ABSTRACT
Glutamate excitotoxicity is a central mechanism contributing to cellular dysfunction and death in various neurological disorders and diseases, such as stroke, traumatic brain injury, epilepsy, schizophrenia, addiction, mood disorders, Huntington's disease, Alzheimer's disease, Parkinson's disease, multiple sclerosis, pathologic pain, and even normal aging-related changes. This detrimental effect emerges from glutamate binding to glutamate receptors, including α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, N-methyl-d-aspartate receptors, kainate receptors, and GluD receptors. Thus, excitotoxicity could be prevented by targeting glutamate receptors and their downstream signaling pathways. However, almost all the glutamate receptor antagonists failed to attenuate excitotoxicity in human patients, mainly due to the limited understanding of the underlying mechanisms regulating excitotoxicity. Transient receptor potential (TRP) channels serve as ancient cellular sensors capable of detecting and responding to both external and internal stimuli. The study of human TRP channels has flourished in recent decades since the initial discovery of mammalian TRP in 1995. These channels have been found to play pivotal roles in numerous pathologic conditions, including excitotoxicity. In this review, our focus centers on exploring the intricate interactions between TRP channels and glutamate receptors in excitotoxicity.
ABSTRACT
N-methyl-D-aspartate receptor (NMDAR)-mediated glutamate excitotoxicity significantly contributes to ischemic neuronal death and post-recanalization infarction expansion. Despite tremendous efforts, targeting NMDARs has proven unsuccessful in clinical trials for mitigating brain injury. Here, we show the discovery of an interaction motif for transient receptor potential melastatin 2 (TRPM2) and protein kinase Cγ (PKCγ) association and demonstrate that TRPM2-PKCγ uncoupling is an effective therapeutic strategy for attenuating NMDAR-mediated excitotoxicity in ischemic stroke. We demonstrate that the TRPM2-PKCγ interaction allows TRPM2-mediated Ca2+ influx to promote PKCγ activation, which subsequently enhances TRPM2-induced potentiation of extrasynaptic NMDAR (esNMDAR) activity. By identifying the PKCγ binding motif on TRPM2 (M2PBM), which directly associates with the C2 domain of PKCγ, an interfering peptide (TAT-M2PBM) is developed to disrupt TRPM2-PKCγ interaction without compromising PKCγ function. M2PBM deletion or TRPM2-PKCγ dissociation abolishes both TRPM2-PKCγ and TRPM2-esNMDAR couplings, resulting in reduced excitotoxic neuronal death and attenuated ischemic brain injury.
Subject(s)
Brain Injuries , TRPM Cation Channels , Humans , Protein Kinases/metabolism , TRPM Cation Channels/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Peptides/metabolismABSTRACT
AIMS: Damage of the blood-brain barrier (BBB) is a hallmark of brain injury during the early stages of ischemic stroke. The subsequent endothelial hyperpermeability drives the initial pathological changes and aggravates neuronal death. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable nonselective cation channel activated by oxidative stress. However, whether TRPM2 is involved in BBB degradation during ischemic stroke remains unknown. We aimed to investigate the role of TRPM2 in BBB degradation during ischemic stroke and the underlying molecular mechanisms. METHODS AND RESULTS: Specific deletion of Trpm2 in endothelial cells using Cdh5 Cre produces a potent protective effect against brain injury in mice subjected to middle cerebral artery occlusion (MCAO), which is characterized by reduced infarction size, mitigated plasma extravasation, suppressed immune cell invasion, and inhibited oxidative stress. In vitro experiments using cultured cerebral endothelial cells (CECs) demonstrated that either Trpm2 deletion or inhibition of TRPM2 activation attenuates oxidative stress, Ca2+ overload, and endothelial hyperpermeability induced by oxygen-glucose deprivation (OGD) and CD36 ligand thrombospondin-1 (TSP1). In transfected HEK293T cells, OGD and TSP1 activate TRPM2 in a CD36-dependent manner. Noticeably, in cultured CECs, deleting Trpm2 or inhibiting TRPM2 activation also suppresses the activation of CD36 and cellular dysfunction induced by OGD or TSP1. CONCLUSIONS: In conclusion, our data reveal a novel molecular mechanism in which TRPM2 and CD36 promote the activation of each other, which exacerbates endothelial dysfunction during ischemic stroke. Our study suggests that TRPM2 in endothelial cells is a promising target for developing more effective and safer therapies for ischemic stroke.
Subject(s)
Brain Injuries , Brain Ischemia , Ischemic Stroke , Stroke , TRPM Cation Channels , Humans , Mice , Animals , Blood-Brain Barrier/metabolism , Ischemic Stroke/metabolism , Endothelial Cells/metabolism , TRPM Cation Channels/metabolism , Calcium/metabolism , HEK293 Cells , Oxygen , Brain Injuries/metabolism , Stroke/metabolism , Brain Ischemia/metabolismABSTRACT
Ischemic stroke is a devastating disease that affects millions of patients worldwide. Unfortunately, there are no effective medications for mitigating brain injury after ischemic stroke. TRP channels are evolutionally ancient biosensors that detect external stimuli as well as tissue or cellular injury. To date, many members of the TRP superfamily have been reported to contribute to ischemic brain injury, including the TRPC subfamily (1, 3, 4, 5, 6, 7), TRPV subfamily (1, 2, 3, 4) and TRPM subfamily (2, 4, 7). These TRP channels share structural similarities but have distinct channel functions and properties. Their activation during ischemic stroke can be beneficial, detrimental, or even both. In this review, we focus on discussing the interesting features of stroke-related TRP channels and summarizing the underlying cellular and molecular mechanisms responsible for their involvement in ischemic brain injury.
ABSTRACT
Voltage-gated calcium channels (VGCCs), especially Cav2.1 and Cav2.2, are the major mediators of Ca2+ influx at the presynaptic membrane in response to neuron excitation, thereby exerting a predominant control on synaptic transmission. To guarantee the timely and precise release of neurotransmitters at synapses, the activity of presynaptic VGCCs is tightly regulated by a variety of factors, including auxiliary subunits, membrane potential, G protein-coupled receptors (GPCRs), calmodulin (CaM), Ca2+-binding proteins (CaBP), protein kinases, various interacting proteins, alternative splicing events, and genetic variations.
Subject(s)
Calcium Channels , Synapses , Humans , Synaptic TransmissionABSTRACT
Cystathionine-ß-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) are an evolutionarily conserved family of magnesium transporters. They promote efflux of Mg2+ ions on their own and influx of divalent cations when expressed with the transient receptor potential ion channel subfamily M member 7 (TRPM7). Recently, ADP-ribosylation factor-like GTPase 15 (ARL15) has been identified as CNNM-binding partner and an inhibitor of divalent cation influx by TRPM7. Here, we characterize ARL15 as a GTP and CNNM-binding protein and demonstrate that ARL15 also inhibits CNNM2 Mg2+ efflux. The crystal structure of a complex between ARL15 and CNNM2 CBS-pair domain reveals the molecular basis for binding and allowed the identification of mutations that specifically block binding. A binding deficient ARL15 mutant, R95A, failed to inhibit CNNM and TRPM7 transport of Mg2+ and Zn2+ ions. Structural analysis and binding experiments with phosphatase of regenerating liver 2 (PRL2 or PTP4A2) showed that ARL15 and PRLs compete for binding CNNM to coordinate regulation of ion transport by CNNM and TRPM7.
Subject(s)
Monomeric GTP-Binding Proteins , TRPM Cation Channels , Cations, Divalent , TRPM Cation Channels/genetics , Protein Binding , Biological TransportABSTRACT
Cystathionine-ß-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) are an evolutionarily conserved family of magnesium transporters. They promote efflux of Mg 2+ ions on their own or uptake of divalent cations when coupled to the transient receptor potential ion channel subfamily M member 7 (TRPM7). Recently, ADP-ribosylation factor-like GTPase 15 (ARL15) has been identified as CNNM binding partner and an inhibitor of divalent cation influx by TRPM7. Here, we characterize ARL15 as a GTP-binding protein and demonstrate that it binds the CNNM CBS-pair domain with low micromolar affinity. The crystal structure of the complex between ARL15 GTPase domain and CNNM2 CBS-pair domain reveals the molecular determinants of the interaction and allowed the identification of mutations in ARL15 and CNNM2 mutations that abrogate binding. Loss of CNNM binding prevented ARL15 suppression of TRPM7 channel activity in support of previous reports that the proteins function as a ternary complex. Binding experiments with phosphatase of regenerating liver 2 (PRL2 or PTP4A2) revealed that ARL15 and PRLs compete for binding CNNM, suggesting antagonistic regulation of divalent cation transport by the two proteins.
ABSTRACT
The vast potential of human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) in preclinical models of cardiac pathologies, precision medicine, and drug screening remains to be fully realized because hiPSC-CMs are immature without adult-like characteristics. Here, we present a method to accelerate hiPSC-CM maturation on a substrate, cardiac mimetic matrix (CMM), mimicking adult human heart matrix ligand chemistry, rigidity, and submicron ultrastructure, which synergistically mature hiPSC-CMs rapidly within 30 days. hiPSC-CMs matured on CMM exhibit systemic transcriptomic maturation toward an adult heart state, are aligned with high strain energy, metabolically rely on oxidative phosphorylation and fatty acid oxidation, and display enhanced redox handling capability, efficient calcium handling, and electrophysiological features of ventricular myocytes. Endothelin-1-induced pathological hypertrophy is mitigated on CMM, highlighting the role of a native cardiac microenvironment in withstanding hypertrophy progression. CMM is a convenient model for accelerated development of ventricular myocytes manifesting highly specialized cardiac-specific functions.
Subject(s)
Induced Pluripotent Stem Cells , Myocytes, Cardiac , Adult , Cell Differentiation/physiology , Cells, Cultured , Humans , Hypertrophy/metabolism , Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/metabolismABSTRACT
Atherosclerosis is the major cause of ischemic heart disease and stroke, the leading causes of mortality worldwide. The central pathological features of atherosclerosis include macrophage infiltration and foam cell formation. However, the detailed mechanisms regulating these two processes remain unclear. Here we show that oxidative stress-activated Ca2+-permeable transient receptor potential melastatin 2 (TRPM2) plays a critical role in atherogenesis. Both global and macrophage-specific Trpm2 deletion protect Apoe -/- mice against atherosclerosis. Trpm2 deficiency reduces oxidized low-density lipoprotein (oxLDL) uptake by macrophages, thereby minimizing macrophage infiltration, foam cell formation and inflammatory responses. Activation of the oxLDL receptor CD36 induces TRPM2 activity, and vice versa. In cultured macrophages, TRPM2 is activated by CD36 ligands oxLDL and thrombospondin-1 (TSP1), and deleting Trpm2 or inhibiting TRPM2 activity suppresses the activation of CD36 signaling cascade induced by oxLDL and TSP1. Our findings establish the TRPM2-CD36 axis as a molecular mechanism underlying atherogenesis, and suggest TRPM2 as a potential therapeutic target for atherosclerosis.
ABSTRACT
Excitotoxicity induced by NMDA receptor (NMDAR) activation is a major cause of neuronal death in ischemic stroke. However, past efforts of directly targeting NMDARs have unfortunately failed in clinical trials. Here, we reveal an unexpected mechanism underlying NMDAR-mediated neurotoxicity, which leads to the identification of a novel target and development of an effective therapeutic peptide for ischemic stroke. We show that NMDAR-induced excitotoxicity is enhanced by physical and functional coupling of NMDAR to an ion channel TRPM2 upon ischemic insults. TRPM2-NMDAR association promotes the surface expression of extrasynaptic NMDARs, leading to enhanced NMDAR activity and increased neuronal death. We identified a specific NMDAR-interacting motif on TRPM2 and designed a membrane-permeable peptide to uncouple the TRPM2-NMDAR interaction. This disrupting peptide protects neurons against ischemic injury in vitro and protects mice against ischemic stroke in vivo. These findings provide an unconventional strategy to mitigate excitotoxic neuronal death without directly targeting NMDARs.
Subject(s)
Brain Injuries , Ischemic Stroke , TRPM Cation Channels , Animals , Mice , N-Methylaspartate/pharmacology , Peptides/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , TRPM Cation Channels/geneticsABSTRACT
Ischemic stroke causes a heavy health burden worldwide, with over 10 million new cases every year. Despite the high prevalence and mortality rate of ischemic stroke, the underlying molecular mechanisms for the common etiological factors of ischemic stroke and ischemic stroke itself remain unclear, which results in insufficient preventive strategies and ineffective treatments for this devastating disease. In this review, we demonstrate that transient receptor potential cation channel, subfamily M, member 2 (TRPM2), a non-selective ion channel activated by oxidative stress, is actively involved in all the important steps in the etiology and pathology of ischemic stroke. TRPM2 could be a promising target in screening more effective prophylactic strategies and therapeutic medications for ischemic stroke.
Subject(s)
Ischemic Stroke , TRPM Cation Channels , Humans , Cell Death , Oxidative Stress , Risk Factors , TRPM Cation Channels/genetics , TRPM Cation Channels/metabolismABSTRACT
The transient receptor potential melastatin 4 (TRPM4) is a Ca2+-activated nonselective monovalent cation channel belonging to the TRP channel superfamily. TRPM4 is widely expressed in various tissues and most abundantly expressed in the heart. TRPM4 plays a critical role in cardiac conduction. Patients carrying a gain-of-function or loss-of-function mutation of TRPM4 display impaired cardiac conduction. Knockout or over-expression of TRPM4 in mice recapitulates conduction defects in patients. Moreover, recent studies have indicated that TRPM4 plays a role in hypertrophy and heart failure. Whereas the role of TRPM4 mediated by cardiac myocytes has been well investigated, little is known about TRPM4 and its role in cardiac fibroblasts. Here we show that in human left ventricular fibroblasts, TRPM4 exhibits typical Ca2+-activation characteristics, linear current-voltage (I-V) relation, and monovalent permeability. TRPM4 currents recorded in fibroblasts from heart failure patients (HF) are more than 2-fold bigger than those from control individuals (CTL). The enhanced functional TRPM4 in HF is not resulted from changed channel properties, as TRPM4 currents from both HF and CTL fibroblasts demonstrate similar sensitivity to intracellular calcium activation and extracellular 9-phenanthrol (9-phen) blockade. Consistent with enhanced TRPM4 activity, the protein level of TRPM4 is about 2-fold higher in HF than that of CTL hearts. Moreover, TRPM4 current in CTL fibroblasts is increased after 24 hours of TGFß1 treatment, implying that TRPM4 in vivo may be upregulated by fibrogenesis promotor TGFß1. The upregulated TRPM4 in HF fibroblasts suggests that TRPM4 may play a role in cardiac fibrogenesis under various pathological conditions.
Subject(s)
Fibroblasts/metabolism , Heart Failure/metabolism , Heart Ventricles/metabolism , TRPM Cation Channels/metabolism , Female , Humans , Male , Middle Aged , Myocytes, Cardiac/metabolism , Up-RegulationABSTRACT
Vascular calcification is an important risk factor for the mortality and morbidity in chronic kidney disease (CKD). Unfortunately, until now there is no certain medication targeting vascular calcification in CKD. In this study, we explored the inhibitory effect of celastrol on high calcium-induced vascular calcification and the underlying molecular mechanisms. Cell proliferation assay showed that celastrol inhibited aortic valve interstitial cell (VIC) and vascular smooth muscle cell (VSMC) proliferation when its concentration was higher than 0.6 µmol/L. 0.8 µmol/L celastrol inhibited the expression of osteogenic genes and calcium deposition induced by high-calcium medium in both AVICs and VSMCs. In mouse vascular calcification model induced by adenine combined with vitamin D, alizarin red and immunostaining showed that celastrol inhibited pro-calcification gene expression and calcium deposition in aortic wall and aortic valve tissues. At the molecular level, celastrol inhibited the increase of BMP2, phosphorylated Smad1/5 (p-Smad1/5) and non-phosphorylated ß-catenin (n-p-ß-catenin) induced by high-calcium medium both in vitro and in vivo. Also, BMP2 overexpression reversed the anti-calcification effects of celastrol by recovering the decrease of p-Smad1/5 and n-p-ß-catenin. Furthermore, celastrol prevented the up-regulation of BMPRII and down-regulation of Smad6 induced by high calcium, and this protectory effect can be abolished by BMP2 overexpression. In conclusion, our data for the first time demonstrate that celastrol attenuates high calcium-induced arterial and valvular calcification by inhibiting BMP2/Smad1/5 signalling, which may provide a novel therapeutic strategy for arterial and valvular calcification in patients with CKD.
Subject(s)
Aortic Valve/drug effects , Bone Morphogenetic Protein 2/metabolism , Pentacyclic Triterpenes/pharmacology , Signal Transduction/drug effects , Smad1 Protein/metabolism , Smad5 Protein/metabolism , Vascular Calcification/metabolism , Animals , Aorta/metabolism , Aortic Valve/physiopathology , Calcium/metabolism , Gene Expression Regulation , Male , Mice , Mice, Inbred C57BL , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Swine , Vitamin D/metabolism , beta Catenin/metabolismABSTRACT
Melatonin (Mel) promotes sleep through G protein-coupled receptors. However, the downstream molecular target(s) is unknown. We identified the Caenorhabditis elegans BK channel SLO-1 as a molecular target of the Mel receptor PCDR-1-. Knockout of pcdr-1, slo-1, or homt-1 (a gene required for Mel synthesis) causes substantially increased neurotransmitter release and shortened sleep duration, and these effects are nonadditive in double knockouts. Exogenous Mel inhibits neurotransmitter release and promotes sleep in wild-type (WT) but not pcdr-1 and slo-1 mutants. In a heterologous expression system, Mel activates the human BK channel (hSlo1) in a membrane-delimited manner in the presence of the Mel receptor MT1 but not MT2 A peptide acting to release free Gßγ also activates hSlo1 in a MT1-dependent and membrane-delimited manner, whereas a Gßλ inhibitor abolishes the stimulating effect of Mel. Our results suggest that Mel promotes sleep by activating the BK channel through a specific Mel receptor and Gßλ.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Large-Conductance Calcium-Activated Potassium Channels/genetics , Melatonin/pharmacology , Sleep/genetics , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Gene Knockout Techniques , Humans , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/genetics , Melatonin/metabolism , Neurons/drug effects , Neurons/metabolism , Receptor, Melatonin, MT2/genetics , Sleep/drug effects , Synaptic Transmission/geneticsABSTRACT
Aortic valve calcification develops in patients with chronic kidney disease who have calcium and phosphate metabolic disorders and poor prognoses. There is no effective treatment except valve replacement. However, metabolic disorders put patients at high risk for surgery. Increased acetylation of histones 3 and 4 is present in interstitial cells from human calcific aortic valves, but whether it is involved in aortic valve calcification has not been studied. In this study, we found that treating cultured porcine aortic valve interstitial cells with a high-calcium/high-phosphate medium induced calcium deposition, apoptosis, and expression of osteogenic marker genes, producing a phenotype resembling valve calcification in vivo. These phenotypic changes were attenuated by the histone acetyltransferase inhibitor C646. C646 treatment increased the levels of class I histone deacetylase members and decreased the acetylation of histones 3 and 4 induced by the high-calcium/high-phosphate treatment. Conversely, the histone deacetylase inhibitor suberoylanilide hydroxamic acid promoted valve interstitial cell calcification. In a mouse model of aortic valve calcification induced by adenine and vitamin D treatment, the levels of acetylated histones 3 and 4 were increased in the calcified aortic valves. Treatment of the models with C646 attenuated aortic valve calcification by restoring the levels of acetylated histones 3 and 4. These observations suggest that increased acetylation of histones 3 and 4 is part of the pathogenesis of aortic valve calcification associated with calcium and phosphate metabolic disorders. Targeting acetylated histones 3 and 4 may be a potential therapy for inoperable aortic valve calcification in chronic kidney disease patients.