Stem cells are defined by their multipotentiality and their long-term ability to self-renew. P107, a member of the pocket protein family of cell cycle regulators has previously been shown in our laboratory to negatively regulate neural precursor cell number and self-renewal (Vanderluit et al., 2004). In this study, we investigated the mechanism by which p107 regulates the neural precursor pool by examining interactions between p107 and the Notch pathway, which has also been shown to regulate the neural stem cell population (Nakamura et al., 2000; Ohtsuka et al., 2001; Hitoshi et al., 2002b). We found an increase in both the transcript and protein levels of Hes1 in p107-/- brains using in situ hybridization and western blot analysis. Examination of the Hes1 promoter revealed three putative E2F binding sites, which were subsequently found to bind E2F3 and E2F4 using chromatin immunoprecipitation. P107 was found to significantly repress Hes1 promoter activity in the luciferase reporter assay, and finally, using the primary neurosphere assay we showed that removal of Hes1 from p107-/- neurospheres restores the number of neurosphere forming cells to wildtype levels. Our results suggest that p107 represses Hes1 transcription through E2F, and demonstrate that an upregulation of Hes1 is responsible for the increased neural precursor pool in p107-/- mice.

Potassium is the most abundant ion species in the mammalian central nervous system. Under normal physiological conditions, most K+ in the brain tissue resides within the cytoplasm of neurons, glia, and axons. However, brain injuries such as trauma or ischemia may cause large amounts of K + to leak out. This sudden increase of K+ in the interstitial space may have severe pathological consequences. Animal models suitable for studying K+-mediated tissue injury are uncommon in the literature. Therefore, the main objectives of this thesis were: (1) to develop an animal model of K+-induced tissue injury and (2) to examine the mechanism and cellular events that are potentially responsible for the observed tissue injury. We chose to study the dentate hilus (DH) of the hippocampus because previous studies have shown that K+ homeostasis in this region is highly sensitive to traumatic brain injury. Approximately 150 adult male rats were used throughout this study. Under anesthesia, a small volume of K+ solution was injected into either the left or right hippocampal hilus. This was followed by an injection of the vehicle solution into the corresponding DH region of the contralateral hippocampus. The animals were then sacrificed at different time points post-K + or drug injections. Tissue sections were collected for immunofluorescent processing and quantification. The main findings are the following: (1) Microinfusion of low concentrations of K+ into the DH produced, within minutes, a large tissue cavity along the subgranular zone (SGZ). The SGZ is mainly composed of the initial segments of mossy fibers from granular cells of the dentate gyrus, interneurons of the hilus and astrocytes. (2) The occurrence of the tissue split was accompanied by rapid injury and destruction of glial cells. There was no detectable death of granular neurons. (3) The K +-induced tissue split can be reliably reproduced by the injection of the Na+,K+-pump inhibitor ouabain, which led to a rapid, endogenous increase of K+ in the extracellular space (ECS). However, injections of equal volume of NaCl, CaCl2 or distilled water did not induce tissue split in the DH. (4) In the presence of K+ channel blockers, both ouabain and exogenously applied K+ were no longer effective in producing the tissue split. Based on the above results, it is concluded that the tissue integrity of hippocampal dentate hilus is extremely vulnerable to high external K +. Glial cell death seems to play an important and early role in the occurrence of K+-induced tissue damage. Since traumatic brain damage as well as temporal lobe epilepsy exhibits similar pathophysiological features to K+-induced tissue injury, we propose that the latter can be used as a useful animal model for future mechanistic and therapeutic investigations.

The midline thalamic paraventricular nucleus (PVT) receives a unique orexinergic innervation. To address possible function, this investigation used patch clamp recordings in rat brain slice preparations to evaluate intrinsic properties of PVT neurons and neuronal responses to bath-applied orexin peptides (A and/or B). PVT neurons displayed distinct state-dependent burst or tonic firing patterns, time dependent and time-independent inward rectification, T-type currents and low threshold spikes, action potential after-hyperpolarizations, spike frequency adaptation and spike broadening. A majority responded to both orexin peptides with slowly rising and prolonged membrane depolarizations, and inward currents that involved closure of potassium channels and/or opening of nonselective cationic channels. These data imply that endogenously released orexins likely act at both types of orexin receptors that can engage two conductances to modulate and increase neuronal excitability in PVT, a role that may be important for 'arousal' and neurotransmission within this midline thalamic nucleus.

Atrial natriuretic factor (ANF) is a hormone that helps maintain fluid homeostasis and has many other physiological roles. Understanding of its regulation may have immense impacts in the treatment and understanding of cardiac diseases. Despite the recent and continuing unravelling of signalling cascades, the intracellular signalling governing ANF secretion from atrial cardiocytes remains mostly unknown. Following recent evidence of the involvement of G proteins in modulating ANF secretion, the role of Gq effector phospholipase C (PLC) and its proximal effectors was investigated in spontaneously beating rat atria. Phospholipase C and protein kinase C inhibitors dramatically increased basal secretion of ANF. Furthermore, although stretch is a potent stimulus for secretion, these inhibitor-mediated increases fell to baseline levels when the stretch of the atria was subsequently introduced. Inositol trisphosphate receptor inhibition did not appear to affect basal secretion but dose-dependently blocked stretch secretion coupling. These results reveal interesting novel phenomena and demonstrate key participation of the PLC cascade in the regulation of ANF secretion.

The activities of two major components involved in drug metabolism, P-glycoprotein (P-gp) and members of the cytochrome P450 oxidase (CYP) family were studied. This investigation specifically examined the influence of polymorphisms within the ABCB1 gene on P-gp efflux activity and expression. Additionally, the role of natural health products (NHPs) on the activity of both P-gp and members of the CYP3A subfamily was also studied. Findings from this study suggest that the haplotype relationship of C3435T, G2677A/T and C1236T within the ABCB1 gene does not influence P-gp efflux activity or expression. Furthermore, some commonly consumed NHPs, including trans-beta-carotene, Arctostaphylos uva-ursi L. (Kinnikinnick), Acorus americanus Raf. and Acorus calamus L. (Acorus), Curcuma longa L. (turmeric powder), Capsicum annuum L. (chilli powder) and Piper nigrum L. (black pepper), are able to influence the activity of P-gp and members of the CYP3A subfamily in vitro. The results suggest that in order for conventional drug therapy to he effective, the genotype of an individual in relation to drug metabolism, as well as the influence of concomitantly consumed NHPs, should be considered in determining how the bioavailability of therapeutic drugs are influenced.

Cortical spreading depression (CSD) induces waves of neuronal depolarization that confer neuroprotection to subsequent ischemic events in the rat brain. To gain insights into the molecular mechanisms elicited by CSD, we used representational difference analysis (RDA) to identify mRNA induced by potassium depolarization in vivo. We have isolated a cDNA encoding a novel bHLH-PAS protein distantly related to SIM2, termed HALO. Our results confirm that HALO mRNA and protein are rapidly and transiently expressed in cortical neurons following CSD but not following short duration ischemia, another form of pre-ischemic conditioning. In the untreated adult brain, HALO is expressed at low levels but is highly expressed during embryonic development in neuronal lineages. Surprisingly, delayed HALO expression is also observed following middle cerebral artery occlusion (MCAO) in rats. Reporter assays show that HALO is a transcriptional activator that associates with the bHLH-PAS sub-class co-factor ARNT2. Adenovirus-mediated expression of epitope-tagged HALO results in the direct induction of the Bax gene and sensitization of cultured cells to cytotoxic stress. Together, our data indicate that HALO is a novel bHLH-PAS transactivator transiently induced by preconditioning and that its sustained expression is detrimental. The identification of HALO may represent an important step in our understanding of the molecular mechanisms of brain preconditioning and injury.

Previous work in our lab identified a novel gene encoding a unique family of tail-anchored alpha-helical coiled-coil proteins termed SLMAPs (sarcolemmal membrane associated proteins) that were highly conserved in rodents and humans. Alternative splicing yields three SLMAP isoforms: a ubiquitously expressed isoform (SLMAP3), and two tissue-specific variants (SLMAP1 and SLMAP2). The salient features of SLMAP are a hydrophobic C-terminal sequence, which targets membranes, a central coiled-coil structure and N-terminal fork head associated domain (FHA). A sequence in fruit flies (corresponding to CG17494 in flybase) was identified that shares 28.6% identity with the full-length mammalian SLMAP3 amino acid sequence. However, a 55.9% identity was identified at the fork head associated domain of Drosophila and SLMAP3 isoform, whereas a 25% identity was shared at the C-terminal transmembrane domain. The rest of the protein was predicted to form a coiled-coil structure. In view of these similarities with the mammalian SLMAP3 isoform, this Drosophila gene was designated Dslmap. In situ hybridization on whole mount embryos revealed a ubiquitous expression of Dslmap mRNA throughout various stages of embryonic development. To study the function of Dslmap during embryonic development, a GAL4-driven hairpin-induced RNA interference (RNAi), as well as overexpression experiments were performed. The Gal4/UAS binary system was used to allow hairpin RNA to conditionally silence Dslmap expression in Drosophila. A pan-neuronal driver (Elav-Gal4) that expresses GAL4 in every cell of the nervous system from embryogenesis onward was mated to transgenic flies carrying the activatable Dslmap RNAi vectors. The resulting phenotypes revealed dramatic defects in the development of axon scaffold in the fly central nervous system (CNS) with particular structural defects within the longitudinal connectives and commissures. An overexpression construct was also made using pUAST vector carrying the full length Dslmap cDNA in frame with a yeast upstream activator sequence (UAS) to generate transgenic lines. Overexpression in the transgenic lines was activated by mating with the CNS Gal4 lines, which resulted in phenotypes that revealed similar defects in the CNS to those observed in the RNAi lines. These data revealed that the Drosophila genome contains a SLMAP homologue, which is ubiquitously expressed and plays a critical role in developing neurons and glia.

Abnormal sympathetic nervous system (SNS) signaling is a synergistic complication of obesity, diabetes mellitus, and congestive heart failure. In vivo biodistribution studies in rats using [11C] meta-hydroxyephedrine ([11C]HED) were performed in obese, lean, type I and type II diabetic animals to delineate deviations in sympathetic nervous integrity at the uptake-1 site (NET-1) as compared to healthy controls. Specific, blockable tracer accumulation was observed in myocardium, lung, brown adipose tissue and pancreas. Obese animals exhibit a time-dependent elevation in uptake-1 specific myocardial [11C]HED retention and time-independent depressed tracer accumulation in brown adipose tissue as compared to lean animals. Type II diabetic rats show a time- and hyperglycaemia-dependent reduction of myocardial tracer uptake and a time- and glycaemia-independent increase in brown adipose tissue uptake-1 specific [11C]HED retention. Positron emission tomography using [11C]HED may prove useful in examining alterations in SNS signaling before, during, and subsequent to therapy for obesity and/or DM.

Zebrafish have the capability to regenerate their fins after injury. Previous studies from our laboratory showed that fin regeneration triggers the re-expression of genes involved in the hedgehog (hh) signaling pathway. One member of the hh family, sonic hedgehog (shh) was suggested to regulate bone patterning based on its expgression pattern and functional analysis. Another member of the hh family, Indian hedgehog (ihha) is expressed in the differentiating scleroblasts, the bone-matrix releasing cells, of each fin ray, and may have a more direct role on bone formation based on its expression pattern and known function in other vertebrates. The present study is aimed at investigating the role of hh signalling in patterning the fin ray regenerate and more specifically its role in ray branching morphogenesis. In a first approach, we used the zebrafish 2.2shh:gfp:ABC transgenic line, in which GFP expression recapitulates the endogenous expression of shh, to ablate the shh-expressing cells using a laser beam. Results show that such ablation leads to a delay of ray bifurcation suggesting that the shh-expressing cells play an important role in branching morphogenesis. In a second approach, we cloned the zebrafish hedgehog interacting protein (hip), a hedgehog antagonist, to investigate the effects of its overexpression on ray patterning. Analysis of hip expression during fin regeneration suggests its involvement in limiting hh signaling on the lateral sides of the dermal bones and in their medial region during branching morphogenesis. Overexpression of hip via in vivo cell transfection in the regenerating fin causes a branching delay, possibly as a result of the altered expression of patched1, the hh receptor and type X collagen, a component of the fin ray. These results suggest that hh signaling is involved in patterning the ray branching during zebrafish fin regeneration. Based on the distinct role of shh and ihha suggested by studies in other systems, ihha may be more involved in the scleroblast proliferation and differentiation, and shh is likely to be responsible to pattern the bone formation by directing the site of scleroblast differentiation or possibly mediating scleroblast migration.

USP4 is a deubiquitinating enzyme whose levels have been shown to be elevated in certain human lung tumors. USP4 is thought to possess oncogenic properties due to its ability to promote tumors in nude mice assays. The lack of an overall effect on ubiquitin levels in overexpression studies has led to the hypothesis that USP4 may act on a few select substrates to edit their ubiquitination status. Although the structure/function relationship is more documented, the physiological substrates and role in vivo are not. In order to elucidate the mechanism by which USP4 could potentially exert its tumorigenic effect, an RNA knockdown approach was undertaken. The effect of changes in USP4 levels was investigated to determine if USP4 plays a role in transcription or mRNA stability. The data suggest that USP4 does not affect levels of mRNAs containing an ARE sequence in NIH-3T3 or Cos-7 cells. Although USP4 was not shown to have an mRNA stabilizing effect, USP4 was found to bind CBP in vivo using an immunoprecipitation experiment and to exert an effect on basal transcription levels. This data suggests that USP4 levels may affect global transcription, perhaps through binding with the transcriptional co-activator CBP.