SHP2 regulates GluA2 tyrosine phosphorylation required for AMPA receptor endocytosis and mGluR-LTD.

Posttranslational modifications regulate the properties and abundance of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that mediate fast excitatory synaptic transmission and synaptic plasticity in the central nervous system. During long-term depression (LTD), protein tyrosine phosphatases (PTPs) dephosphorylate tyrosine residues in the C-terminal tail of AMPA receptor GluA2 subunit, which is essential for GluA2 endocytosis and group I metabotropic glutamate receptor (mGluR)-dependent LTD. However, as a selective downstream effector of mGluRs, the mGluR-dependent PTP responsible for GluA2 tyrosine dephosphorylation remains elusive at Schaffer collateral (SC)-CA1 synapses. In the present study, we find that mGluR5 stimulation activates Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) by increasing phospho-Y542 levels in SHP2. Under steady-state conditions, SHP2 plays a protective role in stabilizing phospho-Y869 of GluA2 by directly interacting with GluA2 phosphorylated at Y869, without affecting GluA2 phospho-Y876 levels. Upon mGluR5 stimulation, SHP2 dephosphorylates GluA2 at Y869 and Y876, resulting in GluA2 endocytosis and mGluR-LTD. Our results establish SHP2 as a downstream effector of mGluR5 and indicate a dual action of SHP2 in regulating GluA2 tyrosine phosphorylation and function. Given the implications of mGluR5 and SHP2 in synaptic pathophysiology, we propose SHP2 as a promising therapeutic target for neurodevelopmental and autism spectrum disorders.

In vitro modulation of mTOR and mGlur5 influence α-synuclein accumulation.

One of the main hallmarks of Parkinson's disease (PD) is abnormal alpha-synuclein (α-syn) aggregation which forms the main component of intracellular Lewy body inclusions. This short report used preformed α-syn fibrils, as well as an A53T mutant α-syn adenovirus to mimic conditions of pathological protein aggregation in dopaminergic human derived SH-SY5Y neural cells. Since there is evidence that the mTOR pathway and glutamatergic signaling each influence protein aggregation, we also assessed the impact of the mTOR inhibitor, rapamycin and the mGluR5 allosteric modulator, CTEP. We found that both rapamycin and CTEP induced a significant reduction of α-syn fibrils in SH-SY5Y cells and this effect was associated with a reduction in mTOR signaling and enhancement in autophagic pathway factors. These data support the possibility that CTEP (or rapamycin) might be a useful pharmacological approach to target abnormal α-syn accumulation by promoting intracellular degradation or enhanced clearance.

Metabotropic glutamate receptor 5 promotes blood-brain barrier recovery after traumatic brain injury.

Blood-brain barrier (BBB) impairment and glutamate release are two pathophysiological features of traumatic brain injury (TBI), contributing to secondary brain damage and neuroinflammation. However, our knowledge of BBB integrity damage and dysfunction are still limited due to the diverse and fluctuating expression of glutamate receptors after trauma. Here, we confirmed the downregulation of metabotropic glutamate receptor 5 (mGluR5) on microvascular endothelial cell within the acute phase of TBI, and the recovered mGluR5 levels on BBB was positively associated with blood perfusion and neurological recovery. In whole body mGluR5-knockout mice, BBB dysfunction and neurological deficiency were exacerbated after TBI compared with wild type mice. In terms of mechanism, the amino acid sequence 201-259 of cytoskeletal protein Alpha-actinin-1 (ACTN1) interacted with mGluR5, facilitating mGluR5 translocation from cytoplasmic compartment to plasma membrane in endothelial cells. Activation of plasma membrane mGluR5 triggers the PLC/PKCµ/c-Jun signaling pathway, leading to increased expression of the tight junction-actin cytoskeleton connecting protein zonula occludens-1 (ZO-1). Our findings uncover a novel mechanism mediated by membrane and cytoplasmic mGluR5 in endothelial cell integrity maintenance and repair, providing the potential therapeutic target for TBI treatment targeting at mGluR5 and mGluR5/ACTN1 complex in BBB.

Metabotropic glutamate receptor 5 (mGluR5) is associated with neurodegeneration and amyloid deposition in Alzheimer's disease: A [18F]PSS232 PET/MRI study.

BACKGROUND: Metabotropic glutamate receptor 5 (mGluR5) is involved in regulating integrative brain function and synaptic transmission. Aberrant mGluR5 signaling and relevant synaptic failure play a key role in the initial pathophysiological mechanism of Alzheimer's disease (AD). The study aims to investigate the association between mGluR5 availability and AD's biomarkers and cognitive function. METHODS: We examined 35 individuals with mGluR5 tracer [18F]PSS232 to assess mGluR5 availability, and with [18F]Florbetapir PET to assess global amyloid deposition, and [18F]FDG PET to assess glucose metabolism. The plasma neurofilament light (NfL) and p-tau181 levels in a subset of individuals were measured (n = 27). The difference in mGluR5 availability between the AD and normal control (NC) groups was explored. The associations of mGluR5 availability with amyloid deposition, glucose metabolism, gray matter volume (GMV), neuropsychological assessment scores, and plasma biomarkers were analyzed. RESULTS: The mGluR5 availability was significantly reduced in AD patients' hippocampus and parahippocampal gyrus compared to NCs. Global amyloid deposition was positively associated with mGluR5 availability in the AD group and reversely associated in the NC group. The mGluR5 availability was positively correlated with regional glucose metabolism in the overall and stratified analyses. The availability of mGluR5 in the hippocampus and parahippocampal gyrus demonstrated a strong relationship with the GMV of the medial temporal lobe, plasma p-tau181 or NfL levels, and global cognitive performance. CONCLUSIONS: [18F]PSS232 PET can quantify the changes of mGluR5 availability in the progression of AD. mGluR5 availability correlated not only with neuropathological biomarkers of AD but also with neurodegenerative biomarkers and cognitive performance. mGluR5 may be a novel neurodegenerative biomarker, and whether mGluR5 could be a potential therapeutic target for AD needs to be further studied.

Inhibiting metabotropic glutamate receptor 5 after stroke restores brain function and connectivity.

Stroke results in local neural disconnection and brain-wide neuronal network dysfunction leading to neurological deficits. Beyond the hyper-acute phase of ischaemic stroke, there is no clinically-approved pharmacological treatment that alleviates sensorimotor impairments. Functional recovery after stroke involves the formation of new or alternative neuronal circuits including existing neural connections. The type-5 metabotropic glutamate receptor (mGluR5) has been shown to modulate brain plasticity and function and is a therapeutic target in neurological diseases outside of stroke. We investigated whether mGluR5 influences functional recovery and network reorganization rodent models of focal ischaemia. Using multiple behavioural tests, we observed that treatment with negative allosteric modulators (NAMs) of mGluR5 (MTEP, fenobam and AFQ056) for 12 days, starting 2 or 10 days after stroke, restored lost sensorimotor functions, without diminishing infarct size. Recovery was evident within hours after initiation of treatment and progressed over the subsequent 12 days. Recovery was prevented by activation of mGluR5 with the positive allosteric modulator VU0360172 and accelerated in mGluR5 knock-out mice compared with wild-type mice. After stroke, multisensory stimulation by enriched environments enhanced recovery, a result prevented by VU0360172, implying a role of mGluR5 in enriched environment-mediated recovery. Additionally, MTEP treatment in conjunction with enriched environment housing provided an additive recovery enhancement compared to either MTEP or enriched environment alone. Using optical intrinsic signal imaging, we observed brain-wide disruptions in resting-state functional connectivity after stroke that were prevented by mGluR5 inhibition in distinct areas of contralesional sensorimotor and bilateral visual cortices. The levels of mGluR5 protein in mice and in tissue samples of stroke patients were unchanged after stroke. We conclude that neuronal circuitry subserving sensorimotor function after stroke is depressed by a mGluR5-dependent maladaptive plasticity mechanism that can be restored by mGluR5 inhibition. Post-acute stroke treatment with mGluR5 NAMs combined with rehabilitative training may represent a novel post-acute stroke therapy.

Neonatal Maternal Separation Induces Sexual Dimorphism in Brain Development: The Influence on Amino Acid Levels and Cognitive Disorders.

Repeated maternal separation (MS) is a useful experimental model in rodents for studying the long-term influence of early-life stress on brain neurophysiology. In our work, we assessed the effect of repeated MS (postnatal day (PND)1-21, 180 min/day) on the postnatal development of rat brain regions involved in memory using proton magnetic resonance spectroscopy (1HMRS) for tissue volume and the level of amino acids such as glutamate, aspartate, glutamine, glycine and gamma-aminobutyric acid (GABA) in the hippocampus. We assessed whether these effects are sex dependent. We also use novel object recognition (NOR) task to examine the effect of MS on memory and the effect of ethanol on it. Finally, we attempted to ameliorate postnatal stress-induced memory deficits by using VU-29, a positive allosteric modulator (PAM) of the metabotropic glutamate type 5 (mGlu5) receptor. In males, we noted deficits in the levels of glutamate, glycine and glutamine and increases in GABA in the hippocampus. In addition, the values of perirhinal cortex, prefrontal cortex and insular cortex and CA3 were decreased in these animals. MS females, in contrast, demonstrated significant increase in glutamate levels and decrease in GABA levels in the hippocampus. Here, the CA1 values alone were increased. VU-29 administration ameliorated these cognitive deficits. Thus, MS stress disturbs amino acids levels mainly in the hippocampus of adult male rats, and enhancement of glutamate neurotransmission reversed recognition memory deficits in these animals.

Plasma endocannabinoids in cocaine dependence and their relation to cerebral metabotropic glutamate receptor 5 density.

Animal models indicate that the endocannabinoid system (ECS) plays a modulatory role in stress and reward processing, both crucially impaired in addictive disorders. Preclinical findings showed endocannabinoid-modulated synaptic plasticity in reward brain networks linked to the metabotropic-glutamate-5 receptor (mGluR5), contributing to drug-reinforcing effects and drug-seeking behavior. Although animal models postulate a link between ECS and cocaine addiction, human translational studies are lacking. Here, we tested previous preclinical findings by investigating plasma endocannabinoids (eCBs) anandamide (AEA), 2-arachidonoylglycerol (2-AG), and the related N-acylethanolamines (NAEs) palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), including their interaction with cerebral mGluR5, in chronic cocaine users (CU). We compared basal plasma concentrations between chronic CU (N = 103; 69 recreational CU and 34 dependent CU) and stimulant-naïve healthy controls (N = 92). Follow-up basal eCB/NAE plasma levels after 12 months were used for reliability and stability check (CU: N = 33; controls: N = 43). In an additional analysis using 11C-ABP688 positron emission tomography (PET) in a male subsample (CU: N = 18; controls: N = 16), we investigated the relationships between eCBs/NAEs and mGluR5 density in the brain. We found higher 2-AG plasma levels in dependent CU compared to controls and recreational CU. 2-AG levels were stable over time across all groups. In the PET-subsample, a positive association between 2-AG and mGluR5 brain density only in CU was found. Our results corroborate animal findings suggesting an alteration of the ECS in cocaine dependence and an association between peripheral 2-AG levels and cerebral mGluR5 in humans. Therefore, the ECS might be a promising pharmaco-therapeutic target for novel treatments of cocaine dependence.

Functional Interaction between Adenosine A2A and mGlu5 Receptors Mediates STEP Phosphatase Activation and Promotes STEP/mGlu5R Binding in Mouse Hippocampus and Neuroblastoma Cell Line.

(1) Background: Recently, we found that adenosine A2A receptor (A2AR) stimulation results in an increase in STEP phosphatase activity. In order to delve into the mechanism through which A2AR stimulation induced STEP activation, we investigated the involvement of mGlu5R since it is well documented that A2AR and mGlu5R physically and functionally interact in several brain areas. (2) Methods: In a neuroblastoma cell line (SH-SY5Y) and in mouse hippocampal slices, we evaluated the enzymatic activity of STEP by using a para-nitrophenyl phosphate colorimetric assay. A co-immunoprecipitation assay and a Western blot analysis were used to evaluate STEP/mGlu5R binding. (3) Results: We found that the A2AR-dependent activation of STEP was mediated by the mGlu5R. Indeed, the A2AR agonist CGS 21680 significantly increased STEP activity, and this effect was prevented not only by the A2AR antagonist ZM 241385, as expected, but also by the mGlu5R antagonist MPEP. In addition, we found that mGlu5R agonist DHPG-induced STEP activation was reversed not only by the mGlu5R antagonist MPEP but also by ZM 241385. Finally, via co-immunoprecipitation experiments, we found that mGlu5R and STEP physically interact when both receptors are activated (4) Conclusions: These results demonstrated a close functional interaction between mGlu5 and A2A receptors in the modulation of STEP activity.

Blunted type-5 metabotropic glutamate receptor-mediated polyphosphoinositide hydrolysis in two mouse models of monogenic autism.

The involvement of the mGlu5 receptors in the pathophysiology of several forms of monogenic autism has been supported by numerous studies following the seminal observation that mGlu5 receptor-dependent long-term depression was enhanced in the hippocampus of mice modeling the fragile-X syndrome (FXS). Surprisingly, there are no studies examining the canonical signal transduction pathway activated by mGlu5 receptors (i.e. polyphosphoinositide - PI - hydrolysis) in mouse models of autism. We have developed a method for in vivo assessment of PI hydrolysis based on systemic injection of lithium chloride followed by treatment with the selective mGlu5 receptor PAM, VU0360172, and measurement of endogenous inositolmonophosphate (InsP) in brain tissue. Here, we report that mGlu5 receptor-mediated PI hydrolysis was blunted in the cerebral cortex, hippocampus, and corpus striatum of Ube3am-/p+ mice modeling Angelman syndrome (AS), and in the cerebral cortex and hippocampus of Fmr1 knockout mice modeling FXS. In vivo mGlu5 receptor-mediated stimulation of Akt on threonine 308 was also blunted in the hippocampus of FXS mice. These changes were associated with a significant increase in cortical and striatal Homer1 levels and striatal mGlu5 receptor and Gαq levels in AS mice, and with a reduction in cortical mGlu5 receptor and hippocampal Gαq levels, and an increase in cortical phospholipase-Cß and hippocampal Homer1 levels in FXS mice. This is the first evidence that the canonical transduction pathway activated by mGlu5 receptors is down-regulated in brain regions of mice modeling monogenic autism.

mGluR5 from Primary Sensory Neurons Promotes Opioid-Induced Hyperalgesia and Tolerance by Interacting with and Potentiating Synaptic NMDA Receptors.

Aberrant activation of presynaptic NMDARs in the spinal dorsal horn is integral to opioid-induced hyperalgesia and analgesic tolerance. However, the signaling mechanisms responsible for opioid-induced NMDAR hyperactivity remain poorly identified. Here, we show that repeated treatment with morphine or fentanyl reduced monomeric mGluR5 protein levels in the dorsal root ganglion (DRG) but increased levels of mGluR5 monomers and homodimers in the spinal cord in mice and rats of both sexes. Coimmunoprecipitation analysis revealed that monomeric and dimeric mGluR5 in the spinal cord, but not monomeric mGluR5 in the DRG, directly interacted with GluN1. By contrast, mGluR5 did not interact with µ-opioid receptors in the DRG or spinal cord. Repeated morphine treatment markedly increased the mGluR5-GluN1 interaction and protein levels of mGluR5 and GluN1 in spinal synaptosomes. The mGluR5 antagonist MPEP reversed morphine treatment-augmented mGluR5-GluN1 interactions, GluN1 synaptic expression, and dorsal root-evoked monosynaptic EPSCs of dorsal horn neurons. Furthermore, CRISPR-Cas9-induced conditional mGluR5 knockdown in DRG neurons normalized mGluR5 levels in spinal synaptosomes and NMDAR-mediated EPSCs of dorsal horn neurons increased by morphine treatment. Correspondingly, intrathecal injection of MPEP or conditional mGluR5 knockdown in DRG neurons not only potentiated the acute analgesic effect of morphine but also attenuated morphine treatment-induced hyperalgesia and tolerance. Together, our findings suggest that opioid treatment promotes mGluR5 trafficking from primary sensory neurons to the spinal dorsal horn. Through dimerization and direct interaction with NMDARs, presynaptic mGluR5 potentiates and/or stabilizes NMDAR synaptic expression and activity at primary afferent central terminals, thereby maintaining opioid-induced hyperalgesia and tolerance.SIGNIFICANCE STATEMENT Opioids are essential analgesics for managing severe pain caused by cancer, surgery, and tissue injury. However, these drugs paradoxically induce pain hypersensitivity and tolerance, which can cause rapid dose escalation and even overdose mortality. This study demonstrates, for the first time, that opioids promote trafficking of mGluR5, a G protein-coupled glutamate receptor, from peripheral sensory neurons to the spinal cord; there, mGluR5 proteins dimerize and physically interact with NMDARs to augment their synaptic expression and activity. Through dynamic interactions, the two distinct glutamate receptors mutually amplify and sustain nociceptive input from peripheral sensory neurons to the spinal cord. Thus, inhibiting mGluR5 activity or disrupting mGluR5-NMDAR interactions could reduce opioid-induced hyperalgesia and tolerance and potentiate opioid analgesic efficacy.

The Metabotropic Glutamate 5 Receptor in Sleep and Wakefulness: Focus on the Cortico-Thalamo-Cortical Oscillations.

Sleep is an essential innate but complex behaviour which is ubiquitous in the animal kingdom. Our knowledge of the distinct neural circuit mechanisms that regulate sleep and wake states in the brain are, however, still limited. It is therefore important to understand how these circuits operate during health and disease. This review will highlight the function of mGlu5 receptors within the thalamocortical circuitry in physiological and pathological sleep states. We will also evaluate the potential of targeting mGlu5 receptors as a therapeutic strategy for sleep disorders that often co-occur with epileptic seizures.

mGluR5 in Astrocytes in the Ventromedial Hypothalamus Regulates Pituitary Adenylate Cyclase-Activating Polypeptide Neurons and Glucose Homeostasis.

The ventromedial hypothalamus (VMH) is a functionally heterogeneous nucleus critical for systemic energy, glucose, and lipid balance. We showed previously that the metabotropic glutamate receptor 5 (mGluR5) plays essential roles regulating excitatory and inhibitory transmission in SF1+ neurons of the VMH and facilitating glucose and lipid homeostasis in female mice. Although mGluR5 is also highly expressed in VMH astrocytes in the mature brain, its role there influencing central metabolic circuits is unknown. In contrast to the glucose intolerance observed only in female mice lacking mGluR5 in VMH SF1 neurons, selective depletion of mGluR5 in VMH astrocytes enhanced glucose tolerance without affecting food intake or body weight in both adult female and male mice. The improved glucose tolerance was associated with elevated glucose-stimulated insulin release. Astrocytic mGluR5 male and female mutants also exhibited reduced adipocyte size and increased sympathetic tone in gonadal white adipose tissue. Diminished excitatory drive and synaptic inputs onto VMH Pituitary adenylate cyclase-activating polypeptide (PACAP+) neurons and reduced activity of these cells during acute hyperglycemia underlie the observed changes in glycemic control. These studies reveal an essential role of astrocytic mGluR5 in the VMH regulating the excitatory drive onto PACAP+ neurons and activity of these cells facilitating glucose homeostasis in male and female mice.SIGNIFICANCE STATEMENT Neuronal circuits within the VMH play chief roles in the regulation of whole-body metabolic homeostasis. It remains unclear how astrocytes influence neurotransmission in this region to facilitate energy and glucose balance control. Here, we explored the role of the metabotropic glutamate receptor, mGluR5, using a mouse model with selective depletion of mGluR5 from VMH astrocytes. We show that astrocytic mGluR5 critically regulates the excitatory drive and activity of PACAP-expressing neurons in the VMH to control glucose homeostasis in both female and male mice. Furthermore, mGluR5 in VMH astrocytes influences adipocyte size and sympathetic tone in white adipose tissue. These studies provide novel insight toward the importance of hypothalamic astrocytes participating in central circuits regulating peripheral metabolism.

Upregulation of Parkinson's disease-associated protein alpha-synuclein suppresses tumorigenesis via interaction with mGluR5 and gamma-synuclein in liver cancer.

Numerous epidemiological studies suggest a link between Parkinson's disease (PD) and cancer, indicating that PD-associated proteins may mediate the development of cancer. Here, we investigated a potential role of PD-associated protein α-synuclein in regulating liver cancer progression in vivo and in vitro. We found the negative correlation of α-synuclein with metabotropic glutamate receptor 5 (mGluR5) and γ-synuclein by analyzing the data from The Cancer Genome Atlas database, liver cancer patients and hepatoma cells with overexpressed α-synuclein. Moreover, upregulated α-synuclein suppressed the growth, migration, and invasion. α-synuclein was found to associate with mGluR5 and γ-synuclein, and the truncated N-terminal of α-synuclein was essential for the interaction. Furthermore, overexpressed α-synuclein exerted the inhibitory effect on hepatoma cells through the degradation of mGluR5 and γ-synuclein via α-synuclein-dependent autophagy-lysosomal pathway (ALP). Consistently, in vivo experiments with rotenone-induced rat model of PD also confirmed that, upregulated α-synuclein in liver cancer tissues through targeting on mGluR5/α-synuclein/γ-synuclein complex inhibited tumorigenesis involving in ALP-dependent degradation of mGluR5 and γ-synuclein. These findings give an insight into an important role of PD-associated protein α-synuclein accompanied by the complex of mGluR5/α-synuclein/γ-synuclein in distant communications between PD and liver cancer, and provide a new strategy in therapeutics for the treatment of liver cancer.

Striatal mGlu5-mediated synaptic plasticity is independently regulated by location-specific receptor pools and divergent signaling pathways.

Metabotropic glutamate receptor 5 (mGlu5) is widely expressed throughout the central nervous system and is involved in neuronal function, synaptic transmission, and a number of neuropsychiatric disorders such as depression, anxiety, and autism. Recent work from this lab showed that mGlu5 is one of a growing number of G protein-coupled receptors that can signal from intracellular membranes where it drives unique signaling pathways, including upregulation of extracellular signal-regulated kinase (ERK1/2), ETS transcription factor Elk-1, and activity-regulated cytoskeleton-associated protein (Arc). To determine the roles of cell surface mGlu5 as well as the intracellular receptor in a well-known mGlu5 synaptic plasticity model such as long-term depression, we used pharmacological isolation and genetic and physiological approaches to analyze spatially restricted pools of mGlu5 in striatal cultures and slice preparations. Here we show that both intracellular and cell surface receptors activate the phosphatidylinositol-3-kinase-protein kinase B-mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, whereas only intracellular mGlu5 activates protein phosphatase 2 and leads to fragile X mental retardation protein degradation and de novo protein synthesis followed by a protein synthesis-dependent increase in Arc and post-synaptic density protein 95. However, both cell surface and intracellular mGlu5 activation lead to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluA2 internalization and chemically induced long-term depression albeit via different signaling mechanisms. These data underscore the importance of intracellular mGlu5 in the cascade of events associated with sustained synaptic transmission in the striatum.

GPCR interactions involving metabotropic glutamate receptors and their relevance to the pathophysiology and treatment of CNS disorders.

Cellular responses to metabotropic glutamate (mGlu) receptor activation are shaped by mechanisms of receptor-receptor interaction. mGlu receptor subtypes form homodimers, intra- or inter-group heterodimers, and heteromeric complexes with other G protein-coupled receptors (GPCRs). In addition, mGlu receptors may functionally interact with other receptors through the ßγ subunits released from G proteins in response to receptor activation or other mechanisms. Here, we discuss the interactions between (i) mGlu1 and GABAB receptors in cerebellar Purkinje cells; (ii) mGlu2 and 5-HT2Aserotonergic receptors in the prefrontal cortex; (iii) mGlu5 and A2A receptors or mGlu5 and D1 dopamine receptors in medium spiny projection neurons of the indirect and direct pathways of the basal ganglia motor circuit; (iv) mGlu5 and A2A receptors in relation to the pathophysiology of Alzheimer's disease; and (v) mGlu7 and A1 adenosine or α- or ß1 adrenergic receptors. In addition, we describe in detail a novel form of non-heterodimeric interaction between mGlu3 and mGlu5 receptors, which appears to be critically involved in mechanisms of activity-dependent synaptic plasticity in the prefrontal cortex and hippocampus. Finally, we highlight the potential implication of these interactions in the pathophysiology and treatment of cerebellar disorders, schizophrenia, Alzheimer's disease, Parkinson's disease, l-DOPA-induced dyskinesias, stress-related disorders, and cognitive dysfunctions. This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".

Rigorous Characterization of Allosteric Modulation of the Human Metabotropic Glutamate Receptor 1 Reveals Probe- and Assay-Dependent Pharmacology.

Allosteric modulation of metabotropic glutamate receptor subtype 1 (mGlu1) represents a viable therapeutic target for treating numerous central nervous system disorders. Although multiple chemically distinct mGlu1 positive (PAMs) and negative (NAMs) allosteric modulators have been identified, drug discovery paradigms have not included rigorous pharmacological analysis. In the present study, we hypothesized that existing mGlu1 allosteric modulators possess unappreciated probe-dependent or biased pharmacology. Using human embryonic kidney 293 (HEK293A) cells stably expressing human mGlu1, we screened mGlu1 PAMs and NAMs from divergent chemical scaffolds for modulation of different mGlu1 orthosteric agonists in intracellular calcium (iCa2+) mobilization and inositol monophosphate (IP1) accumulation assays. Operational models of agonism and allosterism were used to derive estimates for important pharmacological parameters such as affinity, efficacy, and cooperativity. Modulation of glutamate and quisqualate-mediated iCa2+ mobilization revealed probe dependence at the level of affinity and cooperativity for both mGlu1 PAMs and NAMs. We also identified the previously described mGlu5 selective NAM PF-06462894 as an mGlu1 NAM with a different pharmacological profile from other NAMs. Differential profiles were also observed when comparing ligand pharmacology between iCa2+ mobilization and IP1 accumulation. The PAMs Ro67-4853 and CPPHA displayed apparent negative cooperativity for modulation of quisqualate affinity, and the NAMs CPCCOEt and PF-06462894 had a marked reduction in cooperativity with quisqualate in IP1 accumulation and upon extended incubation in iCa2+ mobilization assays. These data highlight the importance of rigorous assessment of mGlu1 modulator pharmacology to inform future drug discovery programs for mGlu1 allosteric modulators. SIGNIFICANCE STATEMENT: Metabotropic glutamate receptor subtype 1 (mGlu1) positive and negative allosteric modulators have therapeutic potential in multiple central nervous system disorders. We show that chemically distinct modulators display differential pharmacology with different orthosteric ligands and across divergent signaling pathways at human mGlu1. Such complexities in allosteric ligand pharmacology should be considered in future mGlu1 allosteric drug discovery programs.

Inhibition of mGluR5 alters BDNF/TrkB and GLT-1 expression in the prefrontal cortex and hippocampus and ameliorates PTSD-like behavior in rats.

RATIONALE AND OBJECTIVE: Post-traumatic stress disorder (PTSD) is a prevalent and debilitating psychiatric disorder. However, its specific etiological mechanism remains unclear. Previous studies have shown that traumatic stress changes metabotropic glutamate receptor 5 (mGluR5) expression in the hippocampus (HIP) and prefrontal cortex (PFC). More importantly, mGluR5 expression is often accompanied by alterations in brain-derived neurotrophic factor (BDNF). Furthermore, BDNF/tropomyosin-associated kinase B (TrkB) signaling plays multiple roles, including roles in neuroplasticity and antidepressant activity, by regulating glutamate transporter-1 (GLT-1) expression. This study aims to explore the effects of inhibiting mGluR5 on PTSD-like behaviors and BDNF, TrkB, and GLT-1 expression in the HIP and PFC of inevitable foot shock (IFS)-treated rats. METHODS: Seven-day IFS was used to establish a PTSD rat model, and 2-methyl-6-(phenylethynyl)-pyridine (MPEP) (10 mg/kg, intraperitoneal injection) was used to inhibit the activity of mGluR5 during IFS in rats. After modeling, behavioral changes and mGluR5, BDNF, TrkB, and GLT-1 expression in the PFC and HIP were examined. RESULTS: First, the IFS procedure induced PTSD-like behavior. Second, IFS increased the expression of mGluR5 and decreased BDNF, TrkB, and GLT-1 expression in the PFC and HIP. Third, the mGluR5 antagonist blocked the above behavioral and molecular alterations. CONCLUSIONS: mGluR5 was involved in IFS-induced PTSD-like behavior by changing BDNF, TrkB, and GLT-1 expression.

Differences in Quantification of the Metabotropic Glutamate Receptor 5 Across Bipolar Disorder and Major Depressive Disorder.

BACKGROUND: Understanding the neurobiology underlying bipolar disorder (BD) versus major depressive disorder (MDD) is crucial for accurate diagnosis and for driving the discovery of novel treatments. A promising target is the metabotropic glutamate receptor 5 (mGluR5), a modulator of glutamate transmission associated with synaptic plasticity. We measured mGluR5 availability in individuals with MDD and BD for the first time using positron emission tomography. METHODS: Individuals with BD (n = 17 depressed; n = 10 euthymic) or MDD (n = 17) and healthy control (HC) individuals (n = 18) underwent imaging with [18F]FPEB positron emission tomography to quantify mGluR5 availability in regions of the prefrontal cortex, which was compared across groups and assessed in relation to depressive symptoms and cognitive function. RESULTS: Prefrontal cortex mGluR5 availability was significantly different across groups (F6,116 = 2.18, p = .050). Specifically, mGluR5 was lower in BD versus MDD and HC groups, with no difference between MDD and HC groups. Furthermore, after dividing the BD group, mGluR5 was lower in both BD-depression and BD-euthymia groups versus both MDD and HC groups across regions of interest. Interestingly, lower dorsolateral prefrontal cortex mGluR5 was associated with worse depression in MDD (r = -0.67, p = .005) but not in BD. Significant negative correlations were observed between mGluR5 and working memory in MDD and BD-depression groups. CONCLUSIONS: This work suggests that mGluR5 could be helpful in distinguishing BD and MDD as a possible treatment target for depressive symptoms in MDD and for cognitive alterations in both disorders. Further work is needed to confirm differentiating roles for mGluR5 in BD and MDD and to probe modulation of mGluR5 as a preventive/treatment strategy.

Spinophilin Limits Metabotropic Glutamate Receptor 5 Scaffolding to the Postsynaptic Density and Cell Type Specifically Mediates Excessive Grooming.

BACKGROUND: Grooming dysfunction is a hallmark of the obsessive-compulsive spectrum disorder trichotillomania. Numerous preclinical studies have utilized SAPAP3-deficient mice for understanding the neurobiology of repetitive grooming, suggesting that excessive grooming is caused by increased metabotropic glutamate receptor 5 (mGluR5) activity in striatal direct- and indirect-pathway medium spiny neurons (MSNs). However, the MSN subtype-specific signaling mechanisms that mediate mGluR5-dependent adaptations underlying excessive grooming are not fully understood. Here, we investigated the MSN subtype-specific roles of the striatal signaling hub protein spinophilin in mediating repetitive motor dysfunction associated with mGluR5 function. METHODS: Quantitative proteomics and immunoblotting were utilized to identify how spinophilin impacts mGluR5 phosphorylation and protein interaction changes. Plasticity and repetitive motor dysfunction associated with mGluR5 action were measured using our novel conditional spinophilin mouse model in which spinophilin was knocked out from striatal direct-pathway MSNs and/or indirect-pathway MSNs. RESULTS: Loss of spinophilin only in indirect-pathway MSNs decreased performance of a novel motor repertoire, but loss of spinophilin in either MSN subtype abrogated striatal plasticity associated with mGluR5 function and prevented excessive grooming caused by SAPAP3 knockout mice or treatment with the mGluR5-specific positive allosteric modulator VU0360172 without impacting locomotion-relevant behavior. Biochemically, we determined that the spinophilin-mGluR5 interaction correlates with grooming behavior and that loss of spinophilin shifts mGluR5 interactions from lipid raft-associated proteins toward postsynaptic density proteins implicated in psychiatric disorders. CONCLUSIONS: These results identify spinophilin as a novel striatal signaling hub molecule in MSNs that cell subtype specifically mediates behavioral, functional, and molecular adaptations associated with repetitive motor dysfunction in psychiatric disorders.

Inhibition of mGluR5 ameliorates lipid accumulation and inflammation in HepG2 cells.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease characterized by ectopic lipid accumulation in hepatocytes. To date, no specific drug has been approved for its treatment. Metabotropic glutamate receptor 5 (mGluR5) has been showed expressed in hepatocytes and related to some liver diseases such as alcoholic steatosis. However, the function of mGluR5 in NAFLD is not clear. This work aims to investigate the effect and potential mechanism of mGluR5 in NAFLD. We found that mGluR5 expression was increased in the livers of HFD-fed mice and in palmitate-treated HepG2 cells. Suppression of mGluR5 by the specific antagonist MPEP could ameliorate palmitate-induced lipid accumulation, whereas the mGluR5 agonist CHPG promoted lipid deposition in the cells. Knockdown of mGluR5 by small interfering RNA further demonstrated that inhibition of mGluR5 could reduce lipid accumulation. Furthermore, our results revealed that mGluR5 regulated lipid metabolism by increasing the gene expression of lipogenesis. Inflammatory factors and phosphorylation levels of NF-κB-p65 and JNK were also tested in treated hepatocytes. mGluR5 promoted the inflammatory reaction and JNK phosphorylation. Inhibition of JNK signaling by JNK-IN-8 rescued CHPG-induced lipogenesis and inflammation. This study showed mGluR5 regulated lipid accumulation and inflammation in palmitic acid-treated HepG2 cells via the JNK signaling pathway. mGluR5 might be a potential drug target for NAFLD.