The atypical 'hippocampal' glutamate receptor coupled to phospholipase D that controls stretch-sensitivity in primary mechanosensory nerve endings is homomeric purely metabotropic GluK2.

A metabotropic glutamate receptor coupled to phospholipase D (PLD-mGluR) was discovered in the hippocampus over three decades ago. Its pharmacology and direct linkage to PLD activation are well established and indicate it is a highly atypical glutamate receptor. A receptor with the same pharmacology is present in spindle primary sensory terminals where its blockade can totally abolish, and its activation can double, the normal stretch-evoked firing. We report here the first identification of this PLD-mGluR protein, by capitalizing on its expression in primary mechanosensory terminals, developing an enriched source, pharmacological profiling to identify an optimal ligand, and then functionalizing it as a molecular tool. Evidence from immunofluorescence, western and far-western blotting indicates PLD-mGluR is homomeric GluK2, since GluK2 is the only glutamate receptor protein/receptor subunit present in spindle mechanosensory terminals. Its expression was also found in the lanceolate palisade ending of hair follicle, also known to contain the PLD-mGluR. Finally, in a mouse model with ionotropic function ablated in the GluK2 subunit, spindle glutamatergic responses were still present, confirming it acts purely metabotropically. We conclude the PLD-mGluR is a homomeric GluK2 kainate receptor signalling purely metabotropically and it is common to other, perhaps all, primary mechanosensory endings.

Feasibility of mobilisation in ICU: a multi-centre point prevalence study of mobility practices in the UK.

BACKGROUND: Early mobilisation in critical care is recommended within clinical guidance; however, mobilisation prevalence across the UK is unknown. The study aimed to determine the proportion of patients mobilised out of bed within 48-72 h, to describe their physiological status, and to compare this to published consensus safety recommendations for out-of-bed activity. METHODS: A UK cross-sectional, multi-centre, observational study of adult critical care mobility practices was conducted. Demographic, physiological and organ support data, mobility level, and rationale for not mobilising out of bed, were collected for all patients on 3rd March 2022. Patients were categorised as: Group 1-mobilised ICU Mobility Scale (IMS) ≥ 3; Group 2-not-mobilised IMS < 3 with physiological reasons; or Group 3-not-mobilised IMS < 3 with non-physiological barriers to mobilisation. Rationale for the decision to not mobilise was collected qualitatively. Regression analysis was used to compare the physiological parameters of Group 1 (mobilised) versus Group 2 (not-mobilised with physiological reasons). Patients were stratified as 'low-risk', 'potential-risk' or 'high-risk' using published risk of adverse event ratings. RESULTS: Data were collected for 960 patients across 84 UK critical care units. Of these 393 (41%) mobilised, 416 (43%) were not-mobilised due to physiological reasons and 151 (16%) were not mobilised with non-physiological reasons. A total of 371 patients had been admitted for ≤ 3 days, of whom 180 (48%) were mobilised, 140 (38%) were not mobilised with physiological reasons, and 51 (14%) were not mobilised with non-physiological reasons. Of the 809 without non-physiological barriers to mobilisation, 367 (45%) had a low risk of adverse event rating and 120 (15%) a potential risk, of whom 309 (84%) and 78 (65%) mobilised, respectively. Mobility was associated with a Richmond Agitation-Sedation Scale of - 1 to + 1, lower doses of vasoactive agents, a lower inspired oxygen requirement. CONCLUSION: Although only 40% of patients mobilised out of bed, 89% of those defined 'low-risk' did so. There is significant overlap in physiological parameters for mobilisation versus non-mobilisation groups, suggesting a comprehensive physiological assessment is vital in decision making rather than relying on arbitrary time points. CLINICAL TRIALS REGISTRATION: NCT05281705 Registered March 16, 2022. Retrospectively registered.

Metabotropic Glutamate Receptors Modulate Exocytotic Tau Release and Propagation.

Using synaptosomes purified from the brains of two transgenic mouse models overexpressing mutated human tau (TgP301S and Tg4510) and brains of patients with sporadic Alzheimer's disease, we showed that aggregated and hyperphosphorylated tau was both present in purified synaptosomes and released in a calcium- and synaptosome-associated protein of 25 kDa (SNAP25)-dependent manner. In all mouse and human synaptosomal preparations, tau release was inhibited by the selective metabotropic glutamate receptor 2/3 (mGluR2/3) agonist LY379268, an effect prevented by the selective mGlu2/3 antagonist LY341495. LY379268 was also able to block pathologic tau propagation between primary neurons in an in vitro microfluidic cellular model. These novel results are transformational for our understanding of the molecular mechanisms mediating tau release and propagation at synaptic terminals in Alzheimer's disease and suggest that these processes could be inhibited therapeutically by the selective activation of presynaptic G protein-coupled receptors. SIGNIFICANCE STATEMENT: Pathological tau release and propagation are key neuropathological events underlying cognitive decline in Alzheimer's disease patients. This paper describes the role of regulated exocytosis, and the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein SNAP25, in mediating tau release from rodent and human synaptosomes. This paper also shows that a selective mGluR2/3 agonist is highly effective in blocking tau release from synaptosomes and tau propagation between neurons, opening the way to the discovery of novel therapeutic approaches to this devastating disease.

Identification and pharmacological profile of SPP1, a potent, functionally selective and brain penetrant agonist at muscarinic M1 receptors.

BACKGROUND AND PURPOSE: We aimed to identify and develop novel, selective muscarinic M1 receptor agonists as potential therapeutic agents for the symptomatic treatment of Alzheimer's disease. EXPERIMENTAL APPROACH: We developed and utilized a novel M1 receptor occupancy assay to drive a structure activity relationship in a relevant brain region while simultaneously tracking drug levels in plasma and brain to optimize for central penetration. Functional activity was tracked in relevant native in vitro assays allowing translational (rat-human) benchmarking of structure-activity relationship molecules to clinical comparators. KEY RESULTS: Using this paradigm, we identified a series of M1 receptor selective molecules displaying desirable in vitro and in vivo properties and optimized key features, such as central penetration while maintaining selectivity and a partial agonist profile. From these compounds, we selected spiropiperidine 1 (SPP1). In vitro, SPP1 is a potent, partial agonist of cortical and hippocampal M1 receptors with activity conserved across species. SPP1 displays high functional selectivity for M1 receptors over native M2 and M3 receptor anti-targets and over a panel of other targets. Assessment of central target engagement by receptor occupancy reveals SPP1 significantly and dose-dependently occupies rodent cortical M1 receptors. CONCLUSIONS AND IMPLICATIONS: We report the discovery of SPP1, a novel, functionally selective, brain penetrant partial orthosteric agonist at M1 receptors, identified by a novel receptor occupancy assay. SPP1 is amenable to in vitro and in vivo study and provides a valuable research tool to further probe the role of M1 receptors in physiology and disease.

In Vitro Pharmacological Characterization and In Vivo Validation of LSN3172176 a Novel M1 Selective Muscarinic Receptor Agonist Tracer Molecule for Positron Emission Tomography.

In the search for improved symptomatic treatment options for neurodegenerative and neuropsychiatric diseases, muscarinic acetylcholine M1 receptors (M1 mAChRs) have received significant attention. Drug development efforts have identified a number of novel ligands, some of which have advanced to the clinic. However, a significant issue for progressing these therapeutics is the lack of robust, translatable, and validated biomarkers. One valuable approach to assessing target engagement is to use positron emission tomography (PET) tracers. In this study we describe the pharmacological characterization of a selective M1 agonist amenable for in vivo tracer studies. We used a novel direct binding assay to identify nonradiolabeled ligands, including LSN3172176, with the favorable characteristics required for a PET tracer. In vitro functional and radioligand binding experiments revealed that LSN3172176 was a potent partial agonist (EC50 2.4-7.0 nM, Emax 43%-73%), displaying binding selectivity for M1 mAChRs (Kd = 1.5 nM) that was conserved across species (native tissue Kd = 1.02, 2.66, 8, and 1.03 at mouse, rat, monkey, and human, respectively). Overall selectivity of LSN3172176 appeared to be a product of potency and stabilization of the high-affinity state of the M1 receptor, relative to other mAChR subtypes (M1 > M2, M4, M5 > M3). In vivo, use of wild-type and mAChR knockout mice further supported the M1-preferring selectivity profile of LSN3172176 for the M1 receptor (78% reduction in cortical occupancy in M1 KO mice). These findings support the development of LSN3172176 as a potential PET tracer for assessment of M1 mAChR target engagement in the clinic and to further elucidate the function of M1 mAChRs in health and disease.

Bitopic Binding Mode of an M1 Muscarinic Acetylcholine Receptor Agonist Associated with Adverse Clinical Trial Outcomes.

The realization of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one), described previously as a potent M1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that "pure" positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses.

Current status of muscarinic M1 and M4 receptors as drug targets for neurodegenerative diseases.

The cholinergic signalling system has been an attractive pathway to seek targets for modulation of arousal, cognition, and attention which are compromised in neurodegenerative and neuropsychiatric diseases. The acetylcholine muscarinic receptor M1 and M4 subtypes which are highly expressed in the central nervous system, in cortex, hippocampus and striatum, key areas of cognitive and neuropsychiatric control, have received particular attention. Historical muscarinic drug development yielded first generation agonists with modest selectivity for these two receptor targets over M2 and M3 receptors, the major peripheral sub-types hypothesised to underlie the dose-limiting clinical side effects. More recent compound screening and medicinal chemistry optimization of orthosteric and allosteric agonists, and positive allosteric modulators binding to sites distinct from the highly homologous acetylcholine binding pocket have yielded a collection of highly selective tool compounds for preclinical validation studies. Several M1 selective ligands have progressed to early clinical development and in time will hopefully lead to useful therapeutics for treating symptoms of Alzheimer's disease and related disorders. This article is part of the Special Issue entitled 'Neuropharmacology on Muscarinic Receptors'.

Phosphodiesterase-1b (Pde1b) knockout mice are resistant to forced swim and tail suspension induced immobility and show upregulation of Pde10a.

RATIONALE: Major depressive disorder is a leading cause of suicide and disability. Despite this, current antidepressants provide insufficient efficacy in more than 60% of patients. Most current antidepressants are presynaptic reuptake inhibitors; postsynaptic signal regulation has not received as much attention as potential treatment targets. OBJECTIVES: We examined the effects of disruption of the postsynaptic cyclic nucleotide hydrolyzing enzyme, phosphodiesterase (PDE) 1b, on depressive-like behavior and the effects on PDE1B protein in wild-type (WT) mice following stress. METHODS: Littermate knockout (KO) and WT mice were tested in locomotor activity, tail suspension (TST), and forced swim tests (FST). FST was also used to compare the effects of two antidepressants, fluoxetine and bupropion, in KO versus WT mice. Messenger RNA (mRNA) expression changes were also determined. WT mice underwent acute or chronic stress and markers of stress and PDE1B expression were examined. RESULTS: Pde1b KO mice exhibited decreased TST and FST immobility. When treated with antidepressants, both WT and KO mice showed decreased FST immobility and the effect was additive in KO mice. Mice lacking Pde1b had increased striatal Pde10a mRNA expression. In WT mice, acute and chronic stress upregulated PDE1B expression while PDE10A expression was downregulated after chronic but not acute stress. CONCLUSIONS: PDE1B is a potential therapeutic target for depression treatment because of the antidepressant-like phenotype seen in Pde1b KO mice.

In vitro pharmacological and rat pharmacokinetic characterization of LY3020371, a potent and selective mGlu2/3 receptor antagonist.

Metabotropic glutamate 2/3 (mGlu2/3) receptors are of considerable interest owing to their role in modulating glutamate transmission via presynaptic, postsynaptic and glial mechanisms. As part of our ongoing efforts to identify novel ligands for these receptors, we have discovered (1S,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid; (LY3020371), a potent and selective orthosteric mGlu2/3 receptor antagonist. In this account, we characterize the effects of LY3020371 in membranes and cells expressing human recombinant mGlu receptor subtypes as well as in native rodent and human brain tissue preparations, providing important translational information for this molecule. In membranes from cells expressing recombinant human mGlu2 and mGlu3 receptor subtypes, LY3020371.HCl competitively displaced binding of the mGlu2/3 agonist ligand [3H]-459477 with high affinity (hmGlu2 Ki = 5.26 nM; hmGlu3 Ki = 2.50 nM). In cells expressing hmGlu2 receptors, LY3020371.HCl potently blocked mGlu2/3 agonist (DCG-IV)-inhibited, forskolin-stimulated cAMP formation (IC50 = 16.2 nM), an effect that was similarly observed in hmGlu3-expressing cells (IC50 = 6.21 nM). Evaluation of LY3020371 in cells expressing the other human mGlu receptor subtypes revealed high mGlu2/3 receptor selectivity. In rat native tissue assays, LY3020371 demonstrated effective displacement of [3H]-459477 from frontal cortical membranes (Ki = 33 nM), and functional antagonist activity in cortical synaptosomes measuring both the reversal of agonist-suppressed second messenger production (IC50 = 29 nM) and agonist-inhibited, K+-evoked glutamate release (IC50 = 86 nM). Antagonism was fully recapitulated in both primary cultured cortical neurons where LY3020371 blocked agonist-suppressed spontaneous Ca2+ oscillations (IC50 = 34 nM) and in an intact hippocampal slice preparation (IC50 = 46 nM). Functional antagonist activity was similarly demonstrated in synaptosomes prepared from epileptic human cortical or hippocampal tissues, suggesting a translation of the mGlu2/3 antagonist pharmacology from rat to human. Intravenous dosing of LY3020371 in rats led to cerebrospinal fluid drug levels that are expected to effectively block mGlu2/3 receptors in vivo. Taken together, these results establish LY3020371 as an important new pharmacological tool for studying mGlu2/3 receptors in vitro and in vivo. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

M1 muscarinic allosteric modulators slow prion neurodegeneration and restore memory loss.

The current frontline symptomatic treatment for Alzheimer's disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR-selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.

Activation of Muscarinic M1 Acetylcholine Receptors Induces Long-Term Potentiation in the Hippocampus.

Muscarinic M1 acetylcholine receptors (M1Rs) are highly expressed in the hippocampus, and their inhibition or ablation disrupts the encoding of spatial memory. It has been hypothesized that the principal mechanism by which M1Rs influence spatial memory is by the regulation of hippocampal synaptic plasticity. Here, we use a combination of recently developed, well characterized, selective M1R agonists and M1R knock-out mice to define the roles of M1Rs in the regulation of hippocampal neuronal and synaptic function. We confirm that M1R activation increases input resistance and depolarizes hippocampal CA1 pyramidal neurons and show that this profoundly increases excitatory postsynaptic potential-spike coupling. Consistent with a critical role for M1Rs in synaptic plasticity, we now show that M1R activation produces a robust potentiation of glutamatergic synaptic transmission onto CA1 pyramidal neurons that has all the hallmarks of long-term potentiation (LTP): The potentiation requires NMDA receptor activity and bi-directionally occludes with synaptically induced LTP. Thus, we describe synergistic mechanisms by which acetylcholine acting through M1Rs excites CA1 pyramidal neurons and induces LTP, to profoundly increase activation of CA1 pyramidal neurons. These features are predicted to make a major contribution to the pro-cognitive effects of cholinergic transmission in rodents and humans.

Synthesis and pharmacological characterization of C4-disubstituted analogs of 1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate: identification of a potent, selective metabotropic glutamate receptor agonist and determination of agonist-bound human mGlu2 and mGlu3 amino terminal domain structures.

As part of our ongoing research to identify novel agents acting at metabotropic glutamate 2 (mGlu2) and 3 (mGlu3) receptors, we have previously reported the identification of the C4α-methyl analog of mGlu2/3 receptor agonist 1 (LY354740). This molecule, 1S,2S,4R,5R,6S-2-amino-4-methylbicyclo[3.1.0]hexane-2,6-dicarboxylate 2 (LY541850), exhibited an unexpected mGlu2 agonist/mGlu3 antagonist pharmacological profile, whereas the C4ß-methyl diastereomer (3) possessed dual mGlu2/3 receptor agonist activity. We have now further explored this structure-activity relationship through the preparation of cyclic and acyclic C4-disubstituted analogs of 1, leading to the identification of C4-spirocyclopropane 5 (LY2934747), a novel, potent, and systemically bioavailable mGlu2/3 receptor agonist which exhibits both antipsychotic and analgesic properties in vivo. In addition, through the combined use of protein-ligand X-ray crystallography employing recombinant human mGlu2/3 receptor amino terminal domains, molecular modeling, and site-directed mutagenesis, a molecular basis for the observed pharmacological profile of compound 2 is proposed.

Pharmacological profiling of the TRPV3 channel in recombinant and native assays.

BACKGROUND AND PURPOSE: Transient receptor potential vanilloid subtype 3 (TRPV3) is implicated in nociception and certain skin conditions. As such, it is an attractive target for pharmaceutical research. Understanding of endogenous TRPV3 function and pharmacology remains elusive as selective compounds and native preparations utilizing higher throughput methodologies are lacking. In this study, we developed medium-throughput recombinant and native cellular assays to assess the detailed pharmacological profile of human, rat and mouse TRPV3 channels. EXPERIMENTAL APPROACH: Medium-throughput cellular assays were developed using a Ca(2+) -sensitive dye and a fluorescent imaging plate reader. Human and rat TRPV3 pharmacology was examined in recombinant cell lines, while the mouse 308 keratinocyte cell line was used to assess endogenous TRPV3 activity. KEY RESULTS: A recombinant rat TRPV3 cellular assay was successfully developed after solving a discrepancy in the published rat TRPV3 protein sequence. A medium-throughput, native, mouse TRPV3 keratinocyte assay was also developed and confirmed using genetic approaches. Whereas the recombinant human and rat TRPV3 assays exhibited similar agonist and antagonist profiles, the native mouse assay showed important differences, namely, TRPV3 activity was detected only in the presence of potentiator or during agonist synergy. Furthermore, the native assay was more sensitive to block by some antagonists. CONCLUSIONS AND IMPLICATIONS: Our findings demonstrate similarities but also notable differences in TRPV3 pharmacology between recombinant and native systems. These findings offer insights into TRPV3 function and these assays should aid further research towards developing TRPV3 therapies.

Pharmacological profiling of native group II metabotropic glutamate receptors in primary cortical neuronal cultures using a FLIPR.

The group II metabotropic glutamate (mGlu) receptors comprised of the mGlu2 and mGlu3 receptor subtypes have gained recognition in recent years as potential targets for psychiatric disorders, including anxiety and schizophrenia. In addition to studies already indicating which subtype mediates the anxiolytic and anti-psychotic effects observed in disease models, studies to help further define the preferred properties of selective group II mGlu receptor ligands will be essential. Comparison of the in vitro properties of these ligands to their in vivo efficacy and tolerance profiles may help provide these additional insights. We have developed a relatively high-throughput native group II mGlu receptor functional assay to aid this characterisation. We have utilised dissociated primary cortical neuronal cultures, which after 7 days in vitro have formed functional synaptic connections and display periodic and spontaneous synchronised calcium (Ca(2+)) oscillations in response to intrinsic action potential bursts. We herein demonstrate that in addition to non-selective group II mGlu receptor agonists, (2R,4R)-APDC, LY379268 and DCG-IV, a selective mGlu2 agonist, LY541850, and mGlu2 positive allosteric modulators, BINA and CBiPES, inhibit the frequency of synchronised Ca(2+) oscillations in primary cultures of rat and mouse cortical neurons. Use of cultures from wild-type, mGlu2(-/-), mGlu3(-/-) and mGlu2/3(-/-) mice allowed us to further probe the contribution of mGlu2 and mGlu3, and revealed LY541850 to be a partial mGlu2 agonist and a full mGlu3 antagonist. Overnight pre-treatment of cultures with these ligands revealed a preferred desensitisation profile after treatment with a positive allosteric modulator. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.