Glial Cell Expressed Mechanosensitive Channel TRPV4 Mediates Infrasound Induced Neuronal Impairment

Background: Infrasound, a type of acoustic oscillation with frequency below 20 Hz, belongs to the low-frequency noise and can be generated by various natural sources such as earthquakes and wind, means of transportation such as automobiles, aircraft, and rail traffic, and numerous industrial sources such as heavy machinery and air compressors. It is reported that the infrasonic wave can disturb normal functions of multiple human organs by triggering biological resonance, due to the fact that the organs’ inherent vibration frequencies(e.g. 8–12 Hz for the head) are just within the range of those of infrasound. As a result, undue infrasound exposure(e.g. noise levels above 100 d B for a long-term period) induces intensive resonance of human organs, causing mechanical displacement and distortion of cell structures and consequently leading to vibroacoustic disease. Thus, infrasonic insults have been increasing as a new public health hazard.The brain is vulnerable to infrasonic insults. Our previous animal experiments showed that exposure to infrasound with a certain frequency and sound pressure level(SPL) affected animal learning and memory abilities, impaired adult neurogenesis in hippocampus, induced neuronal apoptosis of hippocampal cells, and triggered microglial activation in rat brain. Our further studies revealed that an increased Ca2+ influx may account for infrasoundinduced neuronal injury. However, at present we still lack the knowledge about molecular mechanism(s) underlying infrasonic insults, that is, how can infrasound be transformed from acoustic stimulus into an intracellular biological signaling, subsequently impairing neuronal cells?Our previous studies showed that TRPV4, a Ca2+-permeable mechanosensitive cation channel, mainly located in glial cells and exibited a higher expression after infrasound exposure both in protein and m RNA levels. So as an extending study, here we investigated the relationship between TRPV4 protein and infrasound induced neuronal damage.Objective:(1) To identify the roles of glail cells in neuronal damage caused by infrasound exposure;(2) To determine the effect of TRPV4 in infrasound induce glial cell activation and neuronal damage.Methods:(1) Adult rats were exposed to 16Hz/130 d B infrasound for 2h/d with consecutive 1, 3, 5, 7, or 14 days, then neurons in CA1 of hippocampus were labeled with Neu N, and apoptotic neurons were labeled with TUNEL staining. After that, rats stereotaxically microinjected with fluorocitrate or intraperitoneally administered with minocycline to inhibit activition of astrocytes and microglia respectively, and exposed to the same infrasound conditions for 14 days, then neurons in CA1 were labeled with anti-Neu N and TUNNEL staining.(2) Culture supernatants from glial cells were collected at 0, 4, 12, and 24 h after infrasound exposure and then assayed simultaneously for detection of the expression of various cytokines(IL-1α, IL-1β, IL-6, IL-10, and TNF-α) with Luminex system. Then cultured hippocampal neurons were treated with glial cell supernatants collected at 0, 4, 12, and 24 h after 2 h of infrasound exposure, and apoptotic neurons were labeled with TUNEL. As a rescue assay, neutralizing anti-IL-1β and anti-TNF-α antibodies were applied, and 24 h later the cells were subjected to TUNEL staining.(3) The cultured neuron, astrocytes and microglia were collected following infrasound exposure,then labeled with anti-TRPV4 to assess the change of TRPV4 specific fluorescence after infrasound exposure. What is more, total protein of different cells was extracted and TRPV4 expression was measured with Western blot to determine the effect of infrasound to TRPV4 expression.(4)We down-regulated TRPV4 expression in glial cells by trpv4 si RNA, or pharmacologically inhibited TRPV4 activity using its selective antagonist RN1734, then IL-1β and TNF-α amounts were examined with Luminex system. What is more, neuronal apoptosis caused by supernatant collected from trpv4 si RNA-transfected or RN1734-treated microglia were measured with TUNEL.(5) To determine whether infrasound is capable of triggering Ca2+ entry via TRPV4 channel, calcium imaging assay was performed on cultured astrocytes.Then Ca2+ entry was observed in trpv4 si RNA-transfected or RN1734-treated cells to assess the function of TRPV4 in infrasound induced Ca2+ entry.(6) To determine whether TRPV4 may be capable of mediating NF-κB activation to induce the expression of proinflammatory cytokines after infrasound exposure, we examined nuclear translocation of NF-κB subunit p65 and phosphorylation state of IκB, an inhibitor binding to NF-κB in an inactive state, in cultured astrocytes treated with TRPV4 agonist 4α-PDD, TRPV4 antagonist RN1734 or trpv4 si RNA transfection.(7) To gain further insight into which molecules or factors bridge a connection between TRPV4-triggered Ca2+ influx and NF-κB activation after infrasound exposure, we performed pharmacological inhibition experiments by applying a series of antagonists,including calmodulin inhibitor CGS9343 B, PKC inhibitor Ro318220, p38 MAPK inhibitor SB20358, JNK selective inhibitor SP600125, and PI3 K inhibitor LY294002. Enhancement of IκB phosphorylation and nuclear translocation p65 translocation were measured with Western blot in glial cells treated with these inhibitors.Results:(1) Relative long-term exposure to infrasound(7–14 days) caused neuronal cell death in the hippocampus, but administration of minocycline and fluorocitrate significantly decreased the numbers of CA1 apoptotic neurons.(2) Astrocytic and microglial supernatants collected at 4, 12 and 24 h following 2-h exposure contained significant higher levels of proinflammatory cytokines IL-1βand TNF-α than their respective controls. And the supernatants collected at 4, 12, and 24 h post exposure significantly enhanced neuronal cell death. What is more, both IL-1β and TNF-α neutralizing antibodies significantly attenuated microglial supernatant-induced neuronal apoptosis.(3) At 24 h after 2-h exposure, TRPV4 expression levels were markedly increased in microglia and astrocytes but not in neurons.(4) Microglia transfected with trpv4 si RNA or treated with RN1734 released markedly lower levels of IL-1β and TNF-α than those transfected with either p Super empty vectors or than those treated with DMSO. Consistently, the supernatant collected from trpv4 si RNA-transfected or RN1734-treated microglia caused fewer apoptotic neurons. Similar results were also observed in astrocytic culture.(5) 2-h exposure to infrasound induced the enhancement of fluo-4 fluorescence, which was attenuated by trpv4 si RNA transfection or by TRPV4 antagonist RN1734.(6) infrasound caused an overt nuclear translocation of p65 and a significant increase in the phosphorylation level of IκB, which, however, can be mitigated by trpv4 si RNA transfection or by TRPV4 antagonist RN1734. Similar results were also observed in cultured microglia.(7) Western blotting results showed that at 24 h following 2-h exposure of astrocytic culture to infrasound, calmodulin inhibitor CGS9343 B and PKC inhibitor Ro318220, but not p38 MAPK inhibitor SB20358, JNK selective inhibitor SP600125 and PI3 K inhibitor LY294002, attenuated infrasoundinduced enhancement of IκB phosphorylation and nuclear translocation p65 translocation. Similar results were also observed in cultured microglia.These findings suggest that after infrasound exposure,calmodulin and PKC signaling pathways are involved in TRPV4-mediated NF-κB activation.Conclusion: In the present study, we found that TRPV4, a Ca2+-permeable mechanosensitive cation channel mainly expressed in glial cells, may serve as a potential key factor responsible for infrasound-induced neuronal injury. TRPV4 inhibition attenuated Ca2+ influx, blocked NF-κB nuclear translocation, decreased the expression levels of glial cellreleased proinflammatory cytokines(e.g. IL-1β and TNF-α) and ultimately protected against infrasound-induced neuronal injury.