Striatal medium spiny neurons (MSNs) receive glutamatergic afferents from the cerebral cortex that synapse onto the spine head and dopaminergic inputs from the substantia nigra which synapse onto the necks of dendritic spines completes a triadic arrangement. Striatal DA loss, as occurs in Parkinsons Disease (PD), results in dystrophic changes in MSN dendrites, including a loss of dendritic spines. This loss of spines has been suggested to reflect removal of tonic dopamine inhibitory control over corticostriatal glutamatergic drive, with increased glutamate release culminating in MSN spine loss. We tested this hypothesis in two ways. We first determined in vivo if decortication reverses or prevents dopamine depletion-induced spine loss by placing motor cortex lesions four weeks after, or at the time of, 6-hydroxydopamine lesions of the SN; animals were sacrificed four weeks after cortical lesions. Motor cortex lesions significantly reversed the loss of MSN spines elicited by dopamine denervation; a similar effect was observed in the prevention experiment. Because decortication results in a loss of corticostriatal axon terminals, it is possible that signaling molecules in addition to glutamate that are contained in these axons may be the culprit in determining spine changes. We therefore treated cultures (14-15 DIV) with MPP+ to disrupt the striatal DA innervation, and then added a metabotropic glutamate receptor agonist, LY379268, to suppress glutamate release in these cultures. Two weeks later cultures were diOlistically labeled and spine density determined. Treatment of the cultures with the mGluR2/3 agonist completely blocked spine loss in dopamine-denervated cultures. These studies provide the first evidence to show that MSN spine loss associated with Parkinsonism can be reversed, and point to suppression of corticostriatal glutamate release as a means of slowing progression in Parkinsons Disease.

Muscarinic acetylcholine receptors (mAChRs), specifically M1 and M4 subtypes, provide viable targets for the treatment of multiple central nervous system disorders. However, highly selective activators of either M1 or M4 have not been available, making it difficult to determine the in vivo effects of selective activation of these receptors. We have used cheminformatics and medicinal chemistry to develop new, highly selective M1 and M4 positive allosteric modulators (PAMs). VU10010 potentiated the functional M4 response to acetylcholine while having no activity at other mAChR subtypes. Whole-cell patch clamp recordings revealed that VU10010 increased carbachol-induced depression of transmission at excitatory but not inhibitory synapses at the Schaffer collateral-CA1 (SC-CA1) synapse in the hippocampus. Chemical optimization of VU10010 afforded two centrally penetrant analogs, VU0152099 and VU0152100, which are also potent, selective M4 PAMs. Interestingly, these compounds reversed amphetamine-induced hyperlocomotion in rats, a model that is predictive of clinical antipsychotic efficacy in humans. A growing body of literature also supports M1 receptors as a viable target for treatment of disorders involving impaired cognitive function. Data in this thesis reports the molecular characterization of a novel compound, BQCA, which is a potent, highly selective PAM of the rat M1 receptor. BQCA induced a robust inward current and increased spontaneous EPSCs in mPFC layer V pyramidal cells, effects which were absent in acute slices from M1 receptor knockout mice. Furthermore, multiple single-unit recordings were obtained from the mPFC of rats which showed that BQCA increased firing of pyramidal cells in vivo. BQCA also restored discrimination reversal learning in a transgenic mouse model of AD and regulated non-amyloidogenic APP processing in vitro. Together, these studies provide compelling evidence while M4 inhibits excitatory transmission at the SC-CA1 synapse, M1 receptor activation induces a dramatic excitation of PFC neurons. Newly developed highly selective ligands that activate or potentiate M1 and M4 provide exciting tools that will be useful in further delineating the individual roles of these receptors in the efficacy of drugs like acetyl cholinesterase inhibitors and xanomeline.

Dopaminergic neurotransmission is a critical component in the regulation of several behaviors, including cognition, motor function, motivation, and reward. Abnormalities in dopamine (DA) signaling have been linked to several disorders, including schizophrenia, Parkinsons Disease, drug addiction, and eating disorders. An important component in regulating DA neurotransmission is the DA transporter (DAT), which controls the inactivation of DA signaling by uptake of DA from the synapse into the presynaptic bouton. This action depends upon the number of functional transporters expressed at the plasma membrane. As such, regulation of DAT cell surface expression is an important mechanism to modify DA neurotransmission. A growing body of literature indicates that insulin signaling, including the downstream effector protein kinase B (Akt), is an important regulator of DAT function by virtue of fine tuning the transporters cell surface expression. In this dissertation, I first sought to further define the insulin signaling pathway regulating DAT by identifying the isoform of Akt responsible for modulating DAT cell surface expression. The data shown here support that Akt2, the isoform believed to mediate insulin signaling, regulates DAT cell surface expression. Furthermore, we sought to understand whether high fat feeding, diet induced obesity (DIO) would cause a reduction in Akt phosphorylation in brain, and as a consequence, alter DAT function. Here I report that a high fat diet alters insulin signaling, namely Akt activity, and results in a reduction of DAT cell surface expression in the striatum. In addition, I demonstrate a functional reduction in DA clearance in vivo and a reduction in AMPH-induced locomotor activity. Upon viral rescue of insulin signaling via expression of insulin receptor substrate 2 (IRS2), DAT cell surface expression and AMPH-induced locomotor activity are restored. Collectively, these data demonstrate that insulin, by signaling through Akt2, regulates DAT function, and that a high fat diet leads to improper insulin signaling, thus altering DAT cell surface expression. Therefore, dysregulation of DA tone by alterations of DAT function is an important concern for individuals who have developed insulin resistance, and may have mechanistic implications for the co-morbid nature of obesity and neuropsychiatric disorders.

There exist five subtypes of the muscarinic acetylcholine receptor (M1-M5), which are differentially expressed throughout the body and play important roles in numerous physiological processes, including autonomic functions, motor control, cognition, reward, and sleep, among others. A historic lack of small molecules possessing high subtype-selectivity has hampered basic neurobiological research into the roles of each subtype in the central nervous system and has precluded successful therapeutic development of muscarinic ligands targeting a particular receptor subtype. Functional cell-based screening in conjunction with medicinal chemistry techniques were performed in order to discover highly subtype-selective allosteric ligands for M1, M4, and M5. Multiple series of M1 positive allosteric modulators, M1 allosteric agonists, M1 antagonists, M4 positive allosteric modulators, and the first series of selective M5 positive allosteric modulators were successfully discovered, optimized, and characterized.

Complex II superfamily members catalyze two separate reactions in respiration: interconversion of fumarate and succinate in the soluble milieu and interconversion of quinol and quinone in the membrane. Electrons liberated as a result of one reaction become substrates for the second, thermodynamically linking both sites and allowing complex II enzymes to catalyze electron entry to or exit from a respiratory chain. In anaerobic respiration, menaquinol:fumarate oxidoreductase (QFR) catalyzes the final step in the most widely used anaerobic respiratory pathway, fumarate reduction. This work analyzes structural and mechanistic details of function and inhibition at each of the two active sites. The first part examines transition state formation at the active site responsible for fumarate and succinate interconversion, the dicarboxylate site. It was found that two threonine residues, Thr-234 and Thr-244, might play important roles in attaining and stabilizing the transition state. The second part of this work focuses on details of dicarboxylate site inhibition by substrate-like molecules and of substrate activation. It was found that all tight binding ligands or ligands transformed by the enzyme also induce large optical shifts in an FAD cofactor and align an activatable bond along the active C4a-N5 axis of FAD. This bond overlap was proposed to play a part of an orbital steering mechanism to guide substrate and activate it for catalysis. This alignment was found be conserved in a number of flavoenzymes that catalyze dehydrogenation reactions, even with completely unrelated substrates and structural folds. The third part of this work focuses on the second active site, the quinone/quinol interconversion site. The E29L variant QFR was crystallized to study semiquinone formation. Preliminary results reveal that the substrate, menaquinone, may bind in a continuum of active site positions as the substrate undergoes catalysis. Another substrate, ubiquinone was shown to bring about the presence of strong electron density at a site located 13 angstroms away from menaquinone site. The forth and final part of this work examines the structural work done to isolate and stabilize complex formation between QFR and FliG, a part of the flagellar motor.

KCNE4 is a potassium channel modulating protein that can dramatically inhibit distinct potassium currents, but we lack a clear understanding its mechanism for doing so and its physiologic significance. In this project we sought new understanding of the physiological functions of KCNE4, through identification of its protein interacting partners and assessment of the consequences of Kcne4 knockout in mouse cardiac physiology. A membrane-based yeast two-hybrid screen identified 20 putative interacting partners of KCNE4. The hit with the most obvious potential for functional intersection with KCNE4 was calmodulin (CaM), a known ion channel modulator. We subsequently demonstrated a Ca2+-dependent biochemical interaction between KCNE4 and CaM and that KCNQ1 modulation by KCNE4 is impaired both upon mutation of the CaM-interaction site of KCNE4 and by acutely chelating intracellular calcium to displace CaM from KCNE4. These findings suggest a connection between the mechanism of KCNQ1 inhibition by KCNE4 and the activating effect of CaM on the channel. Whereas we had previously assumed that the inhibition of KCNQ1 by KCNE4 is caused by a direct effect of KCNE4 on the channel, these data introduce the new possibility that KCNE4 inhibits KCNQ1 by disrupting CaM-mediated activation. Analysis of a Kcne4-null mouse constituted another approach to investigating the physiologic significance of KCNE4. We hypothesized that Kcne4 may be an endogenous negative regulator of repolarizing currents in the cardiac action potential, and that Kcne4-null mice might display shortened repolarization time, with possible implications for arrhythmia susceptibility or excitation-contraction coupling. Electrocardiographic analysis revealed that Kcne4-null mice under isoflurane anesthesia have a shortened QTc interval compared with wild-type littermates. Our hypothesis was also supported by the observation of shortened action potential duration in isolated ventricular myocytes from Kcne4-null mice. Further, echocardiography studies demonstrated that conscious Kcne4-null mice have increased left ventricular internal diameter during diastole and systole and impaired myocardial contractility. Collectively, these studies suggest KCNE4 may contribute to cardiac physiology as a Ca2+-sensitive modulator of repolarizing currents, with possible downstream effects on excitation-contraction coupling and myocardial contractility.

During vertebrate development, neural crest (NC) cells migrate from the dorsal neural tube and generate a wide variety of cell types throughout the embryo including neurons, glia, melanocytes, smooth muscle, cartilage and bone. The formation of NC progenitors has been extensively studied, yet molecules controlling NC multipotency and self-renewal and factors mediating cell-intrinsic distinctions between multipotent versus fate-restricted progenitors are poorly understood. As part of my thesis work, I show that the transcription factor Foxd3 mediates a fate restriction choice for multipotent NC progenitors with loss of Foxd3 biasing NC toward a mesenchymal fate. Neural derivatives of NC were lost in Foxd3 mutant mouse embryos, whereas abnormally-fated NC-derived vascular smooth muscle cells were ectopically located in the aorta. Cranial NC defects were associated with precocious differentiation towards osteoblast and chondrocyte cell fates, and individual mutant NC from different anterior-posterior regions underwent fate changes, losing neural and increasing myofibroblast potential. During development of the enteric nervous system (ENS) Foxd3 expression is maintained in NC progenitors and glia. Using a novel Ednrb-iCre transgene to delete Foxd3 after NC migrate into the midgut, I demonstrated a late temporal requirement for Foxd3 during ENS development. Fate mapping in Foxd3 mutant embryos revealed a reduction of ENS progenitors throughout the gut and loss of Ednrb-iCre lineage cells in the distal colon. Although mutant mice were viable, defects in ENS patterning were associated with severe reduction of glial cells derived from the Ednrb-iCre lineage. Lineage and differentiation analysis suggested a compensatory population of Foxd3-positive progenitors that did not express Ednrb-iCre in mutant embryos. My findings highlight the roles played by Foxd3 during ENS development including proliferation of ENS progenitors, neural patterning, and glial differentiation. Collectively, these data suggest Foxd3 functions as a critical regulator of NC fate potential and establish novel parallels between NC and other progenitor populations that depend on this functionally conserved stem cell protein to regulate multipotency and self-renewal.

PHARMACOLOGY FORMATION AND METABOLISM OF 15-DEOXY-{Delta}12,14-PROSTAGLANDIN J2 IN VIVO KLARISSA D. HARDY Dissertation under the direction of Professor L. Jackson Roberts, II 15-Deoxy-{Delta}12,14-prostaglandin J2 (15-d-PGJ2) is a reactive cyclopentenone PG generated from the dehydration of cyclooxygenase (COX)-derived PGD2, and this compound has emerged as a putative mediator of numerous biological effects. The cyclopentenone ring of 15-d-PGJ2 contains an electrophilic alpha,beta-unsaturated carbonyl moiety which can readily undergo nucleophilic addition reactions (Michael addition) with cellular thiols, such as glutathione and cysteine residues of proteins. Due to its reactivity, 15-d-PGJ2 can modulate protein function and exerts potent anti-inflammatory and pro-apoptotic activity in various cell types. The biological effects of 15-d-PGJ2 are mediated in part through activation of the nuclear receptor peroxisome proliferator-activated receptor-gamma as well as through direct interaction with other intracellular protein targets, such as transcription factors and signaling molecules. However, evidence for the biosynthesis of 15-d-PGJ2 in vivo is lacking. The goal of this dissertation research is to determine the extent to which 15-d-PGJ2 is formed in vivo and examine the mechanisms that contribute to its formation. We hypothesized that 15-d-PGJ2 may be generated in vivo via the COX and free radical-catalyzed pathways. Through the studies described herein, we have found that 15-d-PGJ2 and a series of 15-d-PGJ2-like compounds (deoxy-J2-IsoPs) are generated in vivo via free radical-initiated lipid peroxidation after induction of oxidative stress, independent of COX. The formation of these reactive compounds has potential relevance to the pathobiology of diseases associated with oxidative stress. This finding provided the impetus for us to examine the systemic generation of 15-d-PGJ2 and deoxy-J2-IsoPs in humans. Due to the reactivity of these molecules, we hypothesized that they undergo rapid adduction to protein and peptide thiols. Thus, we determined the metabolic fate of 15-d-PGJ2 in primary human cells and in vivo in the rat. From these studies, we identified several metabolites of 15-d-PGJ2 in primary human hepatocytes. We also identified the major urinary metabolite of 15-d-PGJ2 in the rat, and our results suggest that this metabolite is formed in humans. Importantly, the identification of this metabolite provides a potential biomarker to quantify the production of 15-d-PGJ2 and related compounds in vivo in humans under various physiological and pathophysiological conditions.

This project is concerned with mechanisms underlying osteoblast differentiation, a critical process during bone remodeling and repair. In this dissertation I explored novel regulators for osteoblast differentiation by identifying functional partners of ATF4, an osteoblast-enriched transcription factor important for osteoblast differentiation and bone formation. Through a combination of biochemical, molecular and cellular experiments, I identified and confirmed vimentin, an intermediate filament protein, as a novel inhibitor for ATF4's transcriptional activity and osteoblast differentiation. Furthermore, through series of genetic and pharmacological interventions in vitro and in vivo, I revealed that the vimentin serves as a downstream mediator that responds TGF beta signaling by binding and inhibiting ATF4 in immure osteoblasts via a non-canonical pathway. Therefore, this research uncovers a novel mechanism by which a cytoskeletal protein, vimentin, acts as a brake on differentiation in immature osteoblasts by its interaction with ATF4.

Huntingtons disease is a fatal autosomal dominant neurodegenerative disease caused by an expansion of CAG repeats in the DNA of the Huntingtin gene. The length of the repeat is inversely related to the age of disease onset, however it only accounts for 60% of the variability in the age of onset. Therefore, other genetic and environmental effects contribute to the remaining 40% in the variability in age of onset. Multiple neurodegenerative diseases exhibit alterations in metal ion homeostasis. Research in our laboratories has demonstrated a disease-toxicant interaction between mutant Huntingtin and manganese exposure in vitro. Manganese is an essential trace metal that is necessary for many physiological processes, including neurotransmitter synthesis. In high levels, manganese can be damaging to the brain. Exposure that leads to this damage is typically encountered in an occupational setting such as when welding, smelting or mining manganese. The research described in this dissertation is the first to describe the complex gene-environment interactions between mutant Huntingtin and manganese exposure in vivo. The neurochemical and morphological changes identified herein are complex and exhibit both positive and negative effects. My research also identified the earliest evidence for striatal dendritic pathology in YAC128 mice that occur between 13 and 16 weeks postnatal. Additionally, this is the first research to show gender-specific changes in MSN morphology following manganese exposure. These alterations in neuron morphology were most prevalent when manganese levels were elevated and were not due to differential striatal manganese accumulation. Taken together, these data lay the groundwork for understanding the gene-environment interaction between mutant Huntingtin and Mn exposure. Future studies will further our understanding of this interaction that may one day lead to therapeutic intervention. These studies also reinforce the need to include animals of both genders in experimentation to further our understanding of disease across both genders.