The role of beta-cell glutamate receptors in pancreatic endocrine function and in the pathogenesis of type 1 diabetes mellitus

Background: Kainate receptors (KARs) are one of the three classes of ionotropic glutamate receptors (iGluRs) expressed primarily in the central nervous system (CNS) where they mediate information transfer and neurotransmitter release. Very little is known about native KARs and their interacting partners outside the CNS. Aim: The aim of this study was to investigate systematically the molecular composition and functional properties of KARs in pancreatic endocrine cells and also to investigate the role of KARs in the pathogenesis of T1DM.Methods: The presence of KAR subunits was investigated in pancreatic clonal β-cells (MIN6 and INS-1), α-cells (α-TC) and primary rat islets of Langerhans using RT-PCR and immunoblotting. The effect of KAR activation on intracellular calcium concentration and insulin secretion in MIN6 was investigated using FURA-2AM epifluorescence imaging and Mercodia insulin assay respectively. The effect of glutamate, kainate and glutamate transporter inhibitor (dihydrokainic acid) on survival and viability of INS1, α-TC and neuroblastoma cells (SH-SY5Y) were investigated using MTT cell viability assay. The presence of autoantibodies against GluR subunits in serum of T1DM patients and controls was investigated using ELISA and immunoblotting. Results: RT-PCR identified mRNAs for GluK2-5 KAR subunits in clonal β-and α-cells and all five subunits (GluK1-5) in primary rat islets of Langerhans. The presence of these subunits was confirmed using immunoblotting with GluK2/3 and GluK5 antibodies. In addition, auxiliary KAR subunits Neto 1 and Neto 2 were also identified in all cell types. FURA-2AM epifluorescence imaging of cultured MIN6 β-cells showed that activation of KARs with kainate induced significant increase in intracellular calcium concentration and insulin secretion. These effects of kainate were blocked by KAR antagonist (NBQX; 30μM) but not by an antagonist (GYKI-53655 hydrochloride; 100μM) of other iGluRs. Chronic exposure to kainate (0.1-0.5mM), glutamate (0.25-12mM) and dihydrokainic acid (0.1mM) cause significantly reduced viability of pancreatic endocrine and neuronal cells. ELISA and immunoblotting showed that serum of T1DM patients and non-diabetic controls react against components of synaptosomal plasma membrane and also against overexpressed GluR subunits. Conclusion: Together, these results indicate that a range of functional KAR subunits and their interacting proteins are expressed in the endocrine pancreas. These KAR subunits identified could assemble as homomeric or heteromeric channels in both β-and α-cells of the pancreas. The activation of these receptors is likely to have an impact on pancreatic hormone secretion and viability of endocrine cells in the islets of Langerhans. Much work ought to be carried out to optimise the methods for identification of autoantibodies against GluR subunits in serum of T1DM patients. This will help to understand the potential role of GluRs in the pathogenesis of T1DM

Targeting the M1 muscarinic acetylcholine receptor in Alzheimer’s disease

Alzheimer’s disease (AD) remains a major cause of morbidity and mortality worldwide, and despite extensive research, only a few drugs are available for management of the disease. One strategy has been to upregulate cholinergic neurotransmission to improve cognitive function, but this approach has dose-limiting adverse effects. To avoid these adverse effects, new drugs that target specific receptor subtypes of the cholinergic system are needed, and the M1 subtype of muscarinic acetylcholine receptor (M1-mAChR) has been shown to be a good target for this approach. By using several strategies, M1-mAChR ligands have been developed and trialled in preclinical animal models and in human studies, with varying degrees of success. This article reviews the different approaches to targeting the M1-mAChR in AD and discusses the advantages and limitations of these strategies. The factors to consider in targeting the M1-mAChR in AD are also discussed

Host tissue proteomics reveal insights into the molecular basis of Schistosoma haematobium-induced bladder pathology.

Urogenital schistosomiasis remains a major public health concern worldwide. In response to egg deposition, the host bladder undergoes gross and molecular morphological changes relevant for disease manifestation. However, limited mechanistic studies to date imply that the molecular mechanisms underlying pathology are not well-defined. We leveraged a mouse model of urogenital schistosomiasis to perform for the first time, proteome profiling of the early molecular events that occur in the bladder after exposure to S. haematobium eggs, and to elucidate the protein pathways involved in urogenital schistosomiasis-induced pathology. Purified S. haematobium eggs or control vehicle were microinjected into the bladder walls of mice. Mice were sacrificed seven days post-injection and bladder proteins isolated and processed for proteome profiling using mass spectrometry. We demonstrate that biological processes including carcinogenesis, immune and inflammatory responses, increased protein translation or turnover, oxidative stress responses, reduced cell adhesion and epithelial barrier integrity, and increased glucose metabolism were significantly enriched in S. haematobium infection. S. haematobium egg deposition in the bladder results in significant changes in proteins and pathways that play a role in pathology. Our findings highlight the potential bladder protein indicators for host-parasite interplay and provide new insights into the complex dynamics of pathology and characteristic bladder tissue changes in urogenital schistosomiasis. The findings will be relevant for development of improved interventions for disease control

Bitopic binding mode of an M1 muscarinic acetylcholine receptor agonist associated with adverse clinical trial outcomes

The realisation of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (M1 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, described previously as a potent M1 receptor allosteric agonist, which showed pro-cognitive 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. We conclude that these properties, whilst imparting beneficial effects on learning and memory, are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data supports 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

Host tissue proteomics reveal insights into the molecular basis of Schistosoma haematobium-induced bladder pathology

Article Authors Metrics Comments Media Coverage Abstract Author summary Introduction Methods Results Discussion Conclusion Supporting information Acknowledgments References Reader Comments Figures Abstract Urogenital schistosomiasis remains a major public health concern worldwide. In response to egg deposition, the host bladder undergoes gross and molecular morphological changes relevant for disease manifestation. However, limited mechanistic studies to date imply that the molecular mechanisms underlying pathology are not well-defined. We leveraged a mouse model of urogenital schistosomiasis to perform for the first time, proteome profiling of the early molecular events that occur in the bladder after exposure to S. haematobium eggs, and to elucidate the protein pathways involved in urogenital schistosomiasis-induced pathology. Purified S. haematobium eggs or control vehicle were microinjected into the bladder walls of mice. Mice were sacrificed seven days post-injection and bladder proteins isolated and processed for proteome profiling using mass spectrometry. We demonstrate that biological processes including carcinogenesis, immune and inflammatory responses, increased protein translation or turnover, oxidative stress responses, reduced cell adhesion and epithelial barrier integrity, and increased glucose metabolism were significantly enriched in S. haematobium infection. S. haematobium egg deposition in the bladder results in significant changes in proteins and pathways that play a role in pathology. Our findings highlight the potential bladder protein indicators for host-parasite interplay and provide new insights into the complex dynamics of pathology and characteristic bladder tissue changes in urogenital schistosomiasis. The findings will be relevant for development of improved interventions for disease control

Biased M1-muscarinic-receptor-mutant mice inform the design of next-generation drugs

Cholinesterase inhibitors, the current frontline symptomatic treatment for Alzheimer’s disease (AD), are associated with low efficacy and adverse effects. M1 muscarinic acetylcholine receptors (M1 mAChRs) represent a potential alternate therapeutic target; however, drug discovery programs focused on this G protein-coupled receptor (GPCR) have failed, largely due to cholinergic adverse responses. Employing novel chemogenetic and phosphorylation-deficient, G protein-biased, mouse models, paired with a toolbox of probe molecules, we establish previously unappreciated pharmacologically targetable M1 mAChR neurological processes, including anxiety-like behaviors and hyper-locomotion. By mapping the upstream signaling pathways regulating these responses, we determine the importance of receptor phosphorylation-dependent signaling in driving clinically relevant outcomes and in controlling adverse effects including ‘epileptic-like’ seizures. We conclude that M1 mAChR ligands that promote receptor phosphorylation-dependent signaling would protect against cholinergic adverse effects in addition to driving beneficial responses such as learning and memory and anxiolytic behavior relevant for the treatment of AD

Inhibition of resistance-refractory P. falciparum kinase PKG delivers prophylactic, blood stage, and transmission-blocking antiplasmodial activity

The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype

Variants of the EAAT2 Glutamate Transporter Gene Promoter Are Associated with Cerebral Palsy in Preterm Infants

© 2017, The Author(s). Preterm delivery is associated with neurodevelopmental impairment caused by environmental and genetic factors. Dysfunction of the excitatory amino acid transporter 2 (EAAT2) and the resultant impaired glutamate uptake can lead to neurological disorders. In this study, we investigated the role of single nucleotide polymorphisms (SNPs; g.-200CCloseSPigtSPiA and g.-181ACloseSPigtSPiC) in the EAAT2 promoter in susceptibility to brain injury and neurodisability in very preterm infants born at or before 32-week gestation. DNA isolated from newborns’ dried blood spots were used for pyrosequencing to detect both SNPs. Association between EAAT2 genotypes and cerebral palsy, cystic periventricular leukomalacia and a low developmental score was then assessed. The two SNPs were concordant in 89.4% of infants resulting in three common genotypes all carrying two C and two A alleles in different combinations. However, in 10.6% of cases, non-concordance was found, generating six additional rare genotypes. The A alleles at both loci appeared to be detrimental and consequently, the risk of developing cerebral palsy increased four- and sixfold for each additional detrimental allele at -200 and -181bp, respectively. The two SNPs altered the regulation of the EAAT2 promoter activity and glutamate homeostasis. This study highlights the significance of glutamate in the pathogenesis of preterm brain injury and subsequent development of cerebral palsy and neurodevelopmental disabilities. Furthermore, the described EAAT2 SNPs may be an early biomarker of vulnerability to neurodisability and may aid the development of targeted treatment strategies

Phosphorylation bar-coding of Free Fatty Acid receptor 2 is generated in a tissue-specific manner

All the source data files associated with the paper "Phosphorylation bar-coding of Free Fatty Acid receptor 2 is generated in a tissue-specific manner

Selective phosphorylation of threonine residues defines GPR84-arrestin interactions of biased ligands

GPR84 is an immune cell-expressed, pro-inflammatory receptor currently being assessed as a therapeutic target in conditions including fibrosis and inflammatory bowel disease. Although it was previously shown that the orthosteric GPR84 activators 2-HTP and 6-OAU promoted its interactions with arrestin-3, a G protein-biased agonist DL-175 did not. Here, we show that replacement of all 21 serine and threonine residues within i-loop 3 of GRP84, but not the 2 serines in the C-terminal tail, eliminated incorporation of [32P] and greatly reduced receptor-arrestin-3 interactions promoted by 2-HTP. We demonstrate that GPR84 was phosphorylated constitutively on residues Ser221 and Ser224 while various other amino acids are phosphorylated in response to 2-HTP. Consistent with this, an antiserum able to identify pSer221/pSer224 recognized GPR84 from cells treated with and without activators, whereas an antiserum able to identify pThr263/pThr264 only recognized GPR84 after exposure to 2-HTP and not DL-175. Two distinct GPR84 antagonists as well as inhibition of G protein-coupled receptor kinase 2/3 prevented phosphorylation of pThr263/pThr264, but neither strategy affected constitutive phosphorylation of Ser221/Ser224. Furthermore, mutation of residues Thr263 and Thr264 to alanine generated a variant of GPR84 also limited in 2-HTP-induced interactions with arrestin-2 and -3. By contrast, this mutant was unaffected in its capacity to reduce cAMP levels. Taken together these results define a key pair of threonine residues, regulated only by subsets of GPR84 small molecule activators and by GRK2/3, that define effective interactions with arrestins and provide novel tools to monitor the phosphorylation and functional status of GPR84