Role of neuropeptide neuromedin U in the nucleus accumbens shell in cocaine self-administration in male rats.

The nucleus accumbens shell (NAcSh) and its afferent and efferent neuronal projections control key aspects of motivation for cocaine. A recently described regulator of γ-aminobutyric acid (GABA) projections from the dorsal raphe nucleus (DRN) to the NAcSh (DRN → NAcSh) is the neuropeptide neuromedin U (NMU). Here, we find that systemic administration of NMU decreases breakpoint for cocaine on a progressive ratio schedule of reinforcement in male rats. Employing a retrograde adeno-associated virus (AAV), we found that RNAi-mediated knockdown of the NMU receptor 2 (NMUR2) in afferent DRN projections to the NAcSh increases the breakpoint for cocaine. Our previous studies demonstrated that NMU regulates GABA release in the NAcSh, and our current investigation found that systemic NMU administration suppresses cocaine-evoked GABA release in the NAcSh and increases phosphorylated c-Fos expression in neurons projecting from the NAcSh to the ventral pallidum (VP). To further probe the impact of NMU/NMUR2 on neuroanatomical pathways regulating motivation for cocaine, we employed multi-viral transsynaptic studies. Using a combination of rabies virus and retrograde AAV helper virus, we mapped the impact of NMU across three distinct brain regions simultaneously and found a direct connection of GABAergic DRN neurons to the NAcSh → VP pathway. Together, these data reveal that NMU/NMUR2 modulates a direct connection within the GABAergic DRN → NAcSh → VP circuit that diminishes breakpoints for cocaine. These findings importantly advance our understanding of the neurochemical underpinnings of pathway-specific regulation of neurocircuitry that may regulate cocaine self-administration, providing a unique therapeutic perspective.

Glutamatergic projections from homeostatic to hedonic brain nuclei regulate intake of highly palatable food.

Food intake is a complex behavior regulated by discrete brain nuclei that integrate homeostatic nutritional requirements with the hedonic properties of food. Homeostatic feeding (i.e. titration of caloric intake), is typically associated with hypothalamic brain nuclei, including the paraventricular nucleus of the hypothalamus (PVN). Hedonic feeding is driven, in part, by the reinforcing properties of highly palatable food (HPF), which is mediated by the nucleus accumbens (NAc). Dysregulation of homeostatic and hedonic brain nuclei can lead to pathological feeding behaviors, namely overconsumption of highly palatable food (HPF), that may drive obesity. Both homeostatic and hedonic mechanisms of food intake have been attributed to several brain regions, but the integration of homeostatic and hedonic signaling to drive food intake is less clear, therefore we aimed to identify the neuroanatomical, functional, and behavioral features of a novel PVN → NAc circuit. Using viral tracing techniques, we determined that PVN → NAc has origins in the parvocellular PVN, and that PVN → NAc neurons express VGLUT1, a marker of glutamatergic signaling. Next, we pharmacogenetically stimulated PVN → NAc neurons and quantified both gamma-aminobutyric acid (GABA) and glutamate release and phospho-cFos expression in the NAc and observed a robust and significant increase in extracellular glutamate and phospho-cFos expression. Finally, we pharmacogenetically stimulated PVN → NAc which decreased intake of highly palatable food, demonstrating that this glutamatergic circuitry regulates aspects of feeding.

Highly Conserved Molecular Features in IgLONs Contrast Their Distinct Structural and Biological Outcomes.

Neuronal growth regulator 1 (NEGR1) and neurotrimin (NTM) are abundant cell-surface proteins found in the brain and form part of the IgLON (Immunoglobulin LSAMP, OBCAM, Neurotrimin) family. In humans, NEGR1 is implicated in obesity and mental disorders, while NTM is linked to intelligence and cognitive function. IgLONs dimerize homophilically and heterophilically, and they are thought to shape synaptic connections and neural circuits by acting in trans (spanning cellular junctions) and/or in cis (at the same side of a junction). Here, we reveal homodimeric structures of NEGR1 and NTM. They assemble into V-shaped complexes via their Ig1 domains, and disruption of the Ig1-Ig1 interface abolishes dimerization in solution. A hydrophobic ridge from one Ig1 domain inserts into a hydrophobic pocket from the opposing Ig1 domain producing an interaction interface that is highly conserved among IgLONs but remarkably plastic structurally. Given the high degree of sequence conservation at the interaction interface, we tested whether different IgLONs could elicit the same biological effect in vivo. In a small-scale study administering different soluble IgLONs directly into the brain and monitoring feeding, only NEGR1 altered food intake significantly. Taking NEGR1 as a prototype, our studies thus indicate that while IgLONs share a conserved mode of interaction and are able to bind each other as homomers and heteromers, they are structurally plastic and can exert unique biological action.

Binge-Type Eating in Rats is Facilitated by Neuromedin U Receptor 2 in the Nucleus Accumbens and Ventral Tegmental Area.

Binge-eating disorder (BED) is the most common eating disorder, characterized by rapid, recurrent overconsumption of highly palatable food in a short time frame. BED shares an overlapping behavioral phenotype with obesity, which is also linked to the overconsumption of highly palatable foods. The reinforcing properties of highly palatable foods are mediated by the nucleus accumbens (NAc) and the ventral tegmental area (VTA), which have been implicated in the overconsumption behavior observed in BED and obesity. A potential regulator of binge-type eating behavior is the G protein-coupled receptor neuromedin U receptor 2 (NMUR2). Previous research demonstrated that NMUR2 knockdown potentiates binge-type consumption of high-fat food. We correlated binge-type consumption across a spectrum of fat and carbohydrate mixtures with synaptosomal NMUR2 protein expression in the NAc and VTA of rats. Synaptosomal NMUR2 protein in the NAc demonstrated a strong positive correlation with binge intake of a "lower"-fat (higher carbohydrate) mixture, whereas synaptosomal NMUR2 protein in the VTA demonstrated a strong negative correlation with binge intake of an "extreme" high-fat (0% carbohydrate) mixture. Taken together, these data suggest that NMUR2 may differentially regulate binge-type eating within the NAc and the VTA.

Incubation of feeding behavior is regulated by neuromedin U receptor 2 in the paraventricular nucleus of the hypothalamus.

A diet of energy-dense food, characterized mainly as a high-fat diet, leads to a persistent excessive consumption defined as overeating. According to the National Institute of Health, more than 2 in 3 adults in the United States are overweight or obese, straining our healthcare system with epidemic proportions. Diets that include abstaining from high-fat foods, ironically, result in an increase in motivation and craving for said high-fat foods, defined as an incubation effect because the behavior aids in developing overeating. Previously, we have shown that modulation of neuromedin U receptor 2 (NMUR2) in the paraventricular nucleus of the hypothalamus (PVN) results in increased food intake and motivation for energy-dense foods. Here, we continue our focus on NMUR2 in the PVN, but in relation to the incubation effect on craving for high-fat food. We employed a model for incubation of craving by having rats abstain from high-fat foods for 30 days before undergoing intake of fatty food on fixed ratio and progressive ratio schedules of reinforcement, and then assess their response to reactivity to cues. Using this model, we compared the feeding behaviors of rats that underwent an mRNA knockdown of the NMUR2 in the PVN to controls after both underwent a 30-day abstinence from high-fat foods. Our results show knockdown of NMUR2 in the PVN blocks the incubation of feeding behavior for food-related cues and high-fat foods.

Cocaine-Evoked Locomotor Activity Negatively Correlates With the Expression of Neuromedin U Receptor 2 in the Nucleus Accumbens.

Cocaine use disorder (CUD) is characterized by repeated cycles of drug seeking and drug taking. Currently, there are no available pharmacotherapies to treat CUD, partially due to a lack of a mechanistic understanding of cocaine-evoked alterations in the brain that drive drug-related behaviors. Repeated cocaine use alters expression of numerous genes in addiction-associated areas of the brain and these alterations are in part driven by inter-subject genetic variability. Recent findings have shown the neuropeptide neuromedin U (NMU) and its receptor NMU receptor 2 (NMUR2) decrease drug-related behaviors, but it is unknown if substances of abuse alter NMU or NMUR2 expression. Here, rats were given twice daily saline or cocaine (15 mg/kg, intraperitoneal (IP)) for 5 days and then 7 days with no treatment. All rats were then given a single cocaine treatment and locomotor activity was measured in the acute (non-sensitized) and repeated drug exposure (sensitized) groups. Immediately following locomotor assay, tissue was taken and we demonstrate that accumbal NMUR2 mRNA expression, but not NMU mRNA expression, is negatively correlated with non-sensitized cocaine-evoked locomotor activity, but the correlation is lost following cocaine sensitization. Furthermore, in a separate cohort NMUR2 protein levels also negatively correlated with cocaine-evoked locomotor activity based on immunohistochemical stereology for NMUR2 protein expression. These findings are the first to demonstrate that repeated cocaine exposure causes dysregulated expression of NMUR2 and highlight the deleterious effects of repeated cocaine exposure on neurobiological receptor systems. Restoring the normal function of NMUR2 could be beneficial to the treatment of CUD.

Small-Molecule Neuromedin U Receptor 2 Agonists Suppress Food Intake and Decrease Visceral Fat in Animal Models.

Obesity is a growing public health concern, with 37.5% of the adult population in need of therapeutics that are more efficacious with a better side effect profile. An innovative target in this regard is neuromedin U, a neuropeptide shown to suppress food intake and attenuate weight gain in animal models. These effects of neuromedin U on feeding behavior are thought to be related to agonism at the centrally expressed neuromedin U receptor 2 (NMUR2). As peptides present unique challenges that limit their therapeutic potential, the discovery of small-molecule NMUR2 agonists is needed to validate the targets therapeutic value, but to date, none have been evaluated in any animal model of disease. We therefore assessed two small-molecule NMUR2 agonists for their in vitro signaling and their in vivo efficacy. The NMUR2 agonists were synthesized and both NMUR2 agonists, NY0116 and NY0128, decreased cAMP while stimulating calcium signaling in stably expressing NMUR2 HEK293 cells. When small-molecule NMUR2 agonists were tested in vivo, acute administration significantly decreased high-fat diet consumption. Repeated administration of the compounds decreased body weight and more specifically, decreased the percentage of visceral adipose tissue (VAT) in obese mice. These results have confirmed small-molecule NMUR2 agonists are efficacious in animal models to decrease fat content, food intake, and body weight, suggesting NMUR2 is a promising therapeutic target for metabolic disorders.

Environmental Enrichment and Social Isolation Mediate Neuroplasticity of Medium Spiny Neurons through the GSK3 Pathway.

Resilience and vulnerability to neuropsychiatric disorders are linked to molecular changes underlying excitability that are still poorly understood. Here, we identify glycogen-synthase kinase 3ß (GSK3ß) and voltage-gated Na+ channel Nav1.6 as regulators of neuroplasticity induced by environmentally enriched (EC) or isolated (IC) conditions-models for resilience and vulnerability. Transcriptomic studies in the nucleus accumbens from EC and IC rats predicted low levels of GSK3ß and SCN8A mRNA as a protective phenotype associated with reduced excitability in medium spiny neurons (MSNs). In vivo genetic manipulations demonstrate that GSK3ß and Nav1.6 are molecular determinants of MSN excitability and that silencing of GSK3ß prevents maladaptive plasticity of IC MSNs. In vitro studies reveal direct interaction of GSK3ß with Nav1.6 and phosphorylation at Nav1.6T1936 by GSK3ß. A GSK3ß-Nav1.6T1936 competing peptide reduces MSNs excitability in IC, but not EC rats. These results identify GSK3ß regulation of Nav1.6 as a biosignature of MSNs maladaptive plasticity.

Gamma-Aminobutyric Acidergic Projections From the Dorsal Raphe to the Nucleus Accumbens Are Regulated by Neuromedin U.

BACKGROUND: Neuromedin U (NMU) is a neuropeptide enriched in the nucleus accumbens shell (NAcSh), a brain region associated with reward. While NMU and its receptor, NMU receptor 2 (NMUR2), have been studied for the ability to regulate food reward, NMU has not been studied in the context of drugs of abuse (e.g., cocaine). Furthermore, the neuroanatomical pathways that express NMUR2 and its ultrastructural localization are unknown. METHODS: Immunohistochemistry was used to determine the synaptic localization of NMUR2 in the NAcSh and characterize which neurons express this receptor (n = 17). The functional outcome of NMU on NMUR2 was examined using microdialysis (n = 16). The behavioral effects of NMU microinjection directly to the NAcSh were investigated using cocaine-evoked locomotion (n = 93). The specific effects of NMUR2 knockdown on cocaine-evoked locomotion were evaluated using viral-mediated RNA interference (n = 40). RESULTS: NMUR2 is localized to presynaptic gamma-aminobutyric acidergic nerve terminals in the NAcSh originating from the dorsal raphe nucleus. Furthermore, NMU microinjection to the NAcSh decreased local gamma-aminobutyric acid concentrations. Next, we evaluated the effects of NMU microinjection on behavioral sensitization to cocaine. When repeatedly administered throughout the sensitization regimen, NMU attenuated cocaine-evoked hyperactivity. Additionally, small hairpin RNA-mediated knockdown of presynaptic NMUR2 in the NAcSh using a retrograde viral vector potentiated cocaine sensitization. CONCLUSIONS: Together, these data reveal that NMUR2 modulates a novel gamma-aminobutyric acidergic pathway from the dorsal raphe nucleus to the NAcSh to influence behavioral responses to cocaine.

Serotonin-2C receptor agonists decrease potassium-stimulated GABA release in the nucleus accumbens.

The serotonin 5-HT2C receptor has shown promise in vivo as a pharmacotherapeutic target for alcoholism. For example, recently, a novel 4-phenyl-2-N,N-dimethylaminotetralin (PAT) drug candidate, that demonstrates 5-HT2C receptor agonist activity together with 5-HT2A/2B receptor inverse agonist activity, was shown to reduce operant responding for ethanol after peripheral administration to rats. Previous studies have shown that the 5-HT2C receptor is found throughout the mesoaccumbens pathway and that 5-HT2C receptor agonism causes activation of ventral tegmental area (VTA) GABA neurons. It is unknown what effect 5-HT2C receptor modulation has on GABA release in the nucleus accumbens core (NAcc). To this end, microdialysis coupled to capillary electrophoresis with laser-induced fluorescence was used to quantify extracellular neurotransmitter concentrations in the NAcc under basal and after potassium stimulation conditions, in response to PAT analogs and other 5-HT2C receptor modulators administered by reverse dialysis to rats. 5-HT2C receptor agonists specifically attenuated stimulated GABA release in the NAcc while 5-HT2C antagonists or inverse agonists had no effect. Agents with activity at 5-HT2A receptors had no effect on GABA release. Thus, in contrast to results reported for the VTA, current results suggest 5-HT2C receptor agonists decrease stimulated GABA release in the NAcc, and provide a possible mechanism of action for 5HT2C -mediated negative modulation of ethanol self-administration.

Fat Preference: a novel model of eating behavior in rats.

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied. To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

The serotonin-2 receptor modulator, (-)-trans-PAT, decreases voluntary ethanol consumption in rats.

Serotonin (5-HT) 5-HT2C receptor agonists have shown promise as novel alcoholism pharmacotherapies, but developing selective agonists has been problematic. Female Sprague Dawley rats were given ethanol in a palatable gel vehicle during operant sessions. 5-HT2C receptor modulators (Ro60-0175, SB242,084, and (-)-trans-PAT) were administered before operant sessions. As a control for the effects of 5-HT2C receptor agonism on caloric intake, drugs were also tested using non-ethanol containing gelatin. Ro60-0175, a 5-HT2 family receptor agonist, decreased both ethanol and vehicle responding while (-)-trans-PAT, a 5-HT2C receptor agonist with 5-HT2A-2B receptor inverse agonist activity, selectively reduced only ethanol responding. The effect of 5-HT2C receptor agonists on self-administration after reinstatement of ethanol after a three week deprivation was also determined. (-)-trans-PAT eliminated increases in ethanol intake following ethanol deprivation whereas Ro60-0175 had no effect. These results emphasize the need for caloric controls and further support the idea that selective modulation of 5-HT2 family receptors is a potential pharmacotherapeutic approach in the treatment of alcoholism.

Wnt inhibitor screen reveals iron dependence of ß-catenin signaling in cancers.

Excessive signaling from the Wnt pathway is associated with numerous human cancers. Using a high throughput screen designed to detect inhibitors of Wnt/ß-catenin signaling, we identified a series of acyl hydrazones that act downstream of the ß-catenin destruction complex to inhibit both Wnt-induced and cancer-associated constitutive Wnt signaling via destabilization of ß-catenin. We found that these acyl hydrazones bind iron in vitro and in intact cells and that chelating activity is required to abrogate Wnt signaling and block the growth of colorectal cancer cell lines with constitutive Wnt signaling. In addition, we found that multiple iron chelators, desferrioxamine, deferasirox, and ciclopirox olamine similarly blocked Wnt signaling and cell growth. Moreover, in patients with AML administered ciclopirox olamine, we observed decreased expression of the Wnt target gene AXIN2 in leukemic cells. The novel class of acyl hydrazones would thus be prime candidates for further development as chemotherapeutic agents. Taken together, our results reveal a critical requirement for iron in Wnt signaling and they show that iron chelation serves as an effective mechanism to inhibit Wnt signaling in humans.

New sources of drugs for hematologic malignancies.

Advancing novel therapeutic agents for the treatment of malignancy into the marketplace is an increasingly costly and lengthy process. As such, new strategies for drug discovery are needed. Drug repurposing represents an opportunity to rapidly advance new therapeutic strategies into clinical trials at a relatively low cost. Known on-patent or off-patent drugs with unrecognized anticancer activity can be rapidly advanced into clinical testing for this new indication by leveraging their known pharmacology, pharmacokinetics, and toxicology. Using this approach, academic groups can participate in the drug discovery field and smaller biotechnology companies can "de-risk" early-stage drug discovery projects. Here, several scientific approaches used to identify drug repurposing opportunities are highlighted, with a focus on hematologic malignancies. In addition, a discussion of the regulatory issues that are unique to drug repurposing and how they impact developing old drugs for new indications is included. Finally, the mechanisms to enhance drug repurposing through increased collaborations between academia, industry, and nonprofit charitable organizations are discussed.

Human cadaveric study of subscapularis muscle innervation and guidelines to prevent denervation.

The upper and lower subscapular nerves provide innervation to the subscapularis muscle. However, the axillary nerve may provide a significant innervation to the lower portion of the muscle. The prevalence and patterns of anomalous innervation of the subscapularis muscle were studied to determine if these variations increased the risk of muscle denervation during open shoulder surgery. Twenty human cadaveric shoulders were dissected, and the innervation to the subscapularis was defined. The distance from the nerve insertion to the shoulder joint was measured in neutral and maximal external rotation. In the most common variation, the lower subscapular nerve arose from the axillary nerve (5 specimens; 25%). Although external rotation of the shoulder brought the nerve insertion significantly more lateral (35.2 to 16.9 mm, P < .001), the origin of the nerve had no significant effect on nerve proximity to the joint. The closeness of the nerve insertions to the shoulder joint warrants care during an anterior approach to the shoulder and dissections on the anterior surface of the muscle. Subscapularis nerve damage or denervation may cause unexplained joint instability and subscapularis dysfunction.