Jonathan D. Hommel

Leptin Receptor Signaling in Midbrain Dopamine Neurons Regulates Feeding

The leptin hormone is critical for normal food intake and metabolism. While leptin receptor (Lepr) function has been well studied in the hypothalamus, the functional relevance of Lepr expression in the ventral tegmental area (VTA) has not been investigated. The VTA contains dopamine neurons that are important in modulating motivated behavior, addiction, and reward. Here, we show that VTA dopamine neurons express Lepr mRNA and respond to leptin with activation of an intracellular JAK-STAT pathway and a reduction in firing rate. Direct administration of leptin to the VTA caused decreased food intake while long-term RNAi-mediated knockdown of Lepr in the VTA led to increased food intake, locomotor activity, and sensitivity to highly palatable food. These data support a critical role for VTA Lepr in regulating feeding behavior and provide functional evidence for direct action of a peripheral metabolic signal on VTA dopamine neurons.

Local gene knockdown in the brain using viral-mediated RNA interference

Conditional mutant techniques that allow spatial and temporal control over gene expression can be used to create mice with restricted genetic modifications. These mice serve as powerful disease models in which gene function in adult tissues can be specifically dissected. Current strategies for conditional genetic manipulation are inefficient, however, and often lack sufficient spatial control. Here we use viral-mediated RNA interference (RNAi) to generate a specific knockdown of Th, the gene encoding the dopamine synthesis enzyme tyrosine hydroxylase, within midbrain neurons of adult mice. This localized gene knockdown resulted in behavioral changes, including a motor performance deficit and reduced response to a psychostimulant. These results underscore the potential of using viral-mediated RNAi for the rapid production and testing of new genetic disease models. Similar strategies may be used in other model species, and may ultimately find applications in human gene therapy.

The hypothalamic neuropeptide melanin-concentrating hormone acts in the nucleus accumbens to modulate feeding behavior and forced-swim performance

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide with a prominent role in feeding and energy homeostasis. The rodent MCH receptor (MCH1R) is highly expressed in the nucleus accumbens shell (AcSh), a region that is important in the regulation of appetitive behavior. Here we establish a role for MCH and MCH1R in mediating a hypothalamic-limbic circuit that regulates feeding and related behaviors. Direct delivery of an MCH1R receptor antagonist to the AcSh blocked feeding and produced an antidepressant-like effect in the forced swim test, whereas intra-AcSh injection of MCH had the opposite effect. Expression studies demonstrated that MCH1R is present in both the enkephalin- and dynorphin-positive medium spiny neurons of the AcSh. Biochemical analysis in AcSh explants showed that MCH signaling blocks dopamine-induced phosphorylation of the AMPA glutamate receptor subunit GluR1 at Ser845. Finally, food deprivation, but not other stressors, stimulated cAMP response element-binding protein-dependent pathways selectively in MCH neurons of the hypothalamus, suggesting that these neurons are responsive to a specific set of physiologically relevant conditions. This work identifies a novel hypothalamic-AcSh circuit that influences appetitive behavior and mediates the antidepressant activity of MCH1R antagonists.

Induction of Inducible cAMP Early Repressor Expression in Nucleus Accumbens by Stress or Amphetamine Increases Behavioral Responses to Emotional Stimuli

Previous research has shown that cAMP response element (CRE)-mediated transcription is activated in the nucleus accumbens, a major brain reward region, by a variety of environmental stimuli and contributes to neuroadaptations to these stimuli. CRE-binding protein (CREB) is the most studied activator of CRE transcription and has been implicated in this brain region as a gating mechanism for behavioral responses to emotional stimuli. Little attention, however, has been given to naturally occurring inhibitors of CRE-mediated transcription, such as the inducible cAMP early repressor (ICER), an inhibitory product of the CRE modulator gene. In the present study, we investigated the extent to which ICER is induced in the nucleus accumbens by two types of environmental stimuli, stress and amphetamine, and characterized how induction of ICER in this region affects complex behavior. We show that stress and amphetamine each induces ICER expression and that overexpression of ICER in the nucleus accumbens, using viral-mediated gene transfer, increases behavioral responses to both rewarding and aversive emotional stimuli. For example, ICER overexpression increases sensitivity to amphetamine-stimulated locomotor activity as well as to natural rewards such as sucrose and social grooming. However, ICER overexpression also increases measures of anxiety in the elevated plus maze and neophobia to novel tastes. Finally, ICER produces an antidepressant-like effect in the forced swim test, further indication of an enhanced active response to stress. These results suggest that ICER is an important mechanism for modulating CRE-mediated transcription in the nucleus accumbens.

Enhanced Leptin Sensitivity, Reduced Adiposity, and Improved Glucose Homeostasis in Mice Lacking Exchange Protein Directly Activated by Cyclic AMP Isoform 1

ABSTRACT The prototypic second messenger cyclic AMP (cAMP) is essential for controlling cellular metabolism, including glucose and lipid homeostasis. In mammals, the majority of cAMP functions are mediated by cAMP-dependent protein kinase (PKA) and exchange proteins directly activated by cAMP (Epacs). To explore the physiological functions of Epac1, we generated Epac1 knockout mice. Here we report that Epac1 null mutants have reduced white adipose tissue and reduced plasma leptin levels but display heightened leptin sensitivity. Epac1-deficient mice are more resistant to high-fat diet-induced obesity, hyperleptinemia, and glucose intolerance. Furthermore, pharmacological inhibition of Epac by use of an Epac-specific inhibitor reduces plasma leptin levels in vivo and enhances leptin signaling in organotypic hypothalamic slices. Taken together, our results demonstrate that Epac1 plays an important role in regulating adiposity and energy balance.

Regulation of Ingestive Behaviors in the Rat by GSK1521498, a Novel μ-Opioid Receptor-Selective Inverse Agonist

μ-Opioid receptor (MOR) agonism induces palatable food consumption principally through modulation of the rewarding properties of food. N-{[3,5-difluoro-3′-(1H-1,2,4-triazol-3-yl)-4-biphenylyl]methyl}-2,3-dihydro-1H-inden-2-amine (GSK1521498) is a novel opioid receptor inverse agonist that, on the basis of in vitro affinity assays, is greater than 10- or 50-fold selective for human or rat MOR, respectively, compared with κ-opioid receptors (KOR) and δ-opioid receptors (DOR). Likewise, preferential MOR occupancy versus KOR and DOR was observed by autoradiography in brain slices from Long Evans rats dosed orally with the drug. GSK1521498 suppressed nocturnal food consumption of standard or palatable chow in lean and diet-induced obese (DIO) Long Evans rats. Both the dose-response relationship and time course of efficacy in lean rats fed palatable chow correlated with μ receptor occupancy and the plasma concentration profile of the drug. Chronic oral administration of GSK1521498 induced body weight loss in DIO rats, which comprised fat mass reduction. The reduction in body weight was equivalent to the cumulative reduction in food consumption; thus, the effect of GSK1521498 on body weight is related to inhibition of food consumption. GSK1521498 suppressed the preference for sucrose-containing solutions in lean rats. In operant response models also using lean rats, GSK1521498 reduced the reinforcement efficacy of palatable food reward and enhanced satiety. In conclusion, GSK1521498 is a potent, MOR-selective inverse agonist that modulates the hedonic aspects of ingestion and, therefore, could represent a pharmacological treatment for obesity and binge-eating disorders.

Neuromedin U receptor 2 knockdown in the paraventricular nucleus modifies behavioral responses to obesogenic high-fat food and leads to increased body weight

Neuromedin U (NMU) is a highly conserved neuropeptide which regulates food intake and body weight. Transgenic mice lacking NMU are hyperphagic and obese, making NMU a novel target for understanding and treating obesity. Neuromedin U receptor 2 (NMUR2) is a high-affinity receptor for NMU found in discrete regions of the central nervous system, in particular the paraventricular nucleus of the hypothalamus (PVN), where it may be responsible for mediating the anorectic effects of NMU. We hypothesized that selective knock down of NMUR2 in the PVN of rats would increase their sensitivity to the reinforcing properties of food resulting in increased intake and preference for high-fat obesogenic food. To this end, we used viral-mediated RNAi to selectively knock down NMUR2 gene expression in the PVN. In rats fed a standard chow, NMUR2 knockdown produced no significant effect on food intake or body weight. However, when the same rats were fed a high-fat diet (45% fat), they consumed significantly more food, gained more body weight, and had increased feed efficiency relative to controls. Furthermore, NMUR2 knockdown rats demonstrated significantly greater binge-type food consumption of the high-fat diet and showed a greater preference for higher-fat food. These results demonstrate that NMUR2 signaling in the PVN regulates consumption and preference for high-fat foods without disrupting feeding behavior associated with non-obesogenic standard chow.

Characterization of a Novel Origin Recognition Complex-Like Complex: Implications for DNA Recognition, Cell Cycle Control, and Locus-Specific Gene Amplification

ABSTRACT The origin recognition complex (ORC) plays a central role in eukaryotic DNA replication. Here we describe a unique ORC-like complex in Tetrahymena thermophila, TIF4, which bound in an ATP-dependent manner to sequences required for cell cycle-controlled replication and gene amplification (ribosomal DNA [rDNA] type I elements). TIF4s mode of DNA recognition was distinct from that of other characterized ORCs, as it bound exclusively to single-stranded DNA. In contrast to yeast ORCs, TIF4 DNA binding activity was cell cycle regulated and peaked during S phase, coincident with the redistribution of the Orc2-related subunit, p69, from the cytoplasm to the macronucleus. Origin-binding activity and nuclear p69 immunoreactivity were further regulated during development, where they distinguished replicating from nonreplicating nuclei. Both activities were lost from germ line micronuclei following the programmed arrest of micronuclear replication. Replicating macronuclei stained with Orc2 antibodies throughout development in wild-type cells but failed to do so in the amplification-defective rmm11 mutant. Collectively, these findings indicate that the regulation of TIF4 is intimately tied to the cell cycle and developmentally programmed replication cycles. They further implicate TIF4 in rDNA gene amplification. As type I elements interact with other sequence-specific single-strand breaks (in vitro and in vivo), the dynamic interplay of Orc-like (TIF4) and non-ORC-like proteins with this replication determinant may provide a novel mechanism for regulation.

Structure–Activity Relationship for Small Molecule Inhibitors of Nicotinamide N-Methyltransferase

Nicotinamide N-methyltransferase (NNMT) is a fundamental cytosolic biotransforming enzyme that catalyzes the N-methylation of endogenous and exogenous xenobiotics. We have identified small molecule inhibitors of NNMT with >1000-fold range of activity and developed comprehensive structure-activity relationships (SARs) for NNMT inhibitors. Screening of N-methylated quinolinium, isoquinolinium, pyrididium, and benzimidazolium/benzothiazolium analogues resulted in the identification of quinoliniums as a promising scaffold with very low micromolar (IC50 ∼ 1 μM) NNMT inhibition. Computer-based docking of inhibitors to the NNMT substrate (nicotinamide)-binding site produced a robust correlation between ligand-enzyme interaction docking scores and experimentally calculated IC50 values. Predicted binding orientation of the quinolinium analogues revealed selective binding to the NNMT substrate-binding site residues and essential chemical features driving protein-ligand intermolecular interactions and NNMT inhibition. The development of this new series of small molecule NNMT inhibitors direct the future design of lead drug-like inhibitors to treat several metabolic and chronic disease conditions characterized by abnormal NNMT activity.

Glycogen synthase kinase 3 beta alters anxiety-, depression-, and addiction-related behaviors and neuronal activity in the nucleus accumbens shell

&NA; Psychiatric disorders such as anxiety, depression and addiction are often comorbid brain pathologies thought to share common mechanistic biology. As part of the cortico‐limbic circuit, the nucleus accumbens shell (NAcSh) plays a fundamental role in integrating information in the circuit, such that modulation of NAcSh circuitry alters anxiety, depression, and addiction‐related behaviors. Intracellular kinase cascades in the NAcSh have proven important mediators of behavior. To investigate glycogen‐synthase kinase 3 (GSK3) beta signaling in the NAcSh in vivo we knocked down GSK3beta expression with a novel adeno‐associated viral vector (AAV2) and assessed changes in anxiety‐ and depression‐like behavior and cocaine self‐administration in GSK3beta knockdown rats. GSK3beta knockdown reduced anxiety‐like behavior while increasing depression‐like behavior and cocaine self‐administration. Correlative electrophysiological recordings in acute brain slices were used to assess the effect of AAV‐shGSK3beta on spontaneous firing and intrinsic excitability of tonically active interneurons (TANs), cells required for input and output signal integration in the NAcSh and for processing reward‐related behaviors. Loose‐patch recordings showed that TANs transduced by AAV‐shGSK3beta exhibited reduction in tonic firing and increased spike half width. When assessed by whole‐cell patch clamp recordings these changes were mirrored by reduction in action potential firing and accompanied by decreased hyperpolarization‐induced depolarizing sag potentials, increased action potential current threshold, and decreased maximum rise time. These results suggest that silencing of GSK3beta in the NAcSh increases depression‐ and addiction‐related behavior, possibly by decreasing intrinsic excitability of TANs. However, this study does not rule out contributions from other neuronal sub‐types. HighlightsSpecific knockdown of GSK3 beta in the NAc shell induces an anxiolytic‐like effect.Knockdown of GSK3 beta in the NAcSh induces depression‐like behavior.Viral‐mediated knockdown of GSK3 beta increases cocaine self‐administration.Knockdown also reduces spontaneous firing and alters intrinsic excitability of TANs.