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Glial cell-expressed mechanosensitive channel TRPV4 mediates infrasound-induced neuronal impairment

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Abstract

Vibroacoustic disease, a progressive and systemic disease, mainly involving the central nervous system, is caused by excessive exposure to low-frequency but high-intensity noise generated by various heavy transportations and machineries. Infrasound is a type of low-frequency noise. Our previous studies demonstrated that infrasound at a certain intensity caused neuronal injury in rats but the underlying mechanism(s) is still largely unknown. Here, we showed that glial cell-expressed TRPV4, a Ca2+-permeable mechanosensitive channel, mediated infrasound-induced neuronal injury. Among different frequencies and intensities, infrasound at 16 Hz and 130 dB impaired rat learning and memory abilities most severely after 7–14 days exposure, a time during which a prominent loss of hippocampal CA1 neurons was evident. Infrasound also induced significant astrocytic and microglial activation in hippocampal regions following 1- to 7-day exposure, prior to neuronal apoptosis. Moreover, pharmacological inhibition of glial activation in vivo protected against neuronal apoptosis. In vitro, activated glial cell-released proinflammatory cytokines IL-1β and TNF-α were found to be key factors for this neuronal apoptosis. Importantly, infrasound induced an increase in the expression level of TRPV4 both in vivo and in vitro. Knockdown of TRPV4 expression by siRNA or pharmacological inhibition of TRPV4 in cultured glial cells decreased the levels of IL-1β and TNF-α, attenuated neuronal apoptosis, and reduced TRPV4-mediated Ca2+ influx and NF-κB nuclear translocation. Finally, using various antagonists we revealed that calmodulin and protein kinase C signaling pathways were involved in TRPV4-triggered NF-κB activation. Thus, our results provide the first evidence that glial cell-expressed TRPV4 is a potential key factor responsible for infrasound-induced neuronal impairment.

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Acknowledgments

The authors thank Ms. Dongyun Feng and Ms. Rui Wu for technical support. This study was supported by grants from the National Natural Science Foundation of China (Grant Nos. 31170801 to M.S., 31070756 to F.D., and 81171236 to X.L.).

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Authors and Affiliations

  1. Department of Neurology, Xijing Hospital, Fourth Military Medical University, Xi’an, 710032, China

    Ming Shi, Fang Du, Yang Liu, Li Li, Jing Cai, Guo-Feng Zhang, Xiao-Fei Xu, Tian Lin, Xue-Dong Liu & Gang Zhao

  2. Lintong Air Force Aeromedical Training Institute, Xi’an, 710060, China

    Hao-Ran Cheng

  3. Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi’an, 710032, China

    Li-Ze Xiong

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  1. Ming Shi
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Correspondence to Ming Shi or Gang Zhao.

Additional information

M. Shi, F. Du, Y. Liu and L. Li contributed equally to this work.

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401_2013_1166_MOESM1_ESM.tif

Supplementary Figure 1. Examination of efficiency and specificity of trpv4 siRNA. Western blot assay revealed that trpv4 siRNA #1 was efficient and specific after U87 cells were transfected with empty pSuper, trpv4 siRNA #1, trpv4 siRNA #2, or trpv4 scramble siRNA. β-Actin was used as an internal control. (TIFF 172 kb)

401_2013_1166_MOESM2_ESM.tif

Supplementary Figure 2. Infrasound does not affect microvascular endothelial cells in hippocampal CA1 region. (a-f) The changes of microvascular endothelial cells as indicated by CD31 immunohistochemistry in controls (a) and rats exposed to infrasound for 1 d (b), 3 d (c), 5 d (d), 7 d (e), or 14 d (f). (g, h) Quantitative analysis of the relative density of CD31 immunoreactivity (g) and the number of branch points of capillary blood vessels (h). Scale bar, 200 μm for a-f. (TIFF 4276 kb)

401_2013_1166_MOESM3_ESM.tif

Supplementary Figure 3. Infrasound activates microglia in vitro. Morphological changes of cultured microglia as revealed by OX42 immunostaining in controls (a, a’, f, f’) and infrasound-exposed cells at 0 h (b, b’, g, g’), 4 h (c, c’, h, h’), 12 h (d, d’, i, i’), or 24 h (e, e’, j, j’) following 1 h (a-e, a’-e’) or 2 h (f-j, f’-j’) exposure to infrasound. Panels (a’-j’) are the magnification views of the boxed areas of respective panels (a-j). Scale bars, 15 μm for a’-j’ insets; 50 μm for a-j. (TIFF 227 kb)

401_2013_1166_MOESM4_ESM.tif

Supplementary Figure 4. The expression levels of IL-1α (a), IL-6 (b), IL-18 (c), IL-10 (d), or INF-γ (e) in the supernatants collected from astrocytic and microglial cultures at 0 h, 4 h, 12 h or 24 h following 2 h exposure to infrasound. (TIFF 931 kb)

401_2013_1166_MOESM5_ESM.tif

Supplementary Figure 5. TRPV4 is expressed in the cultured cells. A low level and a relatively high level of TRPV4 (red) were present in Tuj1+ neurons (green, a-c) and OX42+ microglia (green, d-f) and GFAP+ astrocytes (green, g-i), respectively. Scale bar, 50 μm. (TIFF 3335 kb)

401_2013_1166_MOESM6_ESM.tif

Supplementary Figure 6. TRPV4 inhibition attenuates infrasound-induced neuronal injury. (a) TUNEL staining revealed that RN1734 decreased the number of apoptotic cells in the hippocampi of infrasound-exposed rats. Arrows in (a) indicate apoptotic cells. Scale bar, 200 μm. (b) Quantitative analysis of the number of TUNEL+ cells in hippocampal CA1 region. **p < 0.01, vs. the vehicle. (c, d) Representative fluorescence traces of cultured microglia showed that trpv4 siRNA-transfection (c) and RN1734 (d) markedly attenuated infrasound-induced increased [Ca2+]i. *p < 0.05. (e) Western blotting results showed that compared with the control (astrocytes without infrasound exposure), the expression levels of phosphorylated IκB and nuclear P65 were increased significantly in infrasound-exposed astrocytes treated with DMSO (vehicle), which can be attenuated by TRPV4 antagonist RN1734. (f) Quantitation of the expression levels of IκB, phosphorylated IκB and P65 as shown in (a). β-Actin and TFIIB were used as internal controls for cytosolic and nuclear proteins, respectively. *p < 0.05, vs. the control; #p < 0.05, vs. the vehicle. (TIFF 6564 kb)

401_2013_1166_MOESM7_ESM.tif

Supplementary Figure 7. The involvement of calmodulin, PKC and NF-κB in the production of IL-1β and TNF-α. Compared with vehicle (DMSO), calmodulin inhibitor CGS9343B, PKC inhibitor Ro318220 and NF-κB inhibitor Ro1069920 lowered the levels of IL-1β (a) and TNF-α (b) in astrocytic and microglial supernatants collected at 24 h after 2 h exposure to infrasound. The cells without infrasound exposure were used as the control. *p < 0.05, vs. astrocytic control; #p < 0.05, vs. microglial control. (TIFF 339 kb)

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Shi, M., Du, F., Liu, Y. et al. Glial cell-expressed mechanosensitive channel TRPV4 mediates infrasound-induced neuronal impairment. Acta Neuropathol 126, 725–739 (2013). https://doi.org/10.1007/s00401-013-1166-x

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