Differential activity of mGlu7 allosteric modulators provides evidence for mGlu7/8 heterodimers at hippocampal Schaffer collateral-CA1 synapses.

Glutamate acts at eight metabotropic glutamate (mGlu) receptor subtypes expressed in a partially overlapping fashion in distinct brain circuits. Recent evidence indicates that specific mGlu receptor protomers can heterodimerize and that these heterodimers can exhibit different pharmacology when compared to their homodimeric counterparts. Group III mGlu agonist-induced suppression of evoked excitatory potentials and induction of long-term potentiation at Schaffer collateral-CA1 (SC-CA1) synapses in the rodent hippocampus can be blocked by the selective mGlu7 negative allosteric modulator (NAM), ADX71743. Curiously, a different mGlu7 NAM, 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-c]pyridin-4(5H)-one, failed to block these responses in brain slices despite its robust activity at mGlu7 homodimers in vitro. We hypothesized that this might result from heterodimerization of mGlu7 with another mGlu receptor protomer and focused on mGlu8 as a candidate given the reported effects of mGlu8-targeted compounds in the hippocampus. Here, we used complemented donor acceptor-resonance energy transfer to study mGlu7/8 heterodimer activation in vitro and observed that ADX71743 blocked responses of both mGlu7/7 homodimers and mGlu7/8 heterodimers, whereas 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-c]pyridin-4(5H)-one only antagonized responses of mGlu7/7 homodimers. Taken together with our electrophysiology observations, these results suggest that a receptor with pharmacology consistent with an mGlu7/8 heterodimer modulates the activity of SC-CA1 synapses. Building on this hypothesis, we identified two additional structurally related mGlu7 NAMs that also differ in their activity at mGlu7/8 heterodimers, in a manner consistent with their ability to inhibit synaptic transmission and plasticity at SC-CA1. Thus, we propose that mGlu7/8 heterodimers are a key molecular target for modulating the activity of hippocampal SC-CA1 synapses.

mGluR5 Negative Modulators for Fragile X: Treatment Resistance and Persistence.

Fragile X syndrome (FXS) is caused by silencing of the human FMR1 gene and is the leading monogenic cause of intellectual disability and autism. Abundant preclinical data indicated that negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGluR5) might be efficacious in treating FXS in humans. Initial attempts to translate these findings in clinical trials have failed, but these failures provide the opportunity for new discoveries that will improve future trials. The emergence of acquired treatment resistance ("tolerance") after chronic administration of mGluR5 NAMs is a potential factor in the lack of success. Here we confirm that FXS model mice display acquired treatment resistance after chronic treatment with the mGluR5 NAM CTEP in three assays commonly examined in the mouse model of FXS: (1) audiogenic seizure susceptibility, (2) sensory cortex hyperexcitability, and (3) hippocampal protein synthesis. Cross-tolerance experiments suggest that the mechanism of treatment resistance likely occurs at signaling nodes downstream of glycogen synthase kinase 3α (GSK3α), but upstream of protein synthesis. The rapid emergence of tolerance to CTEP begs the question of how previous studies showed an improvement in inhibitory avoidance (IA) cognitive performance after chronic treatment. We show here that this observation was likely explained by timely inhibition of mGluR5 during a critical period, as brief CTEP treatment in juvenile mice is sufficient to provide a persistent improvement of IA behavior measured many weeks later. These data will be important to consider when designing future fragile X clinical trials using compounds that target the mGluR5-to-protein synthesis signaling cascade.

Selective inhibition of glycogen synthase kinase 3α corrects pathophysiology in a mouse model of fragile X syndrome.

Fragile X syndrome is caused by FMR1 gene silencing and loss of the encoded fragile X mental retardation protein (FMRP), which binds to mRNA and regulates translation. Studies in the Fmr1-/y mouse model of fragile X syndrome indicate that aberrant cerebral protein synthesis downstream of metabotropic glutamate receptor 5 (mGluR5) signaling contributes to disease pathogenesis, but clinical trials using mGluR5 inhibitors were not successful. Animal studies suggested that treatment with lithium might be an alternative approach. Targets of lithium include paralogs of glycogen synthase kinase 3 (GSK3), and nonselective small-molecule inhibitors of these enzymes improved disease phenotypes in a fragile X syndrome mouse model. However, the potential therapeutic use of GSK3 inhibitors has been hampered by toxicity arising from inhibition of both α and ß paralogs. Recently, we developed GSK3 inhibitors with sufficient paralog selectivity to avoid a known toxic consequence of dual inhibition, that is, increased ß-catenin stabilization. We show here that inhibition of GSK3α, but not GSK3ß, corrected aberrant protein synthesis, audiogenic seizures, and sensory cortex hyperexcitability in Fmr1-/y mice. Although inhibiting either paralog prevented induction of NMDA receptor-dependent long-term depression (LTD) in the hippocampus, only inhibition of GSK3α impaired mGluR5-dependent and protein synthesis-dependent LTD. Inhibition of GSK3α additionally corrected deficits in learning and memory in Fmr1-/y mice; unlike mGluR5 inhibitors, there was no evidence of tachyphylaxis or enhanced psychotomimetic-induced hyperlocomotion. GSK3α selective inhibitors may have potential as a therapeutic approach for treating fragile X syndrome.

mGlu7 potentiation rescues cognitive, social, and respiratory phenotypes in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl-CpG binding protein 2 (MECP2) gene. The cognitive impairments seen in mouse models of RTT correlate with deficits in long-term potentiation (LTP) at Schaffer collateral (SC)-CA1 synapses in the hippocampus. Metabotropic glutamate receptor 7 (mGlu7) is the predominant mGlu receptor expressed presynaptically at SC-CA1 synapses in adult mice, and its activation on GABAergic interneurons is necessary for induction of LTP. We demonstrate that pathogenic mutations in MECP2 reduce mGlu7 protein expression in brain tissue from RTT patients and in MECP2-deficient mouse models. In rodents, this reduction impairs mGlu7-mediated control of synaptic transmission. We show that positive allosteric modulation of mGlu7 activity restores LTP and improves contextual fear learning, novel object recognition, and social memory. Furthermore, mGlu7 positive allosteric modulation decreases apneas in Mecp2+/- mice, suggesting that mGlu7 may be a potential therapeutic target for multiple aspects of the RTT phenotype.

Co-Activation of Metabotropic Glutamate Receptor 3 and Beta-Adrenergic Receptors Modulates Cyclic-AMP and Long-Term Potentiation, and Disrupts Memory Reconsolidation.

Activation of ß-adrenergic receptors (ßARs) enhances both the induction of long-term potentiation (LTP) in hippocampal CA1 pyramidal cells and hippocampal-dependent cognitive function. Interestingly, previous studies reveal that coincident activation of group II metabotropic glutamate (mGlu) receptors with ßARs in the hippocampal astrocytes induces a large increase in cyclic-AMP (cAMP) accumulation and release of adenosine. Adenosine then acts on A1 adenosine receptors at neighboring excitatory Schaffer collateral terminals, which could counteract effects of activation of neuronal ßARs on excitatory transmission. On the basis of this, we postulated that activation of the specific mGlu receptor subtype that mediates this response could inhibit ßAR-mediated effects on hippocampal synaptic plasticity and cognitive function. Using novel mGlu receptor subtype-selective allosteric modulators along with knockout mice we now report that the effects of mGlu2/3 agonists on ßAR-mediated increases in cAMP accumulation are exclusively mediated by mGlu3. Furthermore, mGlu3 activation inhibits the ability of the ßAR agonist isoproterenol to enhance hippocampal LTP, and this effect is absent in slices treated with either a glial toxin or an adenosine A1 receptor antagonist. Finally, systemic administration of the mGlu2/3 agonist LY379268 disrupted contextual fear memory in a manner similar to the effect of the ßAR antagonist propranolol, and this effect was reversed by the mGlu3-negative allosteric modulator VU0650786. Taken together, these data suggest that mGlu3 can influence astrocytic signaling and modulate ßAR-mediated effects on hippocampal synaptic plasticity and cognitive function.

ß-Arrestin2 Couples Metabotropic Glutamate Receptor 5 to Neuronal Protein Synthesis and Is a Potential Target to Treat Fragile X.

Synaptic protein synthesis is essential for modification of the brain by experience and is aberrant in several genetically defined disorders, notably fragile X (FX), a heritable cause of autism and intellectual disability. Neural activity directs local protein synthesis via activation of metabotropic glutamate receptor 5 (mGlu5), yet how mGlu5 couples to the intracellular signaling pathways that regulate mRNA translation is poorly understood. Here, we provide evidence that ß-arrestin2 mediates mGlu5-stimulated protein synthesis in the hippocampus and show that genetic reduction of ß-arrestin2 corrects aberrant synaptic plasticity and cognition in the Fmr1-/y mouse model of FX. Importantly, reducing ß-arrestin2 does not induce psychotomimetic activity associated with full mGlu5 inhibitors and does not affect Gq signaling. Thus, in addition to identifying a key requirement for mGlu5-stimulated protein synthesis, these data suggest that ß-arrestin2-biased negative modulators of mGlu5 offer significant advantages over first-generation inhibitors for the treatment of FX and related disorders.

The Role of mGlu Receptors in Hippocampal Plasticity Deficits in Neurological and Psychiatric Disorders: Implications for Allosteric Modulators as Novel Therapeutic Strategies.

Long-term potentiation (LTP) and long-term depression (LTD) are two distinct forms of synaptic plasticity that have been extensively characterized at the Schaffer collateral-CA1 (SCCA1) synapse and the mossy fiber (MF)-CA3 synapse within the hippocampus, and are postulated to be the molecular underpinning for several cognitive functions. Deficits in LTP and LTD have been implicated in the pathophysiology of several neurological and psychiatric disorders. Therefore, there has been a large effort focused on developing an understanding of the mechanisms underlying these forms of plasticity and novel therapeutic strategies that improve or rescue these plasticity deficits. Among many other targets, the metabotropic glutamate (mGlu) receptors show promise as novel therapeutic candidates for the treatment of these disorders. Among the eight distinct mGlu receptor subtypes (mGlu1-8), the mGlu1,2,3,5,7 subtypes are expressed throughout the hippocampus and have been shown to play important roles in the regulation of synaptic plasticity in this brain area. However, development of therapeutic agents that target these mGlu receptors has been hampered by a lack of subtype-selective compounds. Recently, discovery of allosteric modulators of mGlu receptors has provided novel ligands that are highly selective for individual mGlu receptor subtypes. The mGlu receptors modulate the multiple forms of synaptic plasticity at both SC-CA1 and MF synapses and allosteric modulators of mGlu receptors have emerged as potential therapeutic agents that may rescue plasticity deficits and improve cognitive function in patients suffering from multiple neurological and psychiatric disorders.

mGlu5 positive allosteric modulation normalizes synaptic plasticity defects and motor phenotypes in a mouse model of Rett syndrome.

Rett syndrome (RS) is a neurodevelopmental disorder that shares many symptomatic and pathological commonalities with idiopathic autism. Alterations in protein synthesis-dependent synaptic plasticity (PSDSP) are a hallmark of a number of syndromic forms of autism; in the present work, we explore the consequences of disruption and rescue of PSDSP in a mouse model of RS. We report that expression of a key regulator of synaptic protein synthesis, the metabotropic glutamate receptor 5 (mGlu5) protein, is significantly reduced in both the brains of RS model mice and in the motor cortex of human RS autopsy samples. Furthermore, we demonstrate that reduced mGlu5 expression correlates with attenuated DHPG-induced long-term depression in the hippocampus of RS model mice, and that administration of a novel mGlu5 positive allosteric modulator (PAM), termed VU0462807, can rescue synaptic plasticity defects. Additionally, treatment of Mecp2-deficient mice with VU0462807 improves motor performance (open-field behavior and gait dynamics), corrects repetitive clasping behavior, as well as normalizes cued fear-conditioning defects. Importantly, due to the rationale drug discovery approach used in its development, our novel mGlu5 PAM improves RS phenotypes and synaptic plasticity defects without evoking the overt adverse effects commonly associated with potentiation of mGlu5 signaling (i.e. seizures), or affecting cardiorespiratory defects in RS model mice. These findings provide strong support for the continued development of mGlu5 PAMs as potential therapeutic agents for use in RS, and, more broadly, for utility in idiopathic autism.