Parkinson's disease (PD) is caused by the death of dopamine neurons in the basal ganglia and results in motor symptoms such as tremor and bradykinesia. Activation of metabotropic glutamate receptor 4 (mGluR4) has been shown to modulate neurotransmission in the basal ganglia and results in antiparkinsonian effects in rodent PD models. N-Phenyl-7-(hydroxyimino)cyclopropa [b]chromen-1a-carboxamide (PHCCC) is a positive allosteric modulator (PAM) of mGluR4 that has been used to further validate the role of mGluR4 in PD, but the compound suffers from a lack of selectivity, relatively low potency, and poor solubility. Via high-throughput screening, we discovered more than 400 novel PAMs of mGluR4. Compounds derived from a novel chemical scaffold were characterized in vitro at both rat and human mGluR4 using two distinct assays of mGluR4 function. The lead compound was approximately 8-fold more potent than PHCCC, enhanced the potency of glutamate at mGluR4 by 8-fold, and did not show any significant potentiator or antagonist activity at other mGluR subtypes. Resolution of the regioisomers of the lead revealed that the cis regioisomer, (Ϯ)-cis-2-(3,5-dichlorphenylcarbamoyl)cyclohexanecarboxylic acid (VU0155041), contained the majority of the mGluR4 PAM activity and also exhibited partial agonist activity at mGluR4 at a site that was distinct from the glutamate binding site, suggesting that this compound is a mixed allosteric agonist/PAM of mGluR4. VU0155041 was soluble in an aqueous vehicle, and intracerebroventricular administration of 31 to 316 nmol of VU0155041 dose-dependently decreased haloperidol-induced catalepsy and reserpine-induced akinesia in rats. These exciting results provide continued support for mGluR4 as a therapeutic target in PD.Metabotropic glutamate receptors (mGluRs) play important roles in a broad range of central nervous system functions and have therapeutic potential in a variety of neurological and psychiatric disorders (Niswender et al., 2005). mGluRs are G protein-coupled receptors (GPCRs) classified into three major groups, groups I, II, and III, based on their sequence homology, signal transduction profile, and ligand binding specificity. The group III mGluRs (mGluRs 4, 6, 7,

Highly selective positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGluR5) have emerged as a potential approach to treat positive symptoms associated with schizophrenia. mGluR5 plays an important role in both long term potentiation (LTP) and long term depression (LTD), suggesting that mGluR5 PAMs may also have utility in improving impaired cognitive function. However, if mGluR5 PAMs shift the balance of LTP and LTD or induce a state in which afferent activity induces lasting changes in synaptic function that are not appropriate for a given pattern of activity, this could disrupt rather than enhance cognitive function. We determined the effect of selective mGluR5 PAMs on induction of LTP and LTD at the Schaffer collateral – CA1 synapse in the hippocampus. mGluR5-selective PAMs significantly enhanced threshold theta burst stimulation (TBS)-induced LTP. In addition, mGluR5 PAMs enhanced both DHPG-induced LTD and LTD induced by delivery of paired-pulse low frequency stimulation. Selective potentiation of mGluR5 had no effect on LTP induced by suprathreshold TBS or saturated LTP. The finding that potentiation of mGluR5-mediated responses to stimulation of glutamatergic afferents enhances both LTP and LTD supports the hypothesis that activation of mGluR5 by endogenous glutamate contributes to both forms of plasticity. Furthermore, two systemically active mGluR5 PAMs enhanced performance in the Morris water maze, a measure of hippocampus-dependent spatial learning. Discovery of small molecules that enhance both LTP and LTD in an activity-appropriate manner demonstrates a unique action on synaptic plasticity that may provide a novel approach for treatment of impaired cognitive function.

Muscarinic acetylcholine receptors (mAChRs) provide viable targets for the treatment of multiple central nervous system disorders. We have used cheminformatics and medicinal chemistry to develop new, highly selective M4 allosteric potentiators. VU10010, the lead compound, potentiates the M4 response to acetylcholine 47-fold while having no activity at other mAChR subtypes. This compound binds to an allosteric site on the receptor and increases affinity for acetylcholine and coupling to G proteins. Whole-cell patch clamp recordings revealed that selective potentiation of M4 with VU10010 increases carbachol-induced depression of transmission at excitatory but not inhibitory synapses in the hippocampus. The effect was not mimicked by an inactive analog of VU10010 and was absent in M4 knockout mice. Selective regulation of excitatory transmission by M4 suggests that targeting of individual mAChR subtypes could be used to differentially regulate specific aspects of mAChR modulation of function in this important forebrain structure.

The group III metabotropic glutamate receptors (mGluRs) represent a family of presynaptically expressed G-protein-coupled receptors (GPCRs) with enormous therapeutic potential; however, robust cellular assays to study their function have been difficult to develop. We present here a new assay, compatible with traditional high-throughput screening platforms, to detect activity of pharmacological ligands interacting with G i/o -coupled GPCRs, including the group III mGluRs 4, 7, and 8. The assay takes advantage of the ability of the G␤␥ subunits of G i and G o heterotrimers to interact with G-protein regulated inwardly rectifying potassium channels (GIRKs), and we show here that we are able to detect the activity of multiple types of pharmacophores including agonists, antagonists, and allosteric modulators of several distinct GPCRs. Using GIRK-mediated thallium flux, we perform a side-by-side comparison of the activity of a number of commercially available compounds, some of which have not been extensively evaluated because of the previous lack of robust assays at each of the three major group III mGluRs. It is noteworthy that several compounds previously considered to be general group III mGluR antagonists have very weak activity using this assay, suggesting the possibility that these compounds may not effectively inhibit these receptors in native systems. We anticipate that the GIRKmediated thallium flux strategy will provide a novel tool to advance the study of G i/o -coupled GPCR biology and promote ligand discovery and characterization.The metabotropic glutamate receptors (mGluRs) are among the most abundantly expressed receptors in the mammalian central nervous system and are thought to play critical roles in regulating activity in a broad range of CNS circuits (Conn and Pin, 1997). Despite an increasing appreciation of the involvement of mGluRs in CNS function, however, progress in understanding the signaling pathways and functional properties of some members of the mGluR family has been relatively slow. This is especially true for the group III mGluRs, which include mGluRs 4, 6, 7, and 8.

Pharmacological inhibition of the dipeptidyl peptidase-4 (DPP4) enzyme potentiates incretin action and is widely used to treat type 2 diabetes. Nevertheless, the precise cells and tissues critical for incretin degradation and glucose homeostasis remain unknown. Here, we use mouse genetics and pharmacologic DPP4 inhibition to identify DPP4 cell types essential for incretin action. Although enterocyte DPP4 accounted for substantial intestinal DPP4 activity, ablation of enterocyte DPP4 in Dpp4 mice did not produce alterations in plasma DPP4 activity, incretin hormone levels, and glucose tolerance. In contrast, endothelial cell (EC)-derived DPP4 contributed substantially to levels of soluble plasma DPP4 activity, incretin degradation, and glucose control. Surprisingly, DPP4 cells of bone marrow origin mediated the selective degradation of fasting GIP, but not GLP-1. Collectively, these findings identify distinct roles for DPP4 in the EC versus the bone marrow compartment for selective incretin degradation and DPP4i-mediated glucoregulation.

Pharmacological activation of the hypothalamic glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) promotes weight loss and improves glucose tolerance. This demonstrates that the hypothalamic GLP-1R is sufficient but does not show whether it is necessary for the effects of exogenous GLP-1R agonists (GLP-1RA) or endogenous GLP-1 on these parameters. To address this, we crossed mice harboring floxed Glp1r alleles to mice expressing Nkx2.1-Cre to knock down Glp1r expression throughout the hypothalamus (GLP-1RKDΔNkx2.1cre). We also generated mice lacking Glp1r expression specifically in two GLP-1RA–responsive hypothalamic feeding nuclei/cell types, the paraventricular nucleus (GLP-1RKDΔSim1cre) and proopiomelanocortin neurons (GLP-1RKDΔPOMCcre). Chow-fed GLP-1RKDΔNkx2.1cre mice exhibited increased food intake and energy expenditure with no net effect on body weight. When fed a high-fat diet, these mice exhibited normal food intake but elevated energy expenditure, yielding reduced weight gain. None of these phenotypes were observed in GLP-1RKDΔSim1cre and GLP-1RKDΔPOMCcre mice. The acute anorectic and glucose tolerance effects of peripherally dosed GLP-1RA exendin-4 and liraglutide were preserved in all mouse lines. Chronic liraglutide treatment reduced body weight in chow-fed GLP-1RKDΔNkx2.1cre mice, but this effect was attenuated with high-fat diet feeding. In sum, classic homeostatic control regions are sufficient but not individually necessary for the effects of GLP-1RA on nutrient homeostasis.

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SummaryGroup III metabotropic glutamate receptors (mGluRs) reduce synaptic transmission at the Schaffer collateral-CA1 (SC-CA1) synapse in rats by a presynaptic mechanism. Previous studies show that low concentrations of the group III-selective agonist, L-AP4, reduce synaptic transmission in slices from neonatal but not adult rats, whereas high micromolar concentrations reduce transmission in both age groups. L-AP4 activates mGluRs 4 and 8 at much lower concentrations than those required to activate mGluR7, suggesting that the group III mGluR subtype modulating transmission is a high affinity receptor in neonates and a low affinity receptor in adults. The previous lack of subtype selective ligands has made it difficult to test this hypothesis. We have measured fEPSPs in the presence of novel subtype selective agents to address this question. We show that the effects of L-AP4 can be blocked by LY341495 in both neonates and adults, verifying that these effects are mediated by mGluRs. In addition, the selective mGluR8 agonist, DCPG, has a significant effect in slices from neonatal rats but does not reduce synaptic transmission in adult slices. The mGluR4 selective allosteric potentiator, PHCCC, is unable to potentiate the L-AP4-induced effects at either age. Taken together, our data suggest that group III mGluRs regulate transmission at the SC-CA1 synapse throughout development but there is a developmental regulation of the subtypes involved so that that both mGluR8 serves this role in neonates but not adults whereas mGluR7 is involved in regulating transmission at this synapse in throughout postnatal development.