat and PYD domain containing 5 (Nlrp5) deficient embryos fail to develop beyond the two-cell stage. Despite this strong phenotype, little is known of the function of NALP5 and the pathways affected by its deficiency. We showed that Nlrp5 deficient oocytes and embryos exhibit a decrease in caspase activity. In addition, the kinetics of NF-κB-p65 nuclear translocation is altered, which leads to negative downstream effects. Autophagy is known to be regulated downstream of NF-κB and is a key event during the oocyte-to-embryo transition. We observed defective execution of autophagy in Nlrp5 deficient two-cells evidenced by absence of autophagosome formation and abnormal lysosomal maturation. We found that inactivating autophagy leads to an accumulation of lipid droplets and embryos lacking Nlrp5 exhibit this accumulation. Thus, NALP5 may be an integral component responsible for autophagy mediated lipid metabolism, which when compromised causes developmental arrest.

Glucagon-like peptide-2 (GLP-2) is an intestinal hormone that promotes gut growth through an insulin-like growth factor (IGF)-1 and intestinal epithelial (IE)-IGF-1 receptor (R)-dependent pathway. GLP-2 also promotes epithelial barrier function by as yet unknown mechanisms. I hypothesized that GLP-2-mediated effects on barrier function requires the IE-IGF-1R. Chronic GLP-2 treatment enhanced barrier function by decreasing gastrointestinal permeability in vivo and increasing jejunal resistance ex vivo. These responses were abolished in inducible IE-IGF-1R knockout (KO) animals. Additionally, epithelial sealing tight junctional proteins claudin-3 and -7 were upregulated by GLP-2 in control but not KO mice. Moreover, IE-IGF-1R deletion induced a shift in occludin localization from apical to intracellular domains. In contrast, in irinotecan-induced enteritis, GLP-2 normalized epithelial barrier function in control animals, but continued to be ineffective in KO mice. Collectively, the effects of GLP-2 on barrier function are dependent on the IE-IGF-1R and involve modulation of the tight junctional complex.

Multidrug resistance phosphoglycoprotein (P-gp) is expressed in the placenta and fetal blood-brain barrier (BBB) and plays a critical role in reducing fetal accumulation of xenobiotics. In other tissues, P-gp activity is inhibited by selective serotonin reuptake inhibitors (SSRIs) and by lethal doses of LPS modeling a bacterial infection. However, nothing is known with respect to the effects of SSRIs or nonlethal infection on P-gp activity in the placenta or fetal tissues. In the studies presented in this thesis, we hypothesized that (1) the SSRI sertraline and (2) a nonlethal maternal bacterial infection would decrease P-gp activity in the placenta and fetal BBB. The first study shows that sertraline affects P-gp activity at these barrier sites in a tissue-specific manner. The second study shows that nonlethal infection does not significantly affect P-gp activity at either site. However, nonlethal infection may still influence substrate biodistribution by altering hepatic elimination of these substrates.

SIRT1 has emerged as a critical regulator of glucose homeostasis and metabolism in the past decade. Glucose homeostasis is tightly regulated by insulin however, the factors affecting insulin release are still incompletely understood. Relatively recent evidence has shown SIRT1 to be a positive mediator of insulin secretion although its mechanism is largely unknown. Therefore, the aim of this study was to determine how SIRT1 regulates insulin release. Using a pancreatic beta cell-specific Sirt1 knockout mouse model (Sirt1BKO), oral glucose challenge revealed a glucose intolerant phenotype with reduced insulin secretion. Isolated Sirt1BKO islets also secreted less insulin without changes to insulin content or islet morphology. Intracellular defects were localized to the mitochondria and showed suppressed bioenergetics negatively affecting downstream glucose-induced calcium influx. This is the first study using a Sirt1BKO mouse model to show novel mitochondrial genes under SIRT1 regulation and when impaired, results in reduced insulin secretion.

SIRT1 has emerged as a critical regulator of glucose homeostasis and metabolism in the past decade. Glucose homeostasis is tightly regulated by insulin however, the factors affecting insulin release are still incompletely understood. Relatively recent evidence has shown SIRT1 to be a positive mediator of insulin secretion although its mechanism is largely unknown. Therefore, the aim of this study was to determine how SIRT1 regulates insulin release. Using a pancreatic beta cell-specific Sirt1 knockout mouse model (Sirt1BKO), oral glucose challenge revealed a glucose intolerant phenotype with reduced insulin secretion. Isolated Sirt1BKO islets also secreted less insulin without changes to insulin content or islet morphology. Intracellular defects were localized to the mitochondria and showed suppressed bioenergetics negatively affecting downstream glucose-induced calcium influx. This is the first study using a Sirt1BKO mouse model to show novel mitochondrial genes under SIRT1 regulation and when impaired, results in reduced insulin secretion.