SCA44- and SCAR13-associated GRM1 mutations affect metabotropic glutamate receptor 1 function through distinct mechanisms.

BACKGROUND AND PURPOSE: Metabotropic glutamate receptor 1 (mGlu1) is a promising therapeutic target for neurodegenerative CNS disorders including spinocerebellar ataxias (SCAs). Clinical reports have identified naturally-occurring mGlu1 mutations in rare SCA subtypes and linked symptoms to mGlu1 mutations. However, how mutations alter mGlu1 function remains unknown, as does amenability of receptor function to pharmacological rescue. Here, we explored SCA-associated mutation effects on mGlu1 cell surface expression, canonical signal transduction and allosteric ligand pharmacology. EXPERIMENTAL APPROACH: Orthosteric agonists, positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs) were assessed at two functional endpoints (iCa2+ mobilisation and inositol 1-phosphate [IP1] accumulation) in FlpIn Trex HEK293A cell lines expressing five mutant mGlu1 subtypes. Key pharmacological parameters including ligand potency, affinity and cooperativity were derived using operational models of agonism and allostery. KEY RESULTS: mGlu1 mutants exhibited differential impacts on mGlu1 expression, with a C-terminus truncation significantly reducing surface expression. Mutations differentially influenced orthosteric ligand affinity, efficacy and functional cooperativity between allosteric and orthosteric ligands. Loss-of-function mutations L454F and N885del reduced orthosteric affinity and efficacy, respectively. A gain-of-function Y792C mutant mGlu1 displayed enhanced constitutive activity in IP1 assays, which manifested as reduced orthosteric agonist activity. The mGlu1 PAMs restored glutamate potency in iCa2+ mobilisation for loss-of-function mutations and mGlu1 NAMs displayed enhanced inverse agonist activity at Y792C relative to wild-type mGlu1. CONCLUSION AND IMPLICATIONS: Collectively, these data highlight distinct mechanisms by which mGlu1 mutations affect receptor function and show allosteric modulators may present a therapeutic strategy to restore aberrant mGlu1 function in rare SCA subtypes.

Development of Putative Bivalent Dicovalent Ligands for the Adenosine A1 Receptor.

Accumulating evidence suggests that G protein-coupled receptors (GPCRs) can exist and function in homodimer and heterodimer forms. The adenosine A1 receptor (A1R) has been shown to form both homodimers and heterodimers, but there is a lack of chemical tools to study these dimeric receptor populations. This work describes the synthesis and pharmacological evaluation of a novel class of bivalent GPCR chemical tools, where each ligand moiety of the bivalent compound contains a sulfonyl fluoride covalent warhead designed to be capable of simultaneously reacting with each A1R of an A1R homodimer. The novel compounds were characterised using radioligand binding assays, including washout assays, and functionally in cAMP assays. The bivalent dicovalent compounds were competitive A1R antagonists and showed evidence of covalent binding and simultaneous binding across an A1R homodimer. Greater selectivity for A1R over the adenosine A3 receptor was observed for bivalent dicovalent over the equivalent monovalent compounds, indicating subtype selectivity can be achieved with dual occupation by a bivalent dicovalent ligand.

Gpr88 Deletion Impacts Motivational Control Without Overt Disruptions to Striatal Dopamine.

Background: Disrupted motivational control is a common-but poorly treated-feature of psychiatric disorders, arising via aberrant mesolimbic dopaminergic signaling. GPR88 is an orphan G protein-coupled receptor that is highly expressed in the striatum and therefore well placed to modulate disrupted signaling. While the phenotype of Gpr88 knockout mice suggests a role in motivational pathways, it is unclear whether GPR88 is involved in reward valuation and/or effort-based decision making in a sex-dependent manner and whether this involves altered dopamine function. Methods: In male and female Gpr88 knockout mice, we used touchscreen-based progressive ratio, with and without reward devaluation, and effort-related choice tasks to assess motivation and cost/benefit decision making, respectively. To explore whether these motivational behaviors were related to alterations in the striatal dopamine system, we quantified expression of dopamine-related genes and/or proteins and used [18F]DOPA positron emission tomography and GTPγ[35S] binding to assess presynaptic and postsynaptic dopamine function, respectively. Results: We showed that male and female Gpr88 knockout mice displayed greater motivational drive than wild-type mice, which was maintained following reward devaluation. Furthermore, we showed that cost/benefit decision making was impaired in male, but not female, Gpr88 knockout mice. Surprisingly, we found that Gpr88 deletion had no effect on striatal dopamine by any of the measures assessed. Conclusions: Our results highlight that GPR88 regulates motivational control but that disruption of such behaviors following Gpr88 deletion occurs independently of gross perturbations to striatal dopamine at a gene, protein, or functional level. This work provides further insights into GPR88 as a drug target for motivational disorders.

Regulation of CRE-Dependent Transcriptional Activity in a Mouse Suprachiasmatic Nucleus Cell Line.

We evaluated the signalling framework of immortalized cells from the hypothalamic suprachiasmatic nucleus (SCN) of the mouse. We selected a vasoactive intestinal peptide (VIP)-positive sub-clone of immortalized mouse SCN-cells stably expressing a cAMP-regulated-element (CRE)-luciferase construct named SCNCRE. We characterized these cells in terms of their status as neuronal cells, as well as for important components of the cAMP-dependent signal transduction pathway and compared them to SCN ex vivo. SCNCRE cells were treated with agents that modulate different intracellular signalling pathways to investigate their potency and timing for transcriptional CRE-dependent signalling. Several activating pathways modulate SCN neuronal signalling via the cAMP-regulated-element (CRE: TGACGCTA) and phosphorylation of transcription factors such as cAMP-regulated-element-binding protein (CREB). CRE-luciferase activity induced by different cAMP-signalling pathway-modulating agents displayed a variety of substance-specific dose and time-dependent profiles and interactions relevant to the regulation of SCN physiology. Moreover, the induction of the protein kinase C (PKC) pathway by phorbol ester application modulates the CRE-dependent signalling pathway as well. In conclusion, the cAMP/PKA- and the PKC-regulated pathways individually and in combination modulate the final CRE-dependent transcriptional output.

Translation-Focused Approaches to GPCR Drug Discovery for Cognitive Impairments Associated with Schizophrenia.

There are no effective therapeutics for cognitive impairments associated with schizophrenia (CIAS), which includes deficits in executive functions (working memory and cognitive flexibility) and episodic memory. Compounds that have entered clinical trials are inadequate in terms of efficacy and/or tolerability, highlighting a clear translational bottleneck and a need for a cohesive preclinical drug development strategy. In this review we propose hippocampal-prefrontal-cortical (HPC-PFC) circuitry underlying CIAS-relevant cognitive processes across mammalian species as a target source to guide the translation-focused discovery and development of novel, procognitive agents. We highlight several G protein-coupled receptors (GPCRs) enriched within HPC-PFC circuitry as therapeutic targets of interest, including noncanonical approaches (biased agonism and allosteric modulation) to conventional clinical targets, such as dopamine and muscarinic acetylcholine receptors, along with prospective novel targets, including the orphan receptors GPR52 and GPR139. We also describe the translational limitations of popular preclinical cognition tests and suggest touchscreen-based assays that probe cognitive functions reliant on HPC-PFC circuitry and reflect tests used in the clinic, as tests of greater translational relevance. Combining pharmacological and behavioral testing strategies based in HPC-PFC circuit function creates a cohesive, translation-focused approach to preclinical drug development that may improve the translational bottleneck currently hindering the development of treatments for CIAS.

Human serum albumin-based nanoparticle-mediated in vitro gene delivery.

The genetic treatment of neurodegenerative diseases still remains a challenging task since many approaches fail to deliver the therapeutic material in relevant concentrations into the brain. As viral vectors comprise the risk of immune and inflammatory responses, human serum albumin (HSA) nanoparticles were found to represent a safer and more convenient alternative. Their ability to cross the blood-brain barrier (BBB) and deliver drugs into the brain in order to enhance gene-based therapy has been previously demonstrated. The present study deals with the development of pGL3-PEI-coated HSA nanoparticles and subsequent in vitro testing in cerebellar granular and HeLa cells. The luciferase control vector pGL3 was chosen as reporter plasmid encoding for the firefly luciferase protein, linear polyethylenimine (22 kDa) as endosomolytic agent for enhancing the cells' transfection. Studies on particle characteristics, their cellular uptake into aforementioned cell lines and on subcellular localisation, and transfection efficiency in the cerebellar cells proved the feasibility of nanoparticle-based gene delivery.

Progressive retinal degeneration and glial activation in the CLN6 (nclf) mouse model of neuronal ceroid lipofuscinosis: a beneficial effect of DHA and curcumin supplementation.

Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative lysosomal storage disorders characterized by vision loss, mental and motor deficits, and spontaneous seizures. Neuropathological analyses of autopsy material from NCL patients and animal models revealed brain atrophy closely associated with glial activity. Earlier reports also noticed loss of retinal cells and reactive gliosis in some forms of NCL. To study this phenomenon in detail, we analyzed the ocular phenotype of CLN6 (nclf) mice, an established mouse model for variant-late infantile NCL. Retinal morphometry, immunohistochemistry, optokinetic tracking, electroretinography, and mRNA expression were used to characterize retinal morphology and function as well as the responses of Müller cells and microglia. Our histological data showed a severe and progressive degeneration in the CLN6 (nclf) retina co-inciding with reactive Müller glia. Furthermore, a prominent phenotypic transformation of ramified microglia to phagocytic, bloated, and mislocalized microglial cells was identified in CLN6 (nclf) retinas. These events overlapped with a rapid loss of visual perception and retinal function. Based on the strong microglia reactivity we hypothesized that dietary supplementation with immuno-regulatory compounds, curcumin and docosahexaenoic acid (DHA), could ameliorate microgliosis and reduce retinal degeneration. Our analyses showed that treatment of three-week-old CLN6 (nclf) mice with either 5% DHA or 0.6% curcumin for 30 weeks resulted in a reduced number of amoeboid reactive microglia and partially improved retinal function. DHA-treatment also improved the morphology of CLN6 (nclf) retinas with a preserved thickness of the photoreceptor layer in most regions of the retina. Our results suggest that microglial reactivity closely accompanies disease progression in the CLN6 (nclf) retina and both processes can be attenuated with dietary supplemented immuno-modulating compounds.