RESUMEN
The senses of proprioception, touch, hearing, and blood pressure on mechanosensitive ion channels that transduce mechanical stimuli with high sensitivity and speed. This conversion process is usually called mechanotransduction. From nematode MEC-4/10 to mammalian PIEZO1/2, mechanosensitive ion channels have evolved into several protein families that use variant gating models to convert different forms of mechanical force into electrical signals. In addition to the model of channel gating by stretching from lipid bilayers, another potent model is the opening of channels by force tethering: a membrane-bound channel is elastically tethered directly or indirectly between the cytoskeleton and the extracellular molecules, and the tethering molecules convey force to change the channel structure into an activation form. In general, the mechanical stimulation forces the extracellular structure to move relative to the cytoskeleton, deforming the most compliant component in the system that serves as a gating spring. Here we review recent studies focusing on the ion channel mechanically activated by a tethering force, the mechanotransduction-involved cytoskeletal protein, and the extracellular matrix. The mechanosensitive channel PIEZO2, DEG/ENaC family proteins such as acid-sensing ion channels, and transient receptor potential family members such as NompC are discussed. State-of-the-art techniques, such as polydimethylsiloxane indentation, the pillar array, and micropipette-guided ultrasound stimulation, which are beneficial tools for exploring the tether model, are also discussed.
RESUMEN
The mechanism of pain chronicity is largely unknown in lumbar radiculopathy (LR). The anatomical location of nerve injury is one of the important factors associated with pain chronicity of LR. Accumulating evidence has shown constriction distal to the dorsal root ganglion (DRG) caused more severe radiculopathy than constriction proximal to the DRG; thereby, the mechanism of pain chronicity in LR could be revealed by comparing the differences in pathological changes of DRGs between nerve constriction distal and proximal to the DRG. Here, we used 2 rat models of LR with nerve constriction distal or proximal to the DRG to probe how the different nerve injury sites could differentially affect pain chronicity and the pathological changes of DRG neuron subpopulations. As expected, rats with nerve constriction distal to the DRG showed more persistent pain behaviors than those with nerve constriction proximal to the DRG in 50% paw withdraw threshold, weight-bearing test, and acetone test. One day after the operation, distal and proximal nerve constriction showed differential pathological changes of DRG. The ratios of activating transcription factor3 (ATF3)-positive DRG neurons were significantly higher in rats with nerve constriction distal to DRG than those with nerve constriction proximal to DRG. In subpopulation analysis, the ratios of ATF3-immunoreactivity (IR) in neurofilament heavy chain (NFH)-positive DRG neurons significantly increased in distal nerve constriction compared to proximal nerve constriction; although, both distal and proximal nerve constriction presented increased ratios of ATF3-IR in calcitonin gene-related peptide (CGRP)-positive DRG neurons. Moreover, the nerve constriction proximal to DRG caused more hypoxia than did that distal to DRG. Together, ATF3 expression in NHF-positive DRG neurons at the acute stage is a potential bio-signature of persistent pain in rat models of LR.
Asunto(s)
Factor de Transcripción Activador 3/metabolismo , Ganglios Espinales/patología , Región Lumbosacra/patología , Neuronas Aferentes/patología , Dolor/diagnóstico , Radiculopatía/complicaciones , Células Receptoras Sensoriales/patología , Factor de Transcripción Activador 3/genética , Animales , Ganglios Espinales/lesiones , Ganglios Espinales/metabolismo , Masculino , Neuronas Aferentes/metabolismo , Dolor/etiología , Dolor/metabolismo , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/metabolismoRESUMEN
Acid-sensing ion channels (ASICs) are important acid sensors involved in neural modulation in the central nervous system and pain-associated tissue acidosis in the peripheral system. Among ASIC subtypes, ASIC1b is the most selectively expressed in peripheral sensory neurons. However, the role of ASIC1b is still elusive in terms of its functions and expression profile. In this study, we probed the role of ASIC1b in acid-induced muscle pain in Asic1b-knockout (Asic1b -/-) and Asic1b-Cre transgenic (Asic1b Cre ) mice. We tested the effect of ASIC1b knockout in a mouse model of fibromyalgia induced by dual intramuscular acid injections. In this model, a unilateral acid injection to the gastrocnemius muscle induced transient bilateral hyperalgesia in wild-type (Asic1b + / +) but not Asic1b -/- mice; a second acid injection, spaced 1 or 5 days apart, to the same muscle induced chronic hyperalgesia lasting for 4 weeks in Asic1b + / + mice, but the duration of hyperalgesia was significantly shortened in Asic1b -/- mice. Mambalgin-1, an ASIC1b-containing channel inhibitor that was mixed with acid saline at the first injection, dose-dependently blocked the acid-induced transient and chronic hyperalgesia in Asic1b + / + mice. In contrast, psalmotoxin 1 (PcTx1), an ASIC1a-selective antagonist, had no effect on acid-induced transient or chronic hyperalgesia. We used whole-cell patch clamp recording to study the properties of acid-induced currents in ASIC1b-expressing dorsal root ganglia (DRG) neurons from Asic1b Cre -TdTomato reporter mice. Medium- to large-sized ASIC1b-expressing DRG neurons mainly exhibited an amiloride-sensitive ASIC-like biphasic current (I ASIC) in response to acid stimulation, whereas small- to medium-sized ASIC1b-expressing DRG neurons predominantly exhibited an amiloride-insensitive sustained current. Specifically, mambalgin-1 selectively inhibited the I ASIC in most ASIC1b-expressing DRG neurons. However, PcTx1 or APETx2 (an ASIC3-selective antagonist) had only a mild inhibitory effect on I ASIC in about half of the ASIC1b-expressing DRG neurons. In situ hybridization revealed that ASIC1b-positive DRG neurons co-expressed highly with ASIC1a and ASIC2a mRNA and partially with ASIC3 and ASIC2b. Thus, ASIC1b might form a wide variety of heteromeric channels. ASIC1b-containing heteromeric channels might be promising targets for the therapeutic treatment of acid-induced chronic muscle pain.
RESUMEN
Stereotypic and/or repetitive behavior is one of the major symptoms of autism spectrum disorder (ASD). Increase of self-grooming behavior is a behavioral phenotype commonly observed in the mouse models for ASD. Previously, we have shown that knockout of acid-sensing ion channel 3 (ASIC3) led to the increased self-grooming behavior in resident-intruder test. Given the facts that ASIC3 is mainly expressed in the peripheral dorsal root ganglion (DRG) and conditional knockout of ASIC3 in the proprioceptors induced proprioception deficits. We speculate a hypothesis that stereotypic phenotype related to ASD, pararalled with striatal dysfunction, might be caused by proprioception defect in the peripheral sensory neuron origin. Herein, we investigate in depth whether and how ASIC3 is involved in the regulation of self-grooming behavior. First, we observed that Asic3 null mutant mice exhibited increased self-grooming in social interaction during juvenile stage. Similarly, they displayed increased self-grooming behavior in a novel cage in the absence of cagemate. To further understand the mechanism by which ASIC3 affects grooming behavior, we analyzed neurochemical, neuropathological and electrophysiological features in the dorsal striatum of Asic3 null mutant mice. Knockout of Asic3 increased dopamine (DA) activity and phospho-ERK immunoreactivities in the dorsal striatum. Furthermore, we detected a lower paired-pulse ratio (PPR) and impaired long-term potentiation (LTP) in corticostriatal circuits in Asic3 null mutant mice as compared with wild-type (WT) littermates. Moreover, knockout of Asic3 altered the medial spiny neurons in the striatum with defects in presynaptic function and decrease of dendritic spines. Lastly, genetic ablation of Asic3 specifically in parvalbumin-positive (PV+) cells resulted in the increase of self-grooming behavior in mice. These findings suggest knockout of Asic3 in the PV+ neurons alters grooming behavior by co-opting corticostriatal circuits.
RESUMEN
Advillin is a sensory neuron-specific actin-binding protein expressed at high levels in all types of somatosensory neurons in early development. However, the precise role of advillin in adulthood is largely unknown. Here we reveal advillin expression restricted to isolectin B4-positive (IB4+) neurons in the adult dorsal root ganglia (DRG). Advillin knockout (KO) specifically impaired axonal regeneration in adult IB4+ DRG neurons. During axon regeneration, advillin was expressed at the very tips of filopodia and modulated growth cone formation by interacting with and regulating focal-adhesion-related proteins. The advillin-containing focal-adhesion protein complex was shed from neurite tips during neurite retraction and was detectable in cerebrospinal fluid in experimental autoimmune encephalomyelitis, oxaliplatin-induced peripheral neuropathy, and chronic constriction injury of the sciatic nerve. In addition, advillin KO disturbed experimental autoimmune encephalomyelitis-induced neural plasticity in the spinal-cord dorsal horn and aggravated neuropathic pain. Our study highlights a role for advillin in growth cone formation, axon regeneration, and neuropathic pain associated with IB4+ DRG neurons in adulthood.
Asunto(s)
Ganglios Espinales/fisiología , Conos de Crecimiento/fisiología , Proteínas de Microfilamentos/metabolismo , Regeneración/fisiología , Animales , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Adhesiones Focales/genética , Adhesiones Focales/metabolismo , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Síndromes de Compresión Nerviosa/genética , Síndromes de Compresión Nerviosa/metabolismo , Neuralgia/genética , Neuralgia/metabolismo , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/metabolismo , Seudópodos/genética , Seudópodos/metabolismo , Neuropatía Ciática/genética , Neuropatía Ciática/metabolismoRESUMEN
UNLABELLED: Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmuno-precipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication. IMPORTANCE: Catalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme activity executes at the right time and the right place in DNA replication forks, complex formation with other components in the DNA replication machinery provides an important regulation for UDG function. In this study, we provide the mechanism for EBV UDG BKRF3 nuclear targeting and the interacting domains of BKRF3 with viral DNA replication proteins. Through knockout and complementation approaches, we further demonstrate that in addition to UDG activity, the interaction of BKRF3 with viral proteins in the replication compartment is crucial for efficient viral DNA replication.
Asunto(s)
Replicación del ADN/genética , ADN Viral/genética , Herpesvirus Humano 4/genética , Uracil-ADN Glicosidasa/genética , Uracil-ADN Glicosidasa/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Línea Celular , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Genoma Viral/genética , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Células HEK293 , Células HeLa , Herpesvirus Humano 4/metabolismo , Humanos , Replicación Viral/genéticaRESUMEN
The BGLF4 protein of Epstein-Barr virus (EBV) is a serine/threonine protein kinase that phosphorylates several viral and cellular substrates at cellular cyclin-dependent kinase target sites. BGLF4 is required for efficient viral DNA replication and release of mature virions. It also stimulates the transactivation activity of the immediate-early transactivator Zta (BZLF1) and suppresses the transactivation activities of BMRF1 and EBNA-2. This study aimed to characterize further the regulation of BGLF4 expression at the transcriptional and translational levels. It was shown that BGLF4 was expressed with early kinetics and reached maximal levels after DNA replication. The promoter activity of BGLF4 was upregulated mainly by the immediate-early transactivator Rta, rather than Zta, as revealed by Zta-specific short hairpin RNA in EBV-positive cells and by luciferase reporter assays. By rapid amplification of 5' cDNA ends, two major transcriptional start sites were identified at 201 and 255 nt upstream of the first in-frame ATG of BGLF4 in P3HR1 cells. An additional transcript initiated from -468 was detected in Akata cells. The translation initiation site of BGLF4 was confirmed by mutagenesis, in vitro translation and transient transfection. The translation regulatory effect mediated by the long 5'-untranslated region (5'UTR) of BGLF4 was demonstrated by dual reporter assays in 293T and EBV-positive NA cells. These results suggested that different promoter usage and 5'UTR-mediated translation enhancement may ensure the proper expression of BGLF4 at various stages of virus replication.
Asunto(s)
Herpesvirus Humano 4/enzimología , Herpesvirus Humano 4/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Virales/genética , Regiones no Traducidas 5' , Secuencia de Bases , Línea Celular , Línea Celular Tumoral , Replicación del ADN/genética , ADN Viral/genética , Genes Virales , Herpesvirus Humano 4/fisiología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Transactivadores/genética , Transactivadores/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción Genética , Transfección , Replicación Viral/genéticaRESUMEN
The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV), a viral oncogene, is essential for transformation of resting B cells by the virus. We previously demonstrated that LMP1 could repress DNA repair in p53-wild-type and p53-deficient human epithelial cells. In this study, using a host cell reactivation (HCR) assay, we demonstrated that p53-enhanced DNA repair was repressed by LMP1 in p53-deficient cells. Moreover, we found that LMP1 was able to repress p53-dependent transcriptional activity. Regarding the mechanisms of p53 repression by LMP1, we found that LMP1 did not inhibit p53 function through direct interaction, by promoting protein degradation or reducing its DNA-binding ability. Using chimeric proteins in the reporter assay, we demonstrated that LMP1 inhibited p53 transactivation by influencing the N-terminal transactivation domain of p53. Subsequent experiments using various LMP1 deletion mutants indicated that a C-terminus-activating region of LMP1, CTAR1 or CTAR2, is responsible for the repression of p53-mediated DNA repair and p53-dependent transcription, which is correlated with the region responsible for NF-kappaB activation. Furthermore, blockage of NF-kappaB signalling by IkappaB-DeltaN was shown to abolish the repression of p53 by LMP1, suggesting that LMP1 likely repressed p53 function through the NF-kappaB pathway. Based on these results, we propose that inhibition of p53-dependent transcriptional activity and DNA repair by LMP1 results in the loss of p53 activity for maintaining genomic stability, which may contribute to the oncogenesis of LMP1 in human epithelial cells.