Kv1.1 voltage-gated potassium channels serve as key regulators of neuronal function and mutations in Kv1.1 lead to the human movement and epilepsy-related disorder, Episodic Ataxia type-1 (EA1). Transcripts encoding Kv1.1 may be modified by a site-specific adenosine-to-inosine RNA editing event, leading to the expression of an altered protein with an isoleucine to valine change at amino acid 400 (I400V). The non-edited [Kv1.1(I)] and edited [Kv1.1(V)] channels display altered functions in heterologous expression systems, but their significance in normal physiology and with regard to EA1 have not been characterized. We have developed new mouse models which solely express either the non-edited [Kv1.1(I)] or edited [Kv1.1(V)] isoforms of the channel. Our studies have revealed that both mice display conditional postnatal lethality and that the non-edited [Kv1.1(I)] mice exhibit an EA1-like disorder, stress-induced motor dyscoordination. In addition, we observed that editing altered drug-induced seizure-susceptibility for these mutant mouse models. Mice expressing the non-edited [Kv1.1(I)] channel exhibited a lowered threshold for drug-induced seizures, whereas mutant animals expressing the edited [Kv1.1(V)] channel exhibited an increased threshold. To further characterize the collective effects of Kv1.1 editing in transcripts containing human EA1 mutations, we discovered that three mutations (V404I, I407M, and V408A), in close proximity to the editing site, decreased Kv1.1 RNA editing in vitro. Utilizing the V408A/+ mouse model of EA1, we confirmed that the V408A mutation leads to decreases in Kv1.1 RNA editing in vivo. Electrophysiological characterization of the edited EA1 mutant channels also indicated that these channel isoforms display channel dysfunctions unique to each mutation, as compared to their respective non-edited EA1 channels. Thus, Kv1.1 RNA editing has a substantial impact on normal physiology and may be dysregulated in the EA1 disorder, as well as contribute to the wide variety in symptom severity observed in EA1 patients.

MOLECULAR PHYSIOLOGY & BIOPHYSICS Exploring the Function of G6PC2 in Pancreatic Islet Beta Cells Kayla Boortz Dissertation under the direction of Professor Richard OâBrien, Ph.D. G6PC2 encodes an islet-specific glucose-6-phosphatase catalytic subunit. Together with glucokinase, G6PC2 forms a substrate cycle that determines the glucose sensitivity of insulin secretion. Genetic and molecular studies show that elevated G6PC2 expression raises fasting blood glucose (FBG) but because chronically elevated FBG is detrimental, increasing type 2 diabetes risk, it is unclear why G6PC2 evolved. Studies have shown that single nucleotide polymorphisms (SNPs) in G6PC2 associate with variation in FBG and body mass index (BMI) in humans. Previously we confirmed these findings in mice and showed that FBG is repressed in G6pc2 knockout (KO) mice relative to wild type (WT) on a mixed or pure C57BL/6J genetic background. I further confirmed that 129SvEv KO mice also have reduced FBG. Moreover, I identified a glucocorticoid response element in the human and 129SvEv G6pc2 promoter that confers glucocorticoid responsiveness and induction of G6PC2 gene expression following treatment with Dexamethasone (Dex), 11-dehydrocorticosterone (11-DHC) or physical restraint in 129SvEv and C57BL/6J mice. Following stress generated by Dex or physical restraint, FBG is repressed in 6 hour fasted 129SvEv and C57BL/6J KO mice, respectively, enhancing the difference in FBG relative to controls. These data suggest that G6PC2 evolved to modulate FBG under conditions of glucocorticoid-related stress thereby conferring a transient, beneficial modulation of the set point for FBG. This thesis also addresses the observation that the common rs560887 G6PC2 SNP is associated with variation in BMI and adiposity. The results described here indicate that the effect of G6pc2 deletion on the response to diet induced obesity is dependent on genetic modifiers. Finally, an indirect analysis of the effects of human G6PC2 SNPs on protein expression and enzyme activity indicates that mutations at catalytically conserved residues may be critical for proper enzyme function and protein expression.

This project investigated the significance of extramyocellular barriers to the pathogenesis of skeletal muscle insulin resistance and their potential as therapeutic targets. The goals of this dissertation were to determine the impact of capillary rarefaction to muscle insulin resistance, the efficacy of the vasoactive and extracellular matrix degrading hormone relaxin to rescue muscle insulin resistance, and the effects of high fat-feeding on caveolae microstructures within skeletal muscle capillaries. Data from obese patients and animal experiments describe a strong association between peripheral insulin resistance and attenuation of skeletal muscle capillary density. Whether the relationship of capillary density and the development of skeletal muscle insulin resistance are casual remains to be elucidated. We utilized a genetic mouse model with muscle-specific deletion of vascular endothelial growth factor-A (VEGF) to induce capillary rarefaction in otherwise lean, healthy mice. VEGF deficient mice had 60% fewer muscle capillaries inducing impairment in muscle insulin action. Furthermore, we investigated the therapeutic potential of targeting the vascular dysfunction and extracellular matrix remodeling associated with a high fat diet with a pharmacological dose of the endogenous hormone relaxin. A 3 week relaxin intervention in high fat-fed C57BL/6J mice rescued the metabolic and vascular dysfunction caused by the diet. The interplay between vascular and metabolic disease are significant mortality risk factors and the extramyocellular adaptations to an obesogenic diet are viable pharmacological targets. The transport of insulin across the capillary wall is rate-limiting to the onset of skeletal muscle insulin action. Transendothelial insulin transport is dependent on caveolae structures of the endothelial layer of muscle capillaries. Employing transmission electron microscopy, we show for the first time that caveolae number in muscle capillaries of 16 week high-fat fed mice are diminished compared to low fat-fed controls. These data suggest a contribution of diminished caveolae density in transendothelial insulin transport, thus augmenting the barrier for insulin delivery. In conclusion, the experiments performed in this dissertation support the paradigm that extramyocellular barriers to insulin-stimulated muscle glucose uptake are significant to the etiology of insulin resistance and these barriers provide novel targets for the treatment of vascular and metabolic disorders associated with obesity.

Elevated very low-density lipoprotein (VLDL)-triglyceride (TG) secretion from liver contributes to atherogenic dyslipidemia that is associated with obesity, diabetes, and the metabolic syndrome. Numerous models of obesity are characterized by increased central nervous system (CNS) neuropeptide Y (NPY) tone which contributes to positive energy balance and obesity. In fact, an acute, single intracerebroventricular (ICV) administration of NPY in lean fasted rats elevates hepatic VLDL-TG secretion. Thus, our overarching hypothesis is that elevated CNS NPY action contributes to dyslipidemia by activating central circuits that modulate liver lipid metabolism. Our studies focused on identifying molecular determinants in the hypothalamus and the liver by which increased CNS NPY signaling modulates hepatic lipoprotein metabolism. First, we sought to determine if the effects of NPY on feeding and/or obesity are dissociable from effects on hepatic VLDL-TG secretion. ICV NPY-treated chow-fed rats pair-fed to vehicle-treated controls develop hypertriglyceridemia in the absence of increased food intake and body fat accumulation. Acute ICV injection of selective Y1, Y2, Y4, and Y5 receptor agonists all induced hyperphagia in lean ad-libitum fed rats with the Y2 receptor agonist having the most pronounced effect. The NPY Y1 receptor agonist robustly stimulated hepatic VLDL-TG secretion, while a Y2 receptor agonist had a modest effect on VLDL-TGs, and no effect was observed for Y4 and Y5 receptor agonists in lean fasted rats. These findings raise the possibility that NPY regulates feeding and lipoprotein metabolism partially via separate NPY receptor systems and/or mechanisms. Lastly, we sought to identify novel regulatory mechanisms in the liver engaged by CNS NPY signaling. We observed, in ICV NPY- and Y1 agonist-treated lean fasted rats, that oleic and linoleic acid were enriched in the liver phospholipid (PL) pool and secreted into plasma TGs. Furthermore, CNS NPY signaling via the Y1 receptor robustly activated key hepatic regulatory enzymes, ADP-ribosylation factor-1 and lipin-1, involved in remodeling liver PL into TG for VLDL maturation and secretion. Altogether, this body of work has overarching implications in further understanding how obesity-related CNS dysfunction contributes to the pathophysiology of atherogenic dyslipidemia associated with obesity, diabetes, and the metabolic syndrome.

Autism is a severe neurodevelopmental disorder characterized by deficits in language and social interaction, and patterns of repetitive and stereotyped behaviors, interests and activities. Evidence indicates that autism has a predominantly genetic etiology, and that as many as fifteen genes may contribute to disease susceptibility. One model suggests autism may result from oligogenic inheritance, with locus heterogeneity, such that different families or individuals possess a different mix of susceptibility alleles. In this dissertation, I present genome-wide linkage studies of autism and traits comprising the aspects of the broader phenotype to identify autism susceptibility loci. I further document detailed molecular and genetic analyses of candidate genes in regions detected by linkage, and in the case of 15q11-q13, as chromosomal duplications found in 1-3% of autism cases. A unifying theme to my dissertation is the focus of genetic studies on genes acting within candidate neurobiological systems suspected of involvement in autism. Genetic analyses include linkage, linkage refinement, construction of detailed linkage disequilibrium (LD) and corresponding haplotype maps across candidate loci, and tests for transmission disequilibrium of single markers and haplotypes. Molecular studies of select candidates aim to identify functional variation on associated alleles; in the absence of association they seek to identify potential rare disease-related variants considering for example evolutionarily conserved sequence. I hypothesize that there are allelic variants, which underlie genetic linkage and/or association to autism and related traits, and these contribute to autism susceptibility through both direct and interactive effects. The goal of this study is to dissect the genetic etiology of autism by leveraging trait-based phenotypic subsets of autism using the approaches and tools I have outlined here.

Inflammation and insulin resistance are characteristics of endotoxemia. While the role of interleukin-6 (IL-6) in insulin resistant states has been characterized, little is known of its role in the metabolic response to inflammation. To study the role of IL-6, conscious chronically catheterized mice were used. Five days prior to being studied, catheters were implanted in the carotid artery and jugular vein. After a 5 h fast, E.coli (250 microgram/mouse) LPS was injected in IL6-/- (KO; n=13), IL6+/- (HET; n=9), and IL-6+/+ (WT; n=10) littermates. The IL-6 response to LPS was simulated in an additional group of KO mice (KO+IL6; n=10). Glucose turnover (Ra) was assessed using 3-[3H]-D-glucose. IL-6 increased similarly in WT and HET (15±0.7 and 14±0.5 ng/ml) 4h after LPS and was undetectable in KO. IL-6 replacement in KO restored circulating IL-6 to levels observed in the WT group (14±0.3 ng/ml). WT was the only group to experience an early rise in tumor necrosis factor-alpha (TNF-alpha). Interleukin-1beta (IL-1beta) was similar in all groups. KO exhibited a more profound hyperglycemia 30 min after LPS injection and no apparent hypoglycemia at 4h (95±5 mg/dl). Glucose levels in KO+IL6, while decreased (93±4 mg/dl) at 4h, remained higher than WT. Ra was not altered. In summary, the absence of IL-6 protected against LPS induced hypoglycemia. Acute restoration of the IL-6 response to LPS did not potentiate hypoglycemia. Thus, while IL-6 promotes glucose intolerance in insulin resistant states, IL-6 promotes hypoglycemia during inflammation.

It is well known that the arterial plasma insulin/glucagon level, the hepatic glucose load, and the route of glucose delivery (the portal glucose signal) are the three major determinants of NHGU. Recent studies have reported that the ability of insulin and glucose to stimulate splanchnic glucose uptake is impaired in individuals with type 2 diabetes. Thus, in specific aim I of this thesis we explored the ability of first phase insulin release to control the glycemic excursion on the background of basal insulin and a duodenal glucose infusion. It was determined that the pulse of insulin acutely affected net hepatic glucose production and increased NHGU while it still had significant effects on plasma glucose levels 4.5 hours later due to its ability to affect non hepatic glucose clearance. Although it has been demonstrated that the portal glucose signal is an important regulator of NHGU, the mechanism of action by which it exerts its effects is not completely understood. The relative importance of the sympathetic and parasympathetic nerves was not known. Other investigators had suggested that the portal signal may exert its effects through an intrahepatic reflex involving other mediators and neurotransmitters. Thus the remaining portion of this thesis was to further elucidate the potential regulators of the portal glucose signal and NHGU. Our attention first turned towards the role of the sympathetic nerves in the regulation of NHGU. The data from specific aim II suggest that the sympathetic nerves exert a tonic inhibition on NHGU in the presence of hyperglycemia, and that removal of these nerves, results in an increase in NHGU. Our conclusions from specific aim II led us to further investigate the vagus nerve and the role of the vagal afferents in mediating the portal glucose signal. The data from specific aim III suggested that the vagus nerve does not play a role in the regulation of NHGU suggesting that the afferent nerves found in the vagus are not the transmitters of the portal glucose signal. The data from specific aims II and III suggested that the sympathetic nerves exerted a dominant effect on NHGU while the input from the vagus nerve is not essential for the regulation of the portal glucose signal. Thus, the possibility exists that a local effect of the portal signal might be occurring via an intrahepatic reflex. Thus we hypothesized that nitric oxide may be involved in the regulation of NHGU. It is clear that the intraportal administration of the nitric oxide donor SIN-1 significantly decreased NHGU in the presence of the portal signal. This effect may be due to either a direct or an indirect mechanism.

The importance of integration of information from neurocircuits innervating the reward circuitry is becoming increasingly recognized. For instance, the roles of stress/anxiety pathways in modulating specific effects of the reward system are becoming more apparent. Behavioral data suggest the bed nucleus of the stria terminalis (BNST) is involved in integrating stress information and relaying this information to the stress and reward pathways. Moreover, behavioral data suggest glutamatergic transmission in the BNST as well as other regions in the stress/reward pathways plays an integral role in regulating stress and drug addiction behaviors. Changes in synaptic physiology in the BNST could in part underlie the persistent behavioral alterations in generalized anxiety, addiction and post-traumatic stress disorder. Metabotropic glutamate receptors (mGluRs) have been implicated in stress, addiction and synaptic plasticity, but their roles in the BNST are currently unknown. Field recordings and whole-cell patch clamp analysis in an in vitro slice preparation of C57/Bl6j adult male mouse dorsolateral BNST were utilized. Activation of either group I, group II or group III mGluRs in the dBNST causes a depression of excitatory synaptic transmission. Further characterization of mGluR5-dependent long-term depression (LTD) of excitatory synaptic transmission indicates this LTD is associated with a decrease in miniature EPSC frequency but not a change in paired pulse ratios of evoked responses. This suggests a novel maintenance mechanism for this form of LTD. Potential downstream effectors of group I mGluRs that have also been implicated in stress, addiction and synaptic plasticity are the cannabinoid system and extracellular signal regulated kinase (ERK). mGluR5-mediated LTD in the BNST is G-protein dependent, and persists in the cannabinoid receptor 1 knockout mouse. Further, converging pharmacological and genetic approaches suggest the mGluR5-mediated LTD in the BNST is ERK dependent. Consistent with an emerging role for group I mGluRs in drug addiction, mGluR5-mediated LTD in the BNST is attenuated in mice trained to self-administer cocaine.

Carbamyl phosphate synthetase I (CPSI) is the liver-specific enzyme that catalyzes the first and rate-determining step of the urea cycle. Mutations in the CPSI gene cause CPSI Deficiency (CPSID), an autosomal recessive disease characterized by hyperammonemia. During the course of this project, many patient mutations likely causing RNA instability were identified. These genomic variants were present in patient genomic DNA but not detectable in patient RNA, indicating RNA degradation perhaps due to nonsense mediated decay (NMD) or splicing errors. This large number of suspected RNA instability mutations suggests CPSID is a good model to understand the prevalence of this mutation type in genetic disease. However, the ability to study a heterogeneous set of mutations in such a large gene has suffered from the lack of an appropriate model system. A goal of this project was to develop a novel model system for testing the functional consequences of these potential RNA instability mutations. Using two homologous recombination strategies, a BAC clone containing the full CPSI gene was modified to contain the pEHG vector and the CMV promoter upstream of CPSI. These modifications allow for stable transfection, selection and ubiquitous expression in eukaryotic cells. Patient mutations were then incorporated into the resulting BECC model system and these constructs were subsequently tested to assay for splicing changes as well as NMD. These mutations included the intronic substitutions c.654-3T>G and c.1210-1G>T, the exonic c.1893T>G mutation which directly creates a nonsense codon, and c.2388C>A which is a translationally silent exonic mutation with unknown function. Extensive control assays show that the BECC model system properly expressed wild-type CPSI, while each mutant shows decreased expression as measured by quantitative RT/PCR assays. To determine if this decrease in expression was due to NMD, it was inhibited by siRNA mediated knockdown of UPF2. Following NMD inhibition, relative expression levels of each mutant were increased, indicating these mutations cause degradation via the NMD pathway. In addition, cryptic splice sites activated by intronic mutations were identified. This project is significant because using CPSID as a model, it shows the prevalence of RNA instability mutations in a severe metabolic disease and provides a new model system to test any mutation, irrespective of type or location, for functional consequences including the activation of aberrant splicing and nonsense-mediated decay.

Adenovirus (Ad) vectors are promising vehicles for vaccine delivery and gene therapy, but vector development has been hampered by the lack of an atomic structure of the intact virion. In this work, cryoEM single particle reconstruction methods have been applied to study the structure of an adenovirus vector, Ad35F. The 9 Å resolution cryoEM structure of Ad35F was analyzed by docking the crystal structures of two major capsid proteins, hexon and penton base. A long α-helix of ~ 40 residues was observed on the capsid surface and assigned to the C-terminal domain of protein IX recently used as a platform for vector retargeting. In the 6 Å resolution structure of Ad35F α-helices of 10 or more residues are clearly visualized for the first time. This in combination with secondary structure prediction for proteins IIIa, VI, VIII, and IX has led to new and more precise assignments for these capsid proteins. The coiled coil of protein IX is assigned to a tetrameric coiled coil now observed on the outer capsid surface. Protein IIIa, which is predicted to be highly α-helical, is assigned to a cluster of helices below the penton base on the inner capsid surface. Protein VI, a membrane lytic factor, is sequestered within the hexon cavities on the inner capsid surface. The new protein assignments have important implications for vector design, virus assembly, and capsid disassembly during cell entry. First, since protein IIIa, VI, and VIII are all located on the inner capsid surface, our structural work indicates that the best candidate proteins for retargeting are hexon, penton base, fiber and protein IX. With the new interior position of protein IIIa, it is easier to envision how protein IIIa can facilitate DNA packaging during assembly. During cell entry in the low pH environment of the endosome, protein IIIa is well situated to allow rapid release of vertex proteins, including protein VI. The sequestration of protein VI is likely important to prevent membrane lysis until the appropriate time point in the viral life cycle.