G protein-coupled receptors (GPCRs) are a large family of cell surface receptors that play a critical role in nervous system function by transmitting signals between cells and their environment. They are involved in many, if not all, nervous system processes, and their dysfunction has been linked to various neurological disorders representing important drug targets. In this review, we will first discuss the role of the nervous system GPCRs in the modulation of tripartite synapse function and how GPCRs control energy metabolism in the brain. We will then discuss the (patho)physiology and pharmacology of opioid, cannabinoid, acetylcholine, chemokine, and melatonin GPCRs in the nervous system. Furthermore, we will briefly report on adhesion GPCR function in nervous tissues. Finally, we will address orphan GPCRs, their implication in the nervous system function and disease, and the challenges that need to be addressed in the future to deorphanize them.
Background and Purpose L-type amino acid (AA) transporters LAT1 (SLC7A5) and LAT2 (SLC7A8) facilitate the bidirectional transport of branched and aromatic AA across the plasma membrane. While LAT1 is overexpressed in different tumour cells and it is dedicated to deliver AA into growing cells, LAT2 facilitates the transcellular AA transport at biological barriers. Data on dynamic AA transport by LAT1/2 in physiological media are widely lacking and the impact of LAT1-selective inhibitors and mutations on the cellular metabolome is unknown. Experimental Approach The human MDST8 cell line lacking LAT1/2 expression was employed to generate transiently and stably expressing MDST8-LAT1 and MDST8-LAT2 cells. Together with the HT-29 cell line, we depicted metabolic signatures mediated by LAT1 and LAT2 using LC-ESI-MS/MS and characterized potent LAT1/2 inhibitors for their selectivity and mode of action. Moreover, LAT1 mutations associated with autism spectrum disorder (ASD) were functionally evaluated. Key Results LAT1 and LAT2 expression induced the expression of 4F2hc and facilitated differential cellular metabolomic signatures in MDST8 cells. The LAT1(A246V) mutation showed overall reduced aromatic AA uptake and profound alterations of intracellular metabolites and biochemical processes. The equipotent LAT1 inhibitors JPH203 and JX-078 showed intriguing differences on metabolomic effects, uncovering their distinct mode of action. Conclusion and Implications Our study demonstrates that SLC7A transporters mediate differential dynamic cellular AA changes under physiological conditions and reports the broad effects of one human LAT1 mutation associated with ADS. Moreover, the characterization of biased LAT1 inhibitors challenges current concepts about transporter pharmacology and has implications for drug discovery.
Objective: Our study aimed to explore the mechanism network that TLR2/AP-1 combined with SOX10 to activate the MAPK pathway via CTGF in Dox-induced myocardial injury. Methods: Rats with Dox-induced myocardial injury were treated with a TLR2 inhibitor or CTGF silencing lentiviral vector. H9c2 cells were treated with genetic vectors or MAPK pathway activators. Cardiac function was tested using echocardiography and serum markers. H&E, sirius red, and TUNEL staining were used to detect myocardial pathological changes, collagen accumulation, and apoptosis. Western blot was used to detect proteins related to cardiac hypertrophy, fibrosis, apoptosis, and MAPK pathway. H9c2 cell injury was assessed by testing cell viability, LDH release, and mitochondrial membrane potential. Results: TLR2 and CTGF were highly expressed in patients with heart failure, and Dox treatment further increased their expression. Inhibiting TLR2 or silencing CTGF improved cardiac function and reduced myocardial fibrosis and apoptosis in Dox-treated rats. Silencing TLR2 alleviated Dox-induced H9c2 cell injury, which was nullified by CTGF overexpression. TLR2 activated AP-1, which cooperated with SOX10 to promote CTGF transcription. MAPK activation aggravated H9c2 cells against Dox-induced injury. Conclusions: TLR2 activates AP-1 which cooperates with SOX10 to promote CTGF transcription and subsequently activate the MAPK pathway, thereby stimulating Dox-induced myocardial injury.
Background and Purpose: The holotoxin A1, isolated from Apostichopus japonicus, has shown potent antifungal activities with unclear mechanism and efficacy against candidiasis. This study aimed to reveal the antifungal effects and mechanism of holotoxin A1 against Candida albicans in vitro and in candidiasis murine models for the first time. Experimental Approach: The antifungal effect of holotoxin A1 against C. albicans was tested in vitro. To explore the antifungal mechanism of holotoxin A1, the transcriptome, ROS levels, and mitochondrial function of C. albicans was evaluated. The oropharyngeal and intra-abdominal candidiasis mouse models were used to verify the effectiveness and systematic toxicity in vivo. Key Results: Holotoxin A1 was a potent fungicide against C. albicans SC5314, clinical strains and drug-resistant strains. Holotoxin A1 inhibited the oxidative phosphorylation and induced oxidative damage by increasing intracellular accumulation of ROS in C. albicans. Holotoxin A1 caused the disfunction of mitochondria by depolarizing the mitochondrial membrane potential and reducing the production of ATP. Holotoxin A1 directly inhibited the enzymatic activity of mitochondrial complex I (CI) and antagonized with the rotenone, an inhibitor of CI, against C. albicans. Meanwhile, the CI subunit NDH51 null mutants showed the decreased susceptibility to holotoxin A1. Furthermore, holotoxin A1 significantly reduced fungal burden and infections with no significant systemic toxicity in oropharyngeal and intra-abdominal candidiasis murine models. Conclusions and Implications: Holotoxin A1 was a promising candidate for the development of novel antifungal drug against both oropharyngeal and intra-abdominal candidiasis, especially caused by the drug resistant strains.
Background and purpose: Hypertension increases the risk for cognitive impairment and promotes vascular and renal inflammation. We tested if immune cell infiltration occurs in the brain during hypertension and if it is associated with cognitive impairment. Experimental approach: Male C57Bl/6 mice were administered vehicle, angiotensin II (0.7 mg/kg/d S.C.) or aldosterone (0.72 mg/kg/d S.C.) via osmotic minipumps. A subset of mice also received hydralazine (50 mg/kg) in their drinking water after minipump implantation. We measured systolic blood pressure, markers of inflammation, working memory and transcriptomic changes in the brain. Key results: Administration of angiotensin II or aldosterone increased blood pressure and promoted blood-brain barrier dysfunction, leukocyte accumulation and impairment of working memory in mice. When co-administered with angiotensin II, hydralazine prevented the development of these changes. In a separate cohort of mice in which angiotensin II-induced changes were first established, intervention with hydralazine lowered blood pressure but did not reverse brain inflammation or cognitive impairment. Finally, angiotensin II infusion altered the transcriptomic profile of the whole brain, as well as specifically within the hippocampus, and co-treatment with hydralazine modulated these changes. Conclusion and implications: Experimental hypertension leads to brain inflammation and impaired working memory. Cognitive impairment that develops during hypertension can be inhibited, but not readily reversed, by anti-hypertensive therapy.
Background and Purpose Metabotropic Glutamate Receptors (mGlu) regulate multiple functions in the nervous systems and are involved in multiple disorders. However, selectively targeting individual mGlu subtypes with spatiotemporal precision is still an unmet need. Photopharmacology can address this concern by means of photoswitchable compounds such as Optogluram, which is a positive allosteric modulator (PAM) of mGlu4 that enables to optically control physiological responses with a high precision. However, Optogluram is not fully selective and finding mGlu4 PAMs with subtype selectivity may be complicated. Experimental Approach New photoswitchable analogues of Optogluram were synthesised with the aim of obtaining photoswitchable PAMs selective for mGlu4 receptor and with improved photoisomerization properties. The photopharmacological profiles of these new compounds were assessed using spectroscopy, functional IP and cAMP assays and computational modelling. Key Results Optogluram-2 emerged as a new photoswitchable PAM for mGlu4 receptor and offered improved photoswitching properties and was selective for mGlu4. Optogluram-2 had activity as both PAM and allosteric agonist. The π-π stacking of the thiazole ring in the allosteric pocket of mGlu6 is hypothesised to be responsible of the mGlu4 selectivity. Conclusion and Implications. The enhanced photoswitching behaviour and improved selectivity of Optogluram-2 makes it an excellent candidate to study the role of mGlu4 with a high spatiotemporal precision that only photopharmacology can offer. Indeed, the use of Optogluram-2 in tissues where mGlu4 can be co-expressed with other mGlu receptors will help to unravel the complexity of mGlu receptors in neural transmission, pinpointing the role of mGlu4 in such systems.
The GPCR signalling cascade is a key pathway responsible for the signal transduction of a multitude of physical and chemical stimuli, including light, odorants, neurotransmitters, and hormones. Understanding the structural and functional properties of the GPCR cascade requires direct observation of signalling processes in high spatial and temporal resolution with minimal perturbation to endogenous systems. Optical microscopy and spectroscopy techniques are uniquely suited to this purpose because they excel at multiple spatial and temporal scales and can be used in living objects. Here, we review recent developments in microscopy and spectroscopy technologies which enable new insights into GPCR signalling. We focus on advanced techniques with high spatial and temporal resolution, single-molecule methods, labelling strategies, and approaches suitable for endogenous systems and large living objects. This review aims to assist researchers in choosing appropriate microscopy and spectroscopy approaches for a variety of applications in the study of cellular signalling.
Background and purpose Chymotrypsin is a serine protease produced by the pancreas and secreted into the lumen of the small intestine, where it digests food proteins. Due to its presence in the gut lumen, we hypothesized that chymotrypsin activity may be found close to epithelial cells and signals to them via Protease-activated receptors (PARs). We deciphered molecular pharmacology mechanisms for chymotrypsin signaling in intestinal epithelial cells. Experimental approaches The presence and activity of chymotrypsin were evaluated by western blot (WB) and enzymatic activity tests in luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally-tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. To study the pharmacology of chymotrypsin signals, we investigated calcium signaling and ERK1/2 activation using calcium mobilization assays and WB in CMT93 intestinal epithelial cells. Key results We found that chymotrypsin was present and active in the vicinity of the murine and human colonic epithelium. Molecular pharmacology studies evidenced that chymotrypsin cleaved both PARs receptors. While chymotrypsin activated calcium and ERK1/2 signaling pathways through PAR2, it disarmed PAR1, preventing further activation by its canonical agonist thrombin. CONCLUSION Our work suggests that the function of chymotrypsin in the gut lumen goes well beyond a simple digestive role. Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.
Inflammation is a physiological response composed by well-defined and overlapping events that can eliminate pathogens and reestablish homeostasis of tissues. Physiological systems have an elastic capacity to deal with numerous perturbations. Infection may lead to inflammation, tissue damage and disease as consequence of breakdown of tissue resilience. The resolutive phase is a sine qua non condition to achieve homeostasis after acute inflammation. Exuberant or chronic inflammation occurs in diverse infectious diseases. Pro-resolving molecules may be useful for the treatment of certain infections, as these molecules modulate the immune response and avoid the exacerbated/misplaced inflammation unleashed by microbes. Some pro-resolving molecules may also favour pathogen clearance, in addition to decreasing tissue damage. In this review, we discuss the endogenous role and the therapeutic potential of the most relevant pro-resolving molecules in the context of bacterial and viral infections.
Accumulating studies in recent years have revealed that airway remodeling is involved in the occurrence, development, and treatment sensitivity of asthma. Airway epithelial cells (AECs) regulate the activation of epithelial-mesenchymal trophic units (EMTUs) during airway remodeling through secretion of a series of signaling mediators. However, the major trigger and the intrinsic pathogenesis of airway remodeling is still obscure. Here, we show that the expression of CTSK in airway epithelia increased significantly along with the development of airway remodeling in HDM-stressed asthma model. Increased secretion of CTSK from airway epithelia induced the activation of EMTU through the activation of PAR2-mediated pathway. We found that CTSK is a potential biomarker of airway remodeling for asthma patients that can reflect the degree of airway remodeling and the severity of asthma. Blockade of CTSK inhibits EMTU activation and alleviate airway remodeling effectively that is an effective intervention target of airway remodeling. Thus, our findings provide that CTSK is a potential biomarker for airway remodeling which may also be a useful target for the targeted intervention of airway remodeling in asthma patients.
Background and Purposes: Chemotherapy-induced peripheral neuropathy commonly causes neuropathic pain. The pathogenesis of CIPN is unclear, and effective therapies are also lacking. Naringenin, a dihydroflavonoid compound in Rutaceae plants and citrus fruits, has anti-inflammatory, antioxidant, and anti-tumor activities. However, its effect on chemotherapy-induced pain has not been investigated. Experimental Approach: We used Paclitaxel (PTX) to establish a mouse model of chemotherapy-induced pain. Mechanical and thermal pain thresholds, glial activation, calcitonin gene-related peptide (CGRP) expression, c-fos expression, phosphorylation of nuclear factor κB (NF-κB), dorsal root ganglion (DRG) neuron excitability, and cell survival of pancreatic, colorectal, and gastric cancer cell lines were measured. Key Results: Systemic application of Naringenin reduced the mechanical and thermal pain hypersensitivity induced by PTX. Naringenin reduced the activation of glial cells in both DRGs and the spinal dorsal horn of PTX-treated mice. Naringenin decreased the PTX-enhanced CGRP expression in DRG and spinal neurons. Naringenin reversed the PTX-enhanced c-fos expression and excitability of DRG neurons. Naringenin downregulated PTX-elevated NF-κB phosphorylation in the spinal cord. Additionally, co-administration of Naringenin with PTX enhanced the inhibitory effect of PTX on pancreatic and colorectal cancer cell growth, whereas the application of Naringenin alone inhibited the survival of pancreatic cancer cells. Conclusion and Implications: Naringenin alleviates PTX-induced pain and may facilitate PTX’s anti-tumor effect. The mechanism involves the inhibition of glial activation, CGRP production, and neuronal sensitization in PTX-treated mice. Our study suggests the multiple beneficial actions of Naringenin in chemotherapy by mitigating side effects and inhibiting tumor growth.
Background and Purpose: In major depressive disorder (MDD), exploration of biomarkers will be helpful in diagnosing the disorder as well as in choosing a treatment, and predicting the treatment response. Currently, tRNA-derived small ribonucleic acids (tsRNAs) have been established as promising non-invasive biomarker candidates that may enable a more reliable diagnosis or monitoring of various diseases. Herein, we aimed to explore tsRNA expression together with functional activities in MDD development. Experimental Approach: Serum samples were obtained from patients with MDD and healthy controls, and small RNA sequencing (RNA-Seq) was used to profile tsRNA expression. Dysregulated tsRNAs in MDD were validated by quantitative real-time polymerase chain reaction (qRT-PCR). The diagnostic utility of specific tsRNAs and the expression of these tsRNAs after antidepressant treatment was analyzed. Key Results: In total, 38 tsRNAs were significantly differentially expressed in MDD samples relative to healthy individuals (34 upregulated and 4 downregulated). qRT-PCR was used to validate the expression of six tsRNAs that were upregulated in MDD (tiRNA-1:20-chrM. Ser-GCT, tiRNA-1:33-Gly-GCC-1, tRF-1:22-chrM.Ser-GCT, tRF-1:31-Ala-AGC-4-M6, tRF-1:31-Pro-TGG-2, and tRF-1:32-chrM. Gln-TTG). Interestingly, serum tiRNA-Gly-GCC-001 levels exhibited an area under the ROC curve of 0.844. Moreover, tiRNA-Gly-GCC-001 is predicted to suppress brain-derived neurotrophic factor (BDNF) expression. Furthermore, significant tiRNA-Gly-GCC-001 downregulation was evident following an eight-week treatment course and served as a promising baseline predictor of patient response to antidepressant therapy. Conclusion and Implications: Our current work firstly found that tiRNA-Gly-GCC-001 is a promising MDD biomarker candidate that can predict patient responses to antidepressant therapy.
Background & purpose Morphine is important for treatment of acute and chronic pain. However, there is high interpatient variability and often inadequate pain relief and adverse effects. To better understand variability in the dose effect relationships of morphine, we investigated the impact of its nonlinear blood-brain-barrier (BBB) transport on mu-opioid receptor (μ-OR) occupancy in different CNS locations, in conjunction with its main metabolites that bind to the same receptor. Methods CNS exposure profiles for morphine, M3G and M6G for clinically relevant dosing regimens based on intravenous, oral immediate- and extended-release formulations were generated using a CNS PBPK model which incorporated nonlinear BBB transport of morphine. The simulated CNS exposure profiles were then used to derive corresponding μ-OR occupancies at multiple CNS pain matrix locations. Results The simulated CNS exposure profiles for morphine, M3G and M6G, associated with nonlinear BBB transport of morphine resulted in varying μ-OR occupancies between the different dose regimens, formulations, and CNS locations. We find that at lower doses, the μ-OR occupancy of morphine was relatively higher than at higher doses of morphine, due to the relative contribution of M3G and M6G. At such higher doses, M6G showed higher occupancy than morphine, whereas M3G occupancy was low throughout the dose ranges. Conclusion and implications Nonlinear BBB transport of morphine influences the μ-OR occupancy ratios of morphine and its metabolites, depending on dose and route of administration, and CNS location. This may impact the clinical effects of morphine treatment for pain relief.
Decreased aortic compliance is a precursor to numerous cardiovascular diseases. Compliance is regulated by the rigidity of the aortic wall and the vascular smooth muscle cells (VSMCs) within it. Extracellular matrix stiffening, observed during ageing, reduces compliance and contributes to hypertension. In response to increased rigidity, VSMCs generate enhanced contractile forces that result in VSMC stiffening and a further reduction in compliance. Due to a lack of suitable in vitro models, the mechanisms driving VSMC response to matrix rigidity remain poorly defined. Human aortic-VSMCs were seeded onto polyacrylamide hydrogels whose rigidity mimicked either healthy or aged/diseased aortae. VSMC response to contractile agonist stimulation was measured through changes in cell area and volume. VSMCs were pre-treated with pharmacological agents prior to agonist stimulation to identify regulators of VSMC hypertrophy. VSMCs undergo a differential response to contractile agonist stimulation based on matrix rigidity. On pliable matrices, VSMCs contract, decreasing in cell area. Meanwhile, on rigid matrices VSMCs undergo a hypertrophic response, increasing in area and volume. Piezo1 mediated calcium influx drives VSMC hypertrophy by promoting microtubule destabilisation. Pharmacological stabilisation of microtubules or blocking calcium influx prevented VSMC hypertrophy on rigid matrices whilst maintaining contractility on pliable matrices. In response to extracellular matrix rigidity, VSMCs undergo a hypertrophic response driven by piezo1-mediated microtubule destabilisation. Pharmacological targeting of this response blocks matrix rigidity induced VSMC hypertrophy whilst VSMC contractility on healthy mimicking matrices is unimpeded. Through delineating this rigidity-induced mechanism, we identify novel targets whose pharmacological inhibition may prove beneficial against VSMC-driven cardiovascular disease.
Background and Purpose Asthma is characterized by airway inflammation, mucus hypersecretion and airway hyperresponsiveness (AHR). The activation of cholinergic anti‐inflammatory pathway (CAP) through nicotinic agents has been shown to control experimental asthma. Yet, the effects of vagus nerve stimulation (VNS)-induced CAP on allergic inflammation remain unknown. Experimental Approach BALB/c mice were sensitized and challenged with house dust mite (HDM) extract, and treated with active VNS (5Hz, 0.5 ms, 0.1 mA). Bronchoalveolar lavage (BAL) ﬂuid was assessed for total and differential cell counts and cytokine levels. Lungs were examined by histopathology and electron microscopy. AHR in response to methacholine was also measured. Key Results In the HDM mouse asthma model, active but not sham VNS reduced BAL fluid total and differential cell counts, blocked mucus hypersecretion and suppressed choline acetyltransferase (ChAT) expression in bronchial epithelial cells. Besides, active VNS also abated HDM-induced elevation of type 2 cytokines IL-4 and IL-5. Furthermore, goblet cell hyperplasia and collagen deposition were diminished in VNS-treated mice. Mechanistically, VNS was found to block the phosphorylation of transcription factor STAT6 and the level of IRF4 in total lung lysates. Finally, VNS abrogated methacholine-induced AHR in asthma mice. Therapeutic effects of VNS were abolished by prior administration with α-bungarotoxin, a specific inhibitor of α7 nicotinic receptors (α7nAChR). Conclusion Our data revealed the protective effects of VNS on various clinical features in allergic airway inflammation model. VNS, a clinically approved therapy for depression and epilepsy, appears to be a promising new strategy for controlling allergic asthma through α7nAChR.
Background and Purpose: Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by using herbal medicines. Currently, no therapies are available to treat or prevent AAN. Histone deacetylase (HDAC) plays a crucial role in the development and progression of renal disease. We tested whether HDAC inhibitors could prevent AAN and determined the underlying mechanism. Experimental Approach: HDACs expression in the kidneys was examined. Mouse kidney and renal tubular epithelial cell damage were assessed after exposure to HDAC1 and HDAC2 blockade (FK-228). Conditional knock-in of Proline-serine-threonine-phosphatase-interacting protein 2 (PSTPIP2) in the kidney and knockdown of PSTPIP2 expression in PSTPIP2-knockin mice, pathological parameters, and kidney injuries were assessed. Key Results: Aristolochic acid upregulated the expression of HDAC1 and HDAC2 in the kidneys. Notably, the HDAC1 and -2 specific inhibitor, romidepsin (FK228, Depsipeptide), suppressed aristolochic acid-induced kidney injury, epithelial cell pyroptosis, apoptosis, and necroptosis (PANoptosis). Moreover, romidepsin upregulated PSTPIP2 in renal tubular epithelial cells, which was enhanced by aristolochic acid treatment. Conditional knock-in of PSTPIP2 in the kidney protected against AAN. In contrast, the knockdown of PSTPIP2 expression in PSTPIP2-knockin mice restored kidney damage and PANoptosis. PSTPIP2 function was determined in vitro using PSTPIP2 knockdown or overexpression in mTEC. Additionally, PSTPIP2 was found to regulate Caspase-8 in Aristolochic acid nephropathy. Conclusion and Implications: HDAC-mediated silencing of PSTPIP2 may contribute to aristolochic acid nephropathy. Hence, HDAC1 and -2 specific inhibitors or PSTPIP2 could be valuable therapeutic agents for the prevention of aristolochic acid nephropathy.
Investigating the intricate mechanisms of G protein-coupled receptor (GPCR) signalling in living cells is far from trivial. Over the last 20 years, the rise of genetically encoded resonance energy transfer (RET) sensors has shed new light onto the mechanisms of GPCR signalling. Such findings have challenged classical views on GPCR signalling and enhanced our understanding of the spatiotemporal dimensions of GPCR activity, leading to the discovery of endosomal GPCR signalling. This review highlights the use of RET sensors to monitor GPCR signalling in real-time and in live cells, focusing on GPCR activation and trafficking, and second messenger activity. It explores the physiological relevance of illustrative cases of endosomal signalling and discusses potential avenues to improve RET approaches to further explore endogenous GPCR activity in physiologically relevant contexts.
Background and Purpose: Traumatic brain injury (TBI) remains a leading cause of mortality and morbidity in young adults. The role of iron in potentiating neurodegeneration following TBI has gained recent interest since iron deposition has been detected in the injured brain in the weeks to months post-TBI, in both the preclinical and clinical setting. A failure in iron homeostasis can lead to oxidative stress, inflammation and excitotoxicity; and whether this is a cause or consequence of the long-term effects of TBI remains unknown. Experimental approach: We investigated the role of iron, and the effect of therapeutic intervention using a brain-permeable iron chelator, deferiprone, in a controlled cortical impact mouse model of TBI. An extensive assessment of cognitive, motor and anxiety/depressive outcome measures were examined, and neuropathological and biochemical changes, over a 3-month period post-TBI. Key Results: Lesion volume was significantly reduced at 3 months, which was preceded by a reduction in astrogliosis and a preservation of neurons in the injured brain at 2 weeks and/or 1-month post-TBI in mice receiving oral deferiprone. Deferiprone treatment showed significant improvements in neurological severity scores and locomotor/gait performance, and cognitive function, and attenuated anxiety-like symptoms post-TBI. Deferiprone reduced iron levels, oxidative stress and altered expression of neurotrophins in the injured brain over this period. Conclusion and Implications: Our findings support a detrimental role of iron in the injured brain and suggest that deferiprone (or similar iron chelators) may be promising therapeutic approaches to improve survival, functional outcomes and quality of life following TBI.