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1.
Nat Immunol ; 23(2): 157-158, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-35105986
2.
Immunity ; 52(5): 767-781.e6, 2020 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-32277911

RESUMEN

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.


Asunto(s)
Fibroblastos/inmunología , Interferones/inmunología , Proteínas de la Membrana/inmunología , Nucleótidos Cíclicos/inmunología , Canales Aniónicos Dependientes del Voltaje/inmunología , Animales , Antivirales/inmunología , Antivirales/metabolismo , Efecto Espectador , Línea Celular , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Herpes Simple/inmunología , Herpes Simple/virología , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/fisiología , Humanos , Interferones/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleótidos Cíclicos/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Nucleotidiltransferasas/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo
3.
Adv Sci (Weinh) ; 11(36): e2401085, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39056405

RESUMEN

Cytotoxic neuronal swelling and glutamate excitotoxicity are two hallmarks of ischemic stroke. However, the underlying molecular mechanisms are not well understood. Here, it is reported that SWELL1, the essential subunit of the volume-regulated anion channel (VRAC), plays a dual role in ischemic injury by promoting neuronal swelling and glutamate excitotoxicity. SWELL1 expression is upregulated in neurons and astrocytes after experimental stroke in mice. The neuronal SWELL1 channel is activated by intracellular hypertonicity, leading to Cl- influx-dependent cytotoxic neuronal swelling and subsequent cell death. Additionally, the SWELL1 channel in astrocytes mediates pathological glutamate release, indicated by increases in neuronal slow inward current frequency and tonic NMDAR current. Pharmacologically, targeting VRAC with a new inhibitor, an FDA-approved drug Dicumarol, attenuated cytotoxic neuronal swelling and cell death, reduced astrocytic glutamate release, and provided significant neuroprotection in mice when administered either before or after ischemia. Therefore, these findings uncover the pleiotropic effects of the SWELL1 channel in neurons and astrocytes in the pathogenesis of ischemic stroke and provide proof of concept for therapeutically targeting it in this disease.


Asunto(s)
Isquemia Encefálica , Modelos Animales de Enfermedad , Ácido Glutámico , Neuronas , Animales , Ratones , Ácido Glutámico/metabolismo , Ácido Glutámico/toxicidad , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Masculino , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Ratones Endogámicos C57BL , Proteínas de la Membrana
4.
J Clin Invest ; 2024 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-39196784

RESUMEN

Chronic low back pain (LBP) can severely affect daily physical activity. Aberrant osteoclast-mediated resorption leads to porous endplates for the sensory innervation to cause LBP. Here, we report that the expression of proton-activated chloride (PAC) channel is induced during osteoclast differentiation in the porous endplates via a RANKL-NFATc1 signaling pathway. Extracellular acidosis evokes robust PAC currents in osteoclasts. An acidic environment of porous endplates and elevated PAC activation-enhanced osteoclast fusion provoke LBP. Further, we find that genetic knockout of PAC gene Pacc1 significantly reduces endplate porosity and spinal pain in a mouse LBP model, but it does not affect bone development or homeostasis of bone mass in adult mice. Moreover, both osteoclast bone resorptive compartment environment and PAC traffic from the plasma membrane to endosomes to form an intracellular organelle Cl channel have low pH around 5.0. The low pH environment activates PAC channel to increase sialyltransferase St3gal1 expression and sialylation of TLR2 in initiation of osteoclast fusion. Aberrant osteoclast-mediated resorption is also found in most skeletal disorders, including osteoarthritis, ankylosing spondylitis, rheumatoid arthritis, heterotopic ossification, enthesopathy. Thus, elevated Pacc1 expression and PAC activity could be a potential therapeutic target for LBP and osteoclast-associated pain.

5.
Sci Adv ; 9(13): eade9931, 2023 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-36989353

RESUMEN

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.


Asunto(s)
Neuralgia , Traumatismos de los Nervios Periféricos , Ratones , Animales , Microglía , Dicumarol/uso terapéutico , Neuralgia/tratamiento farmacológico , Neuralgia/etiología , Médula Espinal , Adenosina Trifosfato/farmacología , Proteínas de la Membrana
6.
bioRxiv ; 2023 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-36712065

RESUMEN

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.

7.
Science ; 364(6438): 395-399, 2019 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-31023925

RESUMEN

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.


Asunto(s)
Canales de Cloruro/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Calcio/metabolismo , Muerte Celular , Canales de Cloruro/clasificación , Canales de Cloruro/genética , Cloruros/metabolismo , Secuencia Conservada , Evolución Molecular , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/patología , Proteínas de la Membrana/clasificación , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Neuronas/metabolismo , Neuronas/patología , Filogenia , Interferencia de ARN , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Pez Cebra , Proteínas de Pez Cebra/clasificación , Proteínas de Pez Cebra/genética
8.
Neuron ; 102(4): 813-827.e6, 2019 05 22.
Artículo en Inglés | MEDLINE | ID: mdl-30982627

RESUMEN

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.


Asunto(s)
Astrocitos/metabolismo , Ácido Glutámico/metabolismo , Discapacidades para el Aprendizaje/genética , Proteínas de la Membrana/genética , Trastornos de la Memoria/genética , Animales , Isquemia Encefálica , Región CA1 Hipocampal , Tamaño de la Célula , Técnicas de Inactivación de Genes , Células HEK293 , Células HeLa , Humanos , Aprendizaje , Proteínas de la Membrana/metabolismo , Memoria , Ratones Noqueados , Células Piramidales/metabolismo , Accidente Cerebrovascular , Transmisión Sináptica
9.
Front Neurosci ; 12: 324, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29867333

RESUMEN

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.

10.
Nat Commun ; 8(1): 359, 2017 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-28842570

RESUMEN

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.


Asunto(s)
Muerte Celular/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Cultivadas , Cicloheximida/farmacología , Quinasas Quinasa Quinasa PAM/genética , Ratones , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Fosforilación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo
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