In recent years increasing evidence suggests that commensal microbiota may play an important role in health and disease, including cerebrovascular disease. Gut microbes impact physiology, at least in part by metabolizing dietary factors and also host-derived substrates and then generating active compounds including toxins. The purpose of the current review is to highlight the complex interplay between microbiota, their metabolites and essential functions for human health ranging from regulation of the metabolism and the immune system to modulation of brain development and function. We also discuss the role of gut dysbiosis in cerebrovascular disease, specifically in acute and chronic stroke phases and the possible implication of intestinal microbiota in post-stroke cognitive impairment and dementia, and we identify potential therapeutic opportunities of targeting microbiota in this context.

The central nervous system (CNS) has long been considered an immune-privileged site, with minimal interaction between immune cells, particularly of the adaptive immune system. Previously, the presence of immune cells in this organ was primarily linked to events involving disruption of the blood-brain barrier (BBB) or inflammation. However, current research has shown that immune cells are found patrolling CNS under homeostatic conditions. Specifically, T cells of the adaptive immune system are able to cross the BBB and are associated with aging and cognitive impairment. In addition, T-cell infiltration has been observed in pathological conditions, where inflammation correlates with poor prognosis. Despite ongoing research, the role of this population in the aging brain under both physiological and pathological conditions is not yet fully understood. In this review, we provide an overview of the interactions between T cells and other immune and CNS parenchymal cells, and examine the molecular mechanisms by which these interactions may contribute to normal brain function and the scenarios in which disruption of these connections lead to cognitive impairment. A comprehensive understanding of the role of T cells in the aging brain and the underlying molecular pathways under normal conditions could pave the way for new research to better understand brain disorders.

Background and Purpose: Blood-brain barrier (BBB) breakdown is one of the most crucial pathological changes of cerebral ischemia-reperfusion (I/R) injury. Trilobatin (TLB), a naturally occurring food additive, exerts neuroprotective effect against cerebral I/R injury as demonstrated in our previous study. This study was designed to investigate the effect of TLB on disruption of BBB after cerebral I/R injury. Experimental Approach: Rats with focal cerebral ischemia caused by transient middle cerebral artery occlusion (MCAO) and brain microvascular endothelial cells along with human astrocytes to mimic blood brain barrier (BBB) injury caused by oxygen and glucose deprivation (OGD) followed by reoxygenation (OGD/R). Key results: The results showed that TLB effectively maintained the integrity of BBB and inhibited neuronal loss following cerebral I/R challenge. Furthermore, TLB dramatically increased tight junction proteins including ZO-1, occludin and claudin 5, as well as decreased the levels of apolipoprotein E (APOE) 4, cyclophilin A (CypA), and phosphorylated nuclear factor kappa B (NF-κB), thereby reduced proinflammatory cytokines. In addition, TLB also decreased Bax/Bcl-2 ratio and cleaved-caspase 3 level along with reduced the number of apoptotic neurons. Intriguingly, molecular docking and transcriptomics predicted MMP9 was a prominent gene evoked by TLB treatment. Furthermore, the protective effect of TLB on OGD/R-induced the loss of BBB integrity in human brain microvascular endothelial cell and astrocyte co-cultures in vitro was markedly reinforced by knockdown of MMP9. Conclusions and implications: Our findings reveal a novel property of TLB: saving BBB disruption following cerebral I/R via targeting MMP9 and inhibiting APOE4/CypA/NF-κB axis.

Background and Purpose: Myocardial infarction (MI) is the leading cause of mortality globally due in part to the limited ability of cardiomyocytes (CMs) to regenerate. Recently, we demonstrated that overexpression of 4 cell cycle factors, CDK1, CDK4, cyclin B1, and cyclin D1 (4F), induced cell division in ~20% of the post-mitotic CMs overexpressed 4F. The current study aims to identify a small molecule that augments 4F-induced CM cycle induction. Experimental Approach, Key Results: Screening of small molecules with a potential to augment 4F-induced cell-cycle induction in 60-day-old mature human induced pluripotent cardiomyocytes (hiPS-CMs) revealed N-(4,6-Dimethylpyridin-2-yl)-4-(pyridin-4-yl)piperazine-1-carbothioamide (NDPPC), which activates cell cycle progression in 4F-transduced hiPS-CMs. Autodock tool and Autodock vina computational methods showed that NDPPC has a potential interaction with the binding site at the human p38⍺ mitogen-activated protein kinase (p38⍺ MAP kinase), a critical negative regulator of the mammalian cell cycle. A p38⍺ MAP kinase activity assay showed that NDPPC inhibits its activity in a dose-dependent manner. Overexpression of p38⍺ MAP kinase in CMs inhibited 4F cell cycle induction, and treatment with NDPPC reversed the cell cycle inhibitory effect. Conclusion and Implications: NDPPC is a novel inhibitor for p38⍺ MAP kinase and is a promising drug to augment CM cell cycle response to the 4F. NDPPC could become an adjunct treatment with other cell cycle activators for heart failure treatment.

GPR84 is an understudied rhodopsin-like class A G protein-coupled receptor which is arousing particular interest from a therapeutic perspective. Not least this reflects that gpr84 expression is significantly up-regulated following acute inflammatory stimuli and in inflammatory diseases and that receptor activation plays a role in regulating pro-inflammatory responses and migration of cells of the innate immune system such as neutrophils, monocytes, macrophages and microglia. Although most physiological responses of GPR84 reflect receptor coupling to Gαi/o G-proteins, several studies indicate that agonist-activated GPR84 can also recruit arrestin adaptor proteins and this regulates receptor internalisation and desensitisation. To date, very little is known on the patterns of GPR84 phosphorylation and how these might control these processes. Here, we consider what is known on the molecular basis of GPR84 signalling with a focus on how GRK-mediated phosphorylation regulates arrestin protein recruitment and receptor function.

Dopamine transmission in the striatum is a critical mediator of the rewarding and reinforcing effects of commonly misused psychoactive drugs. G protein-coupled receptors (GPCRs) that bind a variety of neuromodulators including dopamine, endocannabinoids, acetylcholine, and endogenous opioid peptides regulate dopamine release by acting on several components of dopaminergic circuitry. Striatal dopamine release can be driven by both somatic action potential firing and local mechanisms that depend on acetylcholine released from striatal cholinergic interneurons. GPCRs that primarily regulate somatic firing of dopamine neurons via direct effects or modulation of synaptic inputs are likely to impact distinct aspects of behavior and psychoactive drug actions compared with GPCRs that primarily regulate local acetylcholine-dependent dopamine release in striatal regions. This review will highlight mechanisms by which GPCRs modulate dopaminergic transmission and the relevance of these findings to psychoactive drugs involved in substance use disorders.

Background and Purpose: The ionotropic purinergic trimeric receptor P2X3 is a new drug target other than the opioid receptor for the treatment of refractory chronic cough (RCC). However, the only marketed P2X3 antagonist, Gefapixant/AF-219, has a side effect of taste disorders due to simultaneous action on the human P2X2/3 (hP2X2/3) heterotrimer. Therefore, selective molecules with high affinity for the hP2X3 homotrimer and low affinity for the hP2X2/3 heterotrimer have potential in iteration 2.0 RCC drug development, such as Sivopixant/S-600918, a clinical phase II RCC candidate with lower taste disturbance than Gefapixant. S-600918 and its analogue (3-(4-((3-chloro-4-isopropoxyphenyl)amino)-3-(4-methylbenzyl)-2,6-dioxo-3,6-dihydro-1,3,5-triazin-1(2H)-yl)propanoic acid (DDTPA) exhibit both high affinity and high selectivity for hP2X3 homotrimers compared to hP2X2/3 heterotrimer. The mechanism of its druggable site and this high selectivity is not clear. Experimental Approach: Here, we reveal a novel allosteric mechanism that distinguishes this drug candidate from other P2X3 inhibitors through chimera construction, site covalent occupation, metadynamics, mutagenesis, and electrophysiology. Key Results: We suggest that the tri-symmetric site adjacent to the upper vestibule determines the high affinity and selectivity of S-600918/DDTPA for hP2X3. Only four amino acids of the hP2X2 upper body domain swapped with hP2X3, allow the hP2X2/3 heterotrimer to gain comparable affinity for S-600918/DDTPA as the hP2X3 homotrimer. Conclusion and Implications: Thus, we have revealed the molecular basis for the cough suppressive effects and reduced side effects of new RCC clinical candidates from the perspective of receptor-ligand recognition, which may provide information critical for the development of new drugs targeting P2X3 for indications such as RCC, idiopathic pulmonary fibrosis (IPF), and primary hypertension.

Schizophrenia remains a sizable socioeconomic burden that continues to be treated with therapeutics based on 70-year old science. All currently approved therapeutics primarily target the dopamine D2 receptor to achieve their efficacy. Whilst dopaminergic dysregulation is a key feature in this disorder, the targeting of dopaminergic machinery has yielded limited efficacy and an appreciable side effect burden. Over the recent decades, numerous drugs that engage non-dopaminergic GPCRs have yielded a promise of efficacy without the deleterious side effect profile, yet none have successfully completed clinical studies and progressed to the market. More recently, there has been increased attention around non-dopaminergic GPCR-targeting drugs, notably KarXT, which demonstrated efficacy in some schizophrenia symptom domains. This provides renewed hope that effective schizophrenia may lay outside of the dopaminergic space. Despite the potential for muscarinic receptor- (and other well-characterised GPCR families) targeting drugs to treat schizophrenia, they are often plagued with complications such as lack of receptor subtype selectivity and peripheral on-target side-effects. Orphan GPCR studies have opened a new avenue of exploration with many demonstrating schizophrenia-relevant mechanisms and a favourable expression profile, thus offering potential for novel drug development. This review discusses centrally-expressed orphan G protein-coupled receptors: GPR3, GPR6, GPR12, GPR52, GPR85, GPR88 and GPR139 and their relationship to schizophrenia. We review their expression, signalling mechanisms and cellular function, in conjunction with small molecule development and structural insights. We seek to provide a snapshot of the growing evidence and development potential of new classes of schizophrenia therapeutics.

A document by Dingxin (Guest Editor) Pan. Click on the document to view its contents.

Background and Purpose Vancomycin is one of the most common antibiotics administered in the hospital setting, yet acute kidney injury is a major limiting factor. Common combinations of antibiotics with vancomycin have been reported to worsen and improve vancomycin-induced kidney injury. We aimed to study the impact of flucloxacillin and imipenem-cilastatin on kidney injury when combined with vancomycin in our translational rat model. Experimental Approach Male Sprague-Dawley rats received allometrically scaled (1) vancomycin (2) flucloxacillin, (3) vancomycin+flucloxacillin, (4) vancomycin+imipenem-cilastatin, or (5) saline for 4 days. Vancomycin was administered intravenously and flucloxacillin or imipenem-cilastatin were administered intraperitoneally. Kidney injury was evaluated via drug accumulation and urinary biomarkers including urinary output, kidney injury molecule-1 (KIM-1), clusterin, and osteopontin. Relationships between vancomycin accumulation in the kidney and urinary kidney injury biomarkers were explored. Key Results Urinary output increased every study day for vancomycin+flucloxacillin; whereas in the vancomycin group it was elevated after the first dose only. In the vancomycin+flucloxacillin group, urinary KIM-1/24h increased on all days compared to vancomycin. In the vancomycin+imipenem-cilastatin group, urinary KIM-1/24h was decreased on days 1 and 2 compared to vancomycin. Similar trends were observed for clusterin. More vancomycin accumulated in the kidney with vancomycin+flucloxacillin compared to vancomycin and vancomycin+imipenem-cilastatin. The accumulation of vancomycin in the kidney tissue correlated with increasing urinary KIM-1 (4-parameter Hill Slope, R2=0.7985). Conclusion and Implications Vancomycin+flucloxacillin caused more kidney injury compared to vancomycin alone and vancomycin+imipenem-cilastatin in a translational rat model as determined by multiple kidney injury biomarkers. The combination of vancomycin+imipenem-cilastatin was nephroprotective.

Ageing is the main risk factor common to most primary neurodegenerative disorders. Indeed, age-related brain alterations have been long considered to predispose to neurodegeneration. Although protein misfolding and the accumulation of toxic protein aggregates have been contemplated as causative events in neurodegeneration, several biological pathways affected by brain ageing are also contributing to pathogenesis. Here, we discuss the evidence showing the involvement of the mechanisms controlling neuronal structure, gene expression, autophagy, cell metabolism, and neuroinflammation in the onset and progression of neurodegenerative disorders. Furthermore, we review the therapeutic strategies currently under development or as future approaches designed to normalize these pathways, which may then boost brain resilience to cope with toxic protein species. Therefore, in addition to therapies targeting the insoluble protein aggregates specifically associated with each neurodegenerative disorder, these novel pharmacological approaches may be part of combined therapies designed to rescue brain function.

Background and Purpose: Overexpression of astrocytic lactoferrin (Lf) was observed in the brains of Alzheimer’s disease (AD) patients, whereas the role of astrocytic Lf in AD progression remains unexplored. In this study, we aimed to evaluate the effects of astrocytic Lf on AD progression. Experimental Approach: The APP/PS1 mice with astrocytes overexpressing human Lf were developed to evaluate the effects of astrocytic Lf on AD progression, and the N2a-sw cells were employed to further uncover the mechanism of astrocytic Lf on β-amyloid (Aβ) production. Key Results: Astrocytic Lf overexpression increased protein phosphatase 2A (PP2A) activity, and reduced amyloid precursor protein (APP) phosphorylation, Aβ burden and tau hyperphosphorylation in APP/PS1 mice. Mechanistically, astrocytic Lf overexpression promoted the astrocytic Lf secretion into neurons in APP/PS1 mice, and the conditional medium from astrocytes overexpressing Lf inhibited the p-APP(Thr668) expression in N2a-sw cells. Furthermore, the recombinant human Lf (hLf) also significantly enhanced PP2A activity and inhibited p-APP expression, while inhibitions of p38 or PP2A activities abrogated the hLf-induced p-APP downregulation in N2a-sw cells. Additionally, hLf promoted the interaction of p38 and PP2A via p38 activation, thereby enhancing PP2A activity; and low-density lipoprotein receptor-related protein 1 (LRP1) knockdown significantly reversed the hLf-induced p38 activation and p-APP downregulation. Conclusions and Implications: Our data suggested that astrocytic Lf promoted neuronal p38 activation via targeting to LRP1, subsequently promoting p38 binds to PP2A to enhance PP2A activity, which finally inhibited Aβ production via APP dephosphorylation. Therefore, promoting astrocytic Lf expression may be a potential strategy against AD.

Background and Purpose: Cardiovascular disease (CVD) affects up to half of the patients with chronic obstructive pulmonary disease (COPD), which exerts deleterious impact on health outcomes and survivability. Vascular endothelial dysfunction marks the onset of cardiovascular disease. The present study examined the effect of a potent NADPH Oxidase (NOX) inhibitor and free-radical scavenger, apocynin, on COPD-related CVD. Experimental Approach: Male BALB/c mice were exposed to either room air (Sham) or cigarette smoke (CS) generated from 9 cigarettes per day, 5 days a week for up to 24 weeks with or without apocynin treatment (5 mg·kg-1·day-1, intraperitoneal injection). Key Results: Eight-weeks of apocynin treatment reduced airway neutrophil infiltration (by 42%) and completely preserved endothelial function and endothelial nitric oxide synthase (eNOS) activity against the oxidative insults of CS exposure. These preservative effects were maintained up until the 24-week time point. 24-week of apocynin treatment exhibited marked benefits on airway inflammation (reduced infiltration of macrophage, neutrophil and lymphocyte) and lung function decline (hyperinflation), and prevented airway collagen deposition by CS exposure. Conclusion and Implications: Limiting NOX activity may slow COPD progression and lower CVD risk, particularly when signs of oxidative stress become evident.

Background and Purpose: How to prevent the damage of ionizing radiation to testis has become an urgent problem to be solved. The present aim is to investigate whether inhibition of p38MAPK signaling can alleviate radiation-induced testicular damage. Experimental Approaches: HE staining was used to measure the morphological changes of epididymis and testis. Immunohistochemistry staining was used to assess the expression of PLZF, SOX9, p-p38MAPK. RNA-Seq was used to profile gene expression. The expression of Mapk14, Atf2, Ddit3 and Ap1m1 genes was detected by qPCR. Western blotting was used to detect the protein expression of p38MAPK and p-p38MAPK. Key Results: There was a dose-response relationship between testicular injury and ionizing radiation. The sperm quality was significantly decreased at 6 and 8 weeks after 6Gy of radiation. Radiation led to the decrease of PLZF+ cells and increase of SOX9+ cells in testis. RNA-Seq data showed radiation induced 969 genes changed in the testis. The expression of genes related to p38MAPK signal pathway enriched by GO was significantly increased by qPCR. The expression of p-p38MAPK in testis was significantly increased at 4 weeks after irradiation. SB203580 treatment increased numbers of spermatozoa, the area and diameter of seminiferous tubules and numbers of SOX9+ cells in irradiated mice, which were consistent with the increased sperm movement rate and density under radiation with SB203580 administration. Conclusions and Implications: Ionizing radiation significantly changes testicular gene expression, in which p38MAPK signal pathway is activated. p38MAPK inhibitor SB203580 partially alleviates the testicular damage caused by radiation and accelerate the recovery of sperm quality.

Substance use disorder (SUD) is a chronic condition with maintained abuse of a substance leading to physiological and psychological alterations and often changes in cognitive and social behaviours. Current therapies mainly consist of psychotherapy coupled with medication; however, alarmingly high relapse rates reveal the shortcomings of the current standard of care. The signalling and expression profile, and neurological function of the serotonin 2C receptor (5-HT2C receptor) make it an ideal candidate of interest for the treatment of SUD. This is further corroborated by pre-clinical and clinical evidence of therapeutically relevant compounds acting at the 5-HT2C receptor. Notwithstanding, drug binding at closely related serotonin receptor subtypes has impeded drug development. More recently, psychedelics, which broadly act at 5-HT2 receptors, have indicated promising potential for the treatment of SUD, implicating in part, the 5-HT2C receptor. The modern psychedelic movement has rekindled therapeutic interest in the 5-HT2C receptor, resulting in an influx of new studies, especially structural analyses. This review delves into the structural, molecular and cellular mechanisms governing the 5-HT2C receptor function, in the context of SUD. This provides the basis of the preclinical and clinical evidence for their role in SUD and highlights the potential for future exploration.

Background and Purpose Neonatal seizures are a clinical emergency. Current anti-seizure medications, however, fail to resolve seizures in ~50% of infants. The P2X7 receptor (P2X7R) is an important driver of inflammation and evidence suggest P2X7R contributing to seizures and epilepsy in adults. To date, however, no genetic proof has been provided to determine the contribution of the P2X7R to neonatal seizures, its effects on inflammatory signalling during neonatal seizures and the therapeutic potential of P2X7R-based treatments on long-lasting brain excitability. Experimental Approach Neonatal seizures were induced via global hypoxia in 7 day-old mouse pups (P7). The role of P2X7Rs during seizures was analyzed in P2X7R overexpressing and knock-out mice. Treatment of wild-type mice post-hypoxia with the P2X7R antagonist JNJ-47965567 was used to determine the effects of the P2X7R on long-lasting brain hyperexcitability. Cell type-specific P2X7R expression was analyzed via P2X7R-EGFP reporter mice. RNA sequencing was used to monitor P2X7R-dependent hippocampal down-stream signalling. Key Results P2X7R deletion reduced seizure severity whereas P2X7R overexpression exacerbated seizure severity and reduced responsiveness to anti-seizure medication. P2X7R deficiency let to an anti-inflammatory phenotype in microglia and treatment of mice with a P2X7R antagonist reduced long-lasting brain hyperexcitability. RNA sequencing identified several pathways altered in P2X7R knock-out mice after neonatal hypoxia including a down-regulation of genes implicated in inflammation and glutamatergic signalling. Conclusion and Implications Treatments based on targeting the P2X7R may represent a novel therapeutic strategy for neonatal seizures with P2X7Rs contributing to the generation of neonatal seizures, driving inflammatory processes and long-term hyperexcitability states.

Autism is a neurodevelopmental condition with a range of symptoms that vary in intensity and severity from person to person. Genetic sequencing has identified thousands of genes containing mutations in autistic individuals, which may contribute to the development of autistic symptoms. Several of these genes encode G protein-coupled receptors (GPCRs) which are cell surface expressed proteins that transduce extracellular messages to the intracellular space. Mutations in GPCRs can impact their function, resulting in aberrant signalling within cells, and across neurotransmitter systems in the brain. This review summarises the current knowledge on autism-associated single nucleotide variations encoding missense mutations in GPCRs, and the impact of these genetic mutations on GPCR function. For some autism-associated mutations, changes in GPCR expression levels, ligand affinity, potency and efficacy have been observed; however, for many the functional consequences remain unknown. Thus, further work to characterise the functional impacts of the genetically identified mutations is required.

Gastrointestinal motility is tightly regulated by the enteric nervous system (ENS). Disruption of coordinated ENS activity can result in dysmotility. Pharmacological treatment options for dysmotility include targeting of G protein-coupled receptors (GPCRs) expressed by neurons of the ENS. Current GPCR-targeting drugs for motility disorders bind to the highly conserved endogenous ligand binding site and promote indiscriminate activation or inhibition of the target receptor throughout the body. This can be associated with significant side-effect liability and a loss of physiological tone. Allosteric modulators of GPCRs bind to a distinct site from the endogenous ligand, which is typically less conserved across multiple receptor subtypes and can modulate endogenous ligand signalling. Allosteric modulation of GPCRs that are important for ENS function may provide effective relief from motility disorders while limiting side-effects. This review will focus on how allosteric modulators of GPCRs may influence gastrointestinal motility, using 5-HT, ACh, and opioid receptors as examples.