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1.
Sci Adv ; 9(13): eade9931, 2023 03 29.
Article in English | MEDLINE | ID: mdl-36989353

ABSTRACT

Following peripheral nerve injury, extracellular adenosine 5'-triphosphate (ATP)-mediated purinergic signaling is crucial for spinal cord microglia activation and neuropathic pain. However, the mechanisms of ATP release remain poorly understood. Here, we show that volume-regulated anion channel (VRAC) is an ATP-releasing channel and is activated by inflammatory mediator sphingosine-1-phosphate (S1P) in microglia. Mice with microglia-specific deletion of Swell1 (also known as Lrrc8a), a VRAC essential subunit, had reduced peripheral nerve injury-induced increase in extracellular ATP in spinal cord. The mutant mice also exhibited decreased spinal microgliosis, dorsal horn neuronal hyperactivity, and both evoked and spontaneous neuropathic pain-like behaviors. We further performed high-throughput screens and identified an FDA-approved drug dicumarol as a novel and potent VRAC inhibitor. Intrathecal administration of dicumarol alleviated nerve injury-induced mechanical allodynia in mice. Our findings suggest that ATP-releasing VRAC in microglia is a key spinal cord determinant of neuropathic pain and a potential therapeutic target for this debilitating disease.


Subject(s)
Neuralgia , Peripheral Nerve Injuries , Mice , Animals , Microglia , Dicumarol/therapeutic use , Neuralgia/drug therapy , Neuralgia/etiology , Spinal Cord , Adenosine Triphosphate/pharmacology , Membrane Proteins
2.
bioRxiv ; 2023 Jan 08.
Article in English | MEDLINE | ID: mdl-36712065

ABSTRACT

Following peripheral nerve injury, extracellular ATP-mediated purinergic signaling is crucial for spinal cord microglia activation and neuropathic pain. However, the mechanisms of ATP release remain poorly understood. Here, we show that volume-regulated anion channel (VRAC) is an ATP-releasing channel and is activated by inflammatory mediator sphingosine-1-phosphate (S1P) in microglia. Mice with microglia-specific deletion of Swell1 (also known as Lrrc8a), a VRAC essential subunit, had reduced peripheral nerve injury-induced increase in extracellular ATP in spinal cord. The mutant mice also exhibited decreased spinal microgliosis, dorsal horn neuronal hyperactivity, and both evoked and spontaneous neuropathic pain-like behaviors. We further performed high-throughput screens and identified an FDA-approved drug dicumarol as a novel and potent VRAC inhibitor. Intrathecal administration of dicumarol alleviated nerve injury-induced mechanical allodynia in mice. Our findings suggest that ATP-releasing VRAC in microglia is a key spinal cord determinant of neuropathic pain and a potential therapeutic target for this debilitating disease.

3.
Nat Immunol ; 23(2): 157-158, 2022 02.
Article in English | MEDLINE | ID: mdl-35105986
4.
Immunity ; 52(5): 767-781.e6, 2020 05 19.
Article in English | MEDLINE | ID: mdl-32277911

ABSTRACT

The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA in infected and malignant cells and catalyzes the formation of 2'3'cGMP-AMP (cGAMP), which in turn triggers interferon (IFN) production via the STING pathway. Here, we examined the contribution of anion channels to cGAMP transfer and anti-viral defense. A candidate screen revealed that inhibition of volume-regulated anion channels (VRACs) increased propagation of the DNA virus HSV-1 but not the RNA virus VSV. Chemical blockade or genetic ablation of LRRC8A/SWELL1, a VRAC subunit, resulted in defective IFN responses to HSV-1. Biochemical and electrophysiological analyses revealed that LRRC8A/LRRC8E-containing VRACs transport cGAMP and cyclic dinucleotides across the plasma membrane. Enhancing VRAC activity by hypotonic cell swelling, cisplatin, GTPγS, or the cytokines TNF or interleukin-1 increased STING-dependent IFN response to extracellular but not intracellular cGAMP. Lrrc8e-/- mice exhibited impaired IFN responses and compromised immunity to HSV-1. Our findings suggest that cell-to-cell transmission of cGAMP via LRRC8/VRAC channels is central to effective anti-viral immunity.


Subject(s)
Fibroblasts/immunology , Interferons/immunology , Membrane Proteins/immunology , Nucleotides, Cyclic/immunology , Voltage-Dependent Anion Channels/immunology , Animals , Antiviral Agents/immunology , Antiviral Agents/metabolism , Bystander Effect , Cell Line , Cells, Cultured , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , HeLa Cells , Herpes Simplex/immunology , Herpes Simplex/virology , Herpesvirus 1, Human/immunology , Herpesvirus 1, Human/physiology , Humans , Interferons/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice, Inbred C57BL , Mice, Knockout , Nucleotides, Cyclic/metabolism , Nucleotidyltransferases/genetics , Nucleotidyltransferases/immunology , Nucleotidyltransferases/metabolism , Voltage-Dependent Anion Channels/metabolism
5.
Science ; 364(6438): 395-399, 2019 04 26.
Article in English | MEDLINE | ID: mdl-31023925

ABSTRACT

Severe local acidosis causes tissue damage and pain, and is one of the hallmarks of many diseases including ischemia, cancer, and inflammation. However, the molecular mechanisms of the cellular response to acid are not fully understood. We performed an unbiased RNA interference screen and identified PAC (TMEM206) as being essential for the widely observed proton-activated Cl- (PAC) currents (I Cl,H). Overexpression of human PAC in PAC knockout cells generated I Cl,H with the same characteristics as the endogenous ones. Zebrafish PAC encodes a PAC channel with distinct properties. Knockout of mouse Pac abolished I Cl,H in neurons and attenuated brain damage after ischemic stroke. The wide expression of PAC suggests a broad role for this conserved Cl- channel family in physiological and pathological processes associated with acidic pH.


Subject(s)
Chloride Channels/metabolism , Membrane Proteins/metabolism , Zebrafish Proteins/metabolism , Animals , Calcium/metabolism , Cell Death , Chloride Channels/classification , Chloride Channels/genetics , Chlorides/metabolism , Conserved Sequence , Evolution, Molecular , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Hypoxia-Ischemia, Brain/metabolism , Hypoxia-Ischemia, Brain/pathology , Membrane Proteins/classification , Membrane Proteins/genetics , Mice , Mice, Knockout , Neurons/metabolism , Neurons/pathology , Phylogeny , RNA Interference , Stroke/metabolism , Stroke/pathology , Zebrafish , Zebrafish Proteins/classification , Zebrafish Proteins/genetics
6.
Neuron ; 102(4): 813-827.e6, 2019 05 22.
Article in English | MEDLINE | ID: mdl-30982627

ABSTRACT

By releasing glutamate, astrocytes actively regulate synaptic transmission and contribute to excitotoxicity in neurological diseases. However, the mechanisms of astrocytic glutamate release have been debated. Here, we report non-vesicular release of glutamate through the glutamate-permeable volume-regulated anion channel (VRAC). Both cell swelling and receptor stimulation activated astrocytic VRAC, which requires its only obligatory subunit, Swell1. Astrocyte-specific Swell1 knockout mice exhibited impaired glutamatergic transmission due to the decreases in presynaptic release probability and ambient glutamate level. Consistently, the mutant mice displayed hippocampal-dependent learning and memory deficits. During pathological cell swelling, deletion of astrocytic Swell1 attenuated glutamate-dependent neuronal excitability and protected mice from brain damage after ischemic stroke. Our identification of a new molecular mechanism for channel-mediated glutamate release establishes a role for astrocyte-neuron interactions in both synaptic transmission and brain ischemia. It provides a rationale for targeting VRAC for the treatment of stroke and other neurological diseases associated with excitotoxicity.


Subject(s)
Astrocytes/metabolism , Glutamic Acid/metabolism , Learning Disabilities/genetics , Membrane Proteins/genetics , Memory Disorders/genetics , Animals , Brain Ischemia , CA1 Region, Hippocampal , Cell Size , Gene Knockout Techniques , HEK293 Cells , HeLa Cells , Humans , Learning , Membrane Proteins/metabolism , Memory , Mice, Knockout , Pyramidal Cells/metabolism , Stroke , Synaptic Transmission
7.
Front Neurosci ; 12: 324, 2018.
Article in English | MEDLINE | ID: mdl-29867333

ABSTRACT

Here we described an experimental protocol for in vivo imaging of macropinocytosis and subsequent intracellular events. By microinjection, we delivered fluorescence dextrans together with or without ATPγS into transparent Drosophila melanogaster embryos. Using a confocal microscope for live imaging, we monitored the generation of dextran-positive macropinosomes and subsequent intracellular events. Our protocol provides a continent and reliable way for investigating macropinocytosis and its underlying mechanisms, especially when combined with genetic strategies.

8.
Nat Commun ; 8(1): 359, 2017 08 25.
Article in English | MEDLINE | ID: mdl-28842570

ABSTRACT

Stimulation of TNFR1 by TNFα can promote three distinct alternative mechanisms of cell death: necroptosis, RIPK1-independent and -dependent apoptosis. How cells decide which way to die is unclear. Here, we report that TNFα-induced phosphorylation of RIPK1 in the intermediate domain by TAK1 plays a key role in regulating this critical decision. Using phospho-Ser321 as a marker, we show that the transient phosphorylation of RIPK1 intermediate domain induced by TNFα leads to RIPK1-independent apoptosis when NF-κB activation is inhibited by cycloheximide. On the other hand, blocking Ser321 phosphorylation promotes RIPK1 activation and its interaction with FADD to mediate RIPK1-dependent apoptosis (RDA). Finally, sustained phosphorylation of RIPK1 intermediate domain at multiple sites by TAK1 promotes its interaction with RIPK3 and necroptosis. Thus, absent, transient and sustained levels of TAK1-mediated RIPK1 phosphorylation may represent distinct states in TNF-RSC to dictate the activation of three alternative cell death mechanisms, RDA, RIPK1-independent apoptosis and necroptosis.TNFα can promote three distinct mechanisms of cell death: necroptosis, RIPK1-independent and dependent apoptosis. Here the authors show that TNFα-induced phosphorylation of RIPK1 in the intermediate domain by TAK1 plays a key role in regulating this decision.


Subject(s)
Cell Death/genetics , MAP Kinase Kinase Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Receptors, Tumor Necrosis Factor, Type I/physiology , Tumor Necrosis Factor-alpha/metabolism , Animals , Cells, Cultured , Cycloheximide/pharmacology , MAP Kinase Kinase Kinases/genetics , Mice , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Phosphorylation , Receptor-Interacting Protein Serine-Threonine Kinases/chemistry , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism
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