TRPV4 channels stimulate Ca2+-induced Ca2+ release in mouse neurons and trigger endoplasmic reticulum stress after intracerebral hemorrhage

Accepted Manuscript Title: TRPV4 channels stimulate Ca2+ -induced Ca2+ release in mouse neurons and trigger endoplasmic reticulum stress after intracerebral hemorrhage Authors: Jingjing Shen, Liu Tu, Di Chen, Ting Tan, Yan Wang, Shali Wang PII: DOI: Reference:

S0361-9230(18)30536-7 https://doi.org/10.1016/j.brainresbull.2018.11.024 BRB 9565

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Brain Research Bulletin

Received date: Revised date: Accepted date:

14 July 2018 24 October 2018 29 November 2018

Please cite this article as: Shen J, Tu L, Chen D, Tan T, Wang Y, Wang S, TRPV4 channels stimulate Ca2+ -induced Ca2+ release in mouse neurons and trigger endoplasmic reticulum stress after intracerebral hemorrhage, Brain Research Bulletin (2018), https://doi.org/10.1016/j.brainresbull.2018.11.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title page Title: TRPV4 channels stimulate Ca2+-induced Ca2+ release in mouse neurons and trigger endoplasmic reticulum stress after intracerebral hemorrhage Author：

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Jingjing Shen , Liu Tu, Di Chen，Ting Tan, Yan Wang, Shali Wang*

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Jingjing Shen, Cerebrovascular Diseases Laboratory, Institute of Neuroscience, Chongqing

medical university, Chongqing, China, 400016. Email: [email protected], Tel ：

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13618331916，Fax：+86-023-68892728

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university, Chongqing , China , 400016.

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Liu Tu, Cerebrovascular Diseases Laboratory, Institute of Neuroscience, Chongqing medical

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Di Chen ， Cerebrovascular Diseases Laboratory, Institute of Neuroscience, Chongqing

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medical university, Chongqing , China , 400016. Ting Tan, Cerebrovascular Diseases Laboratory, Institute of Neuroscience, Chongqing

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medical university, Chongqing , China , 400016. Yan Wang，Cerebrovascular Diseases Laboratory, Institute of Neuroscience, Chongqing

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medical university, Chongqing , China , 400016. Corresponding author:

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Name：Shali Wang Address ： Cerebrovascular Diseases Laboratory, Institute of Neuroscience, Chongqing medical university, No.1, Yixueyuan Road, Yuzhong District, Chongqing , China Postal code: 400016

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Email: [email protected] Tel: 13436031935 Fax: +86-023-68892728

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Declarations of interest: none.

Highlights

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Graphical abstract

Inhibition of TRPV4 channels promoted the neuronal survival after ICH.

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TRPV4 channels triggered Ca2+-induced Ca2+ release from endoplasmic reticulum.

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Blocking TRPV4 receptors inhibited PERK-CHOP-Bcl-2 signaling pathway.

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ICH recruited inositol triphosphate receptors into the TRPV4 protein complex.

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Abstract Individuals with intracerebral hemorrhage (ICH) suffer varying degrees of neurological dysfunction as a result of neuronal apoptosis, and thus, maintenance of neuronal survival may 2

be crucial to prevent ICH brain injury. Here, we report that the expression of transient receptor potential vanilloid 4 (TRPV4) was upregulated in mouse neurons after ICH. The selective TRPV4 agonist GSK1016790A aggravated neuronal death whereas the TRPV4 antagonist HC-067047 promoted neuronal survival after ICH. We found that GSK1016790A

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triggered Ca2+ signals that were amplified and propagated by Ca2+-induced Ca2+ release (CICR) from the endoplasmic reticulum (ER) in the neurons. ICH recruited inositol

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triphosphate receptors (IP3Rs) into the TRPV4 protein complex, which positively regulated

the activity of TRPV4 channels. Excessive activation of TRPV4 channels destroyed Ca2+

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homeostasis and induced ER unfolded protein response (UPR). Blocking TRPV4 receptors

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decreased UPR，inhibited the PERK-CHOP-Bcl-2 signaling pathway and increased neuron

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survival. Overall, these results suggested that overactivation of TRPV4 channels after ICH

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ledto the destruction of Ca2+ homeostasis, which in turn caused UPR and neural apoptosis.

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Inhibition of TRPV4 channels is a promising therapy to promote neurons recover, and to ameliorate disability after ICH.

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Key Words ： intracerebral hemorrhage ； TRPV4 channel; Ca2+-induced Ca2+ release; endoplasmic reticulum stress; neuronal apoptosis

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Abbreviation

Activating transcription factor-6

Bcl-2

B-cell lymphoma 2

CHOP

CCAAT/enhancer-binding protein homologous protein

CICR

Ca2+ induced Ca2+ release

CNS

Central nervous system

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ATF-6

3

Dimethyl sulfoxide

ER

Endoplasmic reticulum

ERS

Endoplasmic reticulum stress

GRP78

Glucose regulated protein 78kD

ICH

Intracerebral hemorrhage

IER1

Inositol-requiring enzyme-l

JNK

c-Jun N-terminal kinase

PERK

PKR-like ER kinase

SDS

Sodium dodecylsulphate-polyacrylamide

TRP

Transient receptor potential

TRPV4

Transient receptor potential vanilloid 4

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DMSO

Terminal deoxynucleotidyl transferase -mediated dUTP nick end labeling

UPR

Unfolded protein response

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TUNEL

1. Introduction Intracerebral hemorrhage (ICH) results from vascular rupture in the brain and has high morbidity and mortality. Hematoma mechanical compression, inflammation, blood-brain

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barrier disruption, hemoglobin, and disorders of blood circulation and metabolism around the hematoma are thought to induce secondary brain damage and neuronal apoptosis (Chen et al., 2014). Various therapeutic strategies for the treatment of ICH are currently applied in clinical practice, but effective drug therapies aimed at neuroprotection are not yet available.

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Transient receptor potential vanilloid 4 (TRPV4), a member of the transient receptor potential superfamily, is broadly expressed in the central nervous system (Kanju and Liedtke,

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2016). The human TRPV4 gene is located on chromosome 12q23-24.1 (Liedtke et al., 2000). TRPV4 channels can be activated by a variety of stimuli, such as moderate temperature,

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mechanical stimulation, cell swelling, and epoxyeicosatrienoic acids and endocannabinoids

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(Vriens et al., 2004). TRPV4 channels bring out an influx of Ca2+ with theirs activation under

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physiological conditions, which may be involved in the regulation of cell volume (Becker et

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al., 2005), the integrity of the blood-cerebrospinal fluid barrier (Narita et al., 2015), the

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excitability of neurons (Shibasaki et al., 2015), and the tension of cerebral vessels (Earley et al., 2005). TRPV4 channels also participate in a series of pathophysiological processes

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including neuronal apoptosis induced by ischemia and hypoxia (Jie et al., 2015), inflammatory response induced by acute lung injury (Balakrishna et al., 2014), and brain

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edema induced by traumatic brain injury (Lu et al., 2017). Recently, it has been reported that it also exerts an effect in intracerebral hemorrhage (Zhao et al., 2018).

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The endoplasmic reticulum (ER) is a large calcium store within a cell. ER stress could

occur when there is an overload of protein synthesis, protein misfolding or depletion of Ca2+ storage (Xu et al., 2005). To deal with ER stress, the unfolded protein response (UPR) is activated to alleviate ER stress and improve cell survival (Hiramatsu et al., 2015). However,

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prolonged activation of UPR causes destruction of calcium hemostasis and brings about programmed cell death (Richter et al., 2016; Chao et al., 2012). It has been found that TRPV4-mediated Ca2+ signals are enlarged and proliferated by Ca2+-induced Ca2+ release (CICR), which forms intracellular Ca2+ oscillation and plays an important role in the

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preservation of intracellular Ca2+ homeostasis (Dunn et al., 2013; Ryskamp et al., 2011; Jo et al., 2015). TRPV4-mediated Ca2+ influx contributes to the endfoot Ca2+ response to neuronal

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activation (Dunn et al., 2013), to apoptosis of mouse retinal ganglion cells (Ryskamp et al., 2011), and to cell volume regulation (Jo et al., 2015). We thus considered whether TRPV4

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could influence Ca2+ homeostasis of the endoplasmic reticulumand then induce neuron

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apoptosis after ICH.

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In the present study, we investigated whether TRPV4 channels exerted a function, and the

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mechanism behind this function, in autologous blood infusion mouse models of ICH. Our

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findings showed that the expression level of TRPV4 is upregulated in mouse neurons after ICH. Inhibition of TRPV4 channels improves neuronal survival. The activation of TRPV4

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leads to the disturbance of Ca2+ homeostasis, which in turn gives rise to UPR and neural apoptosis after ICH. Our data suggest that TRPV4 is involved in the development of brain

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injury after ICH, implicating TRPV4 as a new therapeutic target for the treatment of hemorrhagic brain injury.

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2. Materials and Methods 2.1. Animal experiments All of the animal experiments were approved by the ethical guidelines of the Chongqing Medical University Animal Research Committee, and complied with the NIH Guide for the

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Care and Use of Laboratory Animals. In all of the experiments, animals were randomly assigned to groups. All of the behavioral and histological analyses were performed by an experimenter blinded to the identity of the groups. ICH models were induced by autologous blood infusion in male C57BL/6J mice (8~13 weeks of age, 18-25g–25 g). We established

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mouse ICH models using a double injection method (Deinsberger et al., 1996). After intraperitoneal injection of 38 mg/kg chloral hydrate, mice were positioned prone and placed

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in a stereotaxic frame. A burr hole was drilled in the right parietal bone of the skull (2.0 mm

lateral to the midline, 0.4 mm anterior to the bregma). The mixed (arterial and venous) blood

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was collected by cutting the tip of the tail and then quickly transferred it into a 50-μl glass

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syringe. The needle was inserted through the burr hole and advanced 3.5 mm below the skull.

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After 5 μl autologous blood was injected, the injection was terminated five minutes. The

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needle was then lowered to 4.0 mm in depth, and 25 μl autologous blood was injected into the

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right striatum (Krafft et al., 2014). Sham animals were subjected to the same manipulations as the ICH mice, but no blood was injected.

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The drugs (GSK1016790A, HC-067047, 2-APB, thapsigargin, SKF96365) were dissolved in DMSO (Sigma, D8418), and the final diluted concentration of DMSO was less than 0.1%

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when used. GSK1016790A and HC-067047 were injected immediately into the lateral ventricle (stereotaxic coordinates: 1.0 mm lateral to the midline, 0.3 mm posterior to the

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bregma, and 2.5 mm below the skull) after operation. 2-APB was applied through intracerebroventricular injection 1 hour before surgery. Except for special hints, both GSK1016790A and HC-067047 were injected immediately into lateral ventricle after ICH. 2.2. Behavioral tests

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Neurological and sensorimotor functions were evaluated via the neurological deficit scoring system and rope grip test at 24 hours after surgery. In the neurological deficit scoring system, mice were scored using the composite Garcia neuroscore (Krafft et al., 2014). The tests included spontaneous activity, axial sensation,

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vibrissae proprioception, symmetry of limb movement, lateral turning, forelimb outstretching, and climbing. The final neuroscore is the sum of the seven individual test scores. The

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minimum score is 3 and the maximum is 21.

In the rope grip test (Munakata et al., 2013), mice were placed midway on a string between 2

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supports and rated as follows: 0, falls off; 1, hangs onto string by one or both forepaws; 2,

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same as for 1, but attempts to climb onto string; 3, hangs onto string by one or both forepaws

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plus one or both hindpaws; 4, hangs onto string by forepaws and hindpaws plus tail wrapped

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around string; and 5 escapes to the supports. The final score was the average of 5 trials.

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2.3. Evaluation of brain water content

Brain water content was measured via the wet weight/dry weight method. Mice were

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anesthetized with 3.8% chloral hydrate and killed at 24 hours after surgery. Ipsilateral brain tissues were separated and weighed in order to obtain the wet weight. The brain tissues were

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then dried at 100℃ for 24 hours and weighed to obtain the dry weight. Brain water content (%) was calculated as (wet weight – dry weight)/wet weight × 100% (Krafft et al., 2014).

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2.4. Immunohistochemistry and TUNEL staining After 3.8% chloral hydrate intraperitoneal anesthesia, the animals were perfused with 0.9% saline and 4% paraformaldehyde in the left ventricle; the brains were rapidly removed and embedded in the paraffin. Coronal paraffin sections were made, and immunohistochemistry

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and TUNEL staining were performed. Immunohistochemistry was performed on sections using rabbit polyclonal anti-TRPV4 (1:200; Abcam, ab39260) and mouse monoclonal anti-NeuN (1:200; Abcam, ab104224). Alexa Fluor 555 goat anti-rabbit IgG (1:1000; Beyotime, P0179) and Alexa Fluor 488 goat anti-mouse IgG (1:1000; Beyotime, P0188) were

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used as secondary antibodies. Fluorescent signals were detected with a fluorescence microscope (Olympus, DP70). For NeuN and TUNEL co-staining, the slices were incubated

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with NeuN antibody and an in situ cell death detection kit (Roche, 11684817910). Fluorescence analysis and cell count were performed using Image J software.

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2.5. Cell cultures

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Brain tissues were isolated from the E17 mice. Cells were dissociated using 0.125% trypsin

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(Gibco, 13824) and planted with the densities of 100-150 cells/mm on glass-bottom dishes for

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Ca2+ imaging. Cells were placed in fresh serum-free Neurobasal medium (Gibco, 21103) plus

10 days.

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2.6. Ca2+ imaging

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2% B27 (Gibco, 17504), fed every 4 days with fresh media, and used for Ca2+ imaging after

Neurons were washed twice with HBSS (Gibco, 14175) and then loaded with fluo-3 AM by

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incubation in HBSS for 30 min at 37℃. After that, the cells were washed twice and perfused with extracellular fluid (140 mM NaCl, 5 mM KCl, 1 mM MgCl2·6H2O, 2 mM CaCl2 , 10

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mM D-glucose, 10 mM HEPES, pH 7.4). The [Ca2+]i levels were examined under a Nikon-A1R microscope. Analysis of single-cell integrated signal density was performed on computers running NIH Image software (NIS 4.3). 2.7. Co-IP and Western blots

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The perihematoma brain tissues were homogenized and split in ice-cold lysis buffer containing (mM): 50 Tris-HCl (pH7.4), 150 NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 EDTA, 1 sodium orthovanadate, and 1 phenylmethanesulfonyl fluoride. After centrifuging at 12,000 × g at 4°C, protein concentration in the extracts was

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determined by BCA protein assay (Beyotime, P0010). For the coimmunoprecipitation，the perihematoma brain tissues were split in a mild lysis

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buffer containing (mM): 20 Tris-HCl (pH7.5), 150 NaCl, 1% Triton X-100, 1% sodium pyrophosphate, 2 EDTA, 1 Na3VO4, and 1 phenylmethanesulfonyl fluoride (Beyotime,

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P0013). The extracts (~1 mg protein) were incubated with nonspecitic IgG (1 μg) or

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polyclonal rabbit anti-TRPV4 (1 μg, Alomone Labs ， acc-034) or anti-IP3R (1 μg,

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Abcam,ab190239) overnight at 4°C, followed by the addition of 40 μl protein A/G agarose for

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3 hours at 4°C. The precipitates were washed 4 times with lysis buffer and denatured with

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SDS sample buffer.

The samples were separated by 8% SDS-PAGE and transferred to polyvinylidene difluoride

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membranes. The transfer membranes were incubated with 5% nonfat dried milk dissolved in TBST, and then incubated overnight at 4°C with the primary antibodies. Membranes were

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washed three times with TBST buffer and incubated with the appropriate secondary antibodies for 2 hours. The protein bands were detected by enhanced chemiluminescence

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(Millipore, WBKLS0500). The gray value of the bands was determined using Image J software (IBM). 2.8. Antibodies and reagents The following antibodies were used: TRPV4 (Abcam, ab39260), GRP78 (Cell Signaling

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Technology, 3183), PERK (Cell Signaling Technology, 3192), P-PERK (Cell Signaling Technology, 3179), CHOP (Abcam, ab11419), CHOP (Cell Signaling Technology, 2895T), Bcl-2 (Cell Signaling Technology, 3498T), TRPV4 (Alomone Labs，acc-034), IP3R (Abcam, ab190239). The following chemicals were used: Thapsigargin (Sigma, T9033), SKF96365

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(Sigma, S7809), GSK1016790A (Sigma, G0798), HC-067047 (Sigma, SML0143), and 2-APB (Tocris, 1224).

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2.9. Statistical analysis

All of the data were presented as mean ± SD. Data analysis was performed using SPSS 19

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(IBM). Graph drawing was performed using Prism 6. Statistical comparisons were made

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using one-way ANOVA following LSD post hoc test, paired- samples t test or

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independent-samples t Test. Differences in the mean values were considered to be significant

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at P < 0.05.

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3. Results

3.1. The TRPV4 protein in the ipsilateral brain tissues was specifically upregulated after

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intracerebral hemorrhage

We examined the protein levels of TRPV4 in a mouse model of intracerebral hemorrhage

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induced by autologous blood infusion. The protein levels of TRPV4 in the ipsilateral brain tissues after ICH were gradually increased (Fig. 1A) compared with those in the sham group.

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The expression of TRPV4 protein was gradually increased from 6 hours after ICH, reached the highest level at 12 hours and remained stable for up to 24 hours (Fig. 1B). There was no significant difference in the expression of TRPV4 protein between 12 hours and 24 hours. TRPV4 protein expression gradually decreased to a nearly normal level at 48 hours to 72

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hours after ICH. In addition, TRPV4 immunoreactivity in the ipsilateral brain tissues at 24 hours after ICH was markedly increased compared with thosethat in the sham group (Fig. 1C). At the same time, we observed a decrease of neurons in the ICH group compared with the sham group (Fig. 1D). This suggested that neuronal injury could be related to the upregulation

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of TRPV4 expression. 3.2. Effect of TRPV4 on neurological deficits in the autologous blood infusion ICH

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model

To evaluate whether TRPV4 channels have a role in brain injury after ICH, GSK1016790A (a

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selective TRPV4 agonist, 0.1 μM, 0.3 μM,1 μM, 3 μM/2μL) or HC-067047 (a selective

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TRPV4 antagonist, 0.1 μM, 0.3 μM, 1 μM, 3 μM/2μL) was administered to the lateral

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ventricle after operation. The evaluation of the behavioral performance was carried on at 24

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hours after ICH. The composite Garcia neuroscore, indicated that GSK1016790A aggravated

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the neurological deficiency, but HC-067047 improved it in a concentration-dependent manner (Fig. 2A). Similarly, the rope grip score was lower in the GSK1016790A-treated group than

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in the vehicle group, but was higher in the HC-067047-treated group than in the vehicle group (Fig. 2B). In addition, we examined the effect of delayed HC-067047 treatment after ICH (Fig.

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2C). We administered HC-067047 intracerebroventricularly at 2 h, 6 h and 12 h. Neurological function tests were performed at 24 h after ICH. The results showed that treatment with

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HC-067047 at 2 h and 6 h after ICH was effective. However, there was no effect when HC-067047 was administered at 12 h after ICH. 3.3. TRPV4 inhibition improved the brain edema after intracerebral hemorrhage After the ICH model was established, 1 μM GSK1016790A or 3 μM HC-067047 was

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immediately administered through the lateral ventricle. Brain morphological changes in the perihematoma brain tissues at 24 h after ICH were detected using hematoxylin and eosin (HE). In the sham-operated group, the structure of mouse brain tissue was clear and complete, the nucleus was clear and the nucleolus was centered. The gap around the cells was small. There

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was an obvious brain edema at 24 h after ICH, which was identified by the dissolved nuclei and vacuolar degeneration of cells, and by the widened intercellular space along with

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interstitial edema. The brain edema was clearly aggravated in the GSK1016790A-treated group, which was characterized by the enlarged area of edema and increased number of

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the number of vacuolar degeneration cells (Fig. 3A).

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vacuolar degeneration cells. On the contrary, HC-067047 relieved brain edema and recovered

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Brain edema was measured via the wet weight/dry weight method 24 h after ICH. The water

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content of brain tissue increased after ICH vs the sham group. The water content of brain

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tissue increased significantly in the GSK1016790A-treated group, and decreased in the HC-067047-treated group (Fig. 3B).

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3.4. Blockage of TRPV4 channels improved neuronal apoptosis at 24 h after intracerebral hemorrhage

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As a neural characteristic structure, the number of Nissl bodies reflects the state of neurons. In the sham-operated group, the Nissl bodies were abundant, showing that the function of

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neuronal protein synthesis was strong. A large number of neuronal cells showed a decrease in Nissl bodies, which was identified by the cells dyed light in the vehicle group, indicating that neuronal cells were damaged. In the GSK1016790A-treated group (1 μM), the number of Nissl bodies was reduced or even disappeared, and some neurons formed vacuoles, indicating

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that neuronal apoptosis increased. On the contrary, by blocking TRPV4 channels, HC-067047 (3 μM) could recover the number of Nissl bodies, indicating that neuronal apoptosis was improved (Fig. 4A). The quantification of Nissl staining also showed that HC-067047 (3 μM) treatment recovered the number of Nissl bodies (Fig. 4B).

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We examined the apoptosis of neurons at 24 h after ICH by double staining with NeuN and TUNEL labeling (Fig. 4C). The proportion of TUNEL-positive cells increased to 61.69% ±

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2.79% after ICH. The proportion of TUNEL-positive cells increased further to 86.08% ± 4.15% in the GSK1016790A-treated group. When TRPV4 channels were blocked by HC-067047,

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the TUNEL-positive cell rate dropped to 27.69% ± 2.68% (Fig. 4D).

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3.5. Activation of TRPV4 channels triggered Ca2+ signals that were amplified and

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propagated by Ca2+-induced Ca2+ release from the endoplasmic reticulum in neurons

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GSK1016790A induced large and often oscillatory Ca2+ signals in mouse primary cultured

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cortical neurons. Individual cells, that responded to GSK1016790A (1 μM), expressed two kinds of Ca2+ oscillatory forms: one was an elevated Ca2+ concentration throughout the

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stimulus period and the other was an oscillatory increase (Fig. 5A, B). The results showed that the intracellular calcium fluorescence intensity increased significantly after TRPV4 channels

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were activated (Fig. 5C).

To confirm the Ca2+ signals amplified by the release of Ca2+ from internal stores, we

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depleted endoplasmic reticulum Ca2+ stores in the control group by opening IP3 gated channels with thapsigargin (10 μM); in addition, we blocked potentiation channels with SKF 96365 (20 μM) in order to prevent Ca2+ replenishment. Under these conditions, GSK1016790A (1 μM) failed to further increase calcium concentration, but produced a

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slightly elevated background level of Ca2+-induced fluorescence (Fig. 5D, E, F). These results suggest that TRPV4 channels trigger Ca2+ release from the endoplasmic reticulum. We next detected whether Ca2+ release from the endoplasmic reticulum was triggered by Ca2+ entering the neurons through open TRPV4 channels. When loaded with fluo-3 AM in

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extracellular solution without Ca2+, the neurons displayed only low baseline fluorescence. After Ca2+ was added again, the intracellular Ca2+ concentration increased immediately (Fig.

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5G, H, I).

3.6. Intracerebral hemorrhage recruits IP3Rs into TRPV4 protein complex

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To explore whether IP3Rs can combine with TRPV4 channels, we used an anti-IP3R antibody

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to precipitate the IP3R complex in the extracts from the ipsilateral brain tissues that had been

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subjected to ICH or sham operation. The precipitates from both groups were separated on the

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SDS-PAGE. The antibody against TRPV4 (Abcam, ab39260) probed the precipitates and

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made ensured the existence of endogenous TRPV4 in the IP3R complex (Fig. 6A). We also carried out the reciprocal coimmunoprecipitation, in which the antibody against IP3R was

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used to precipitate TRPV4 (Fig. 6B). The results showed that IP3Rs was physically associated with TRPV4 receptors, and ICH promoted the IP3Rs binding with TRPV4 receptors.

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3.7. Neuronal apoptosis induced by ICH was associated with activation of the PERK-CHOP-Bcl-2 pathway

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We examined whether TRPV4 channels induced ER stress after ICH. Indeed, we found that ICH induced a UPR, which was clearly evidenced by increased expression of GRP78, p-PERK, and CHOP, along with decreased expression of Bcl-2. Activating TRPV4 channels by GSK1016790A increased phospho-PERK and its downstream effector CHOP. In contrast,

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blocking of TRPV4 channels by HC-067047 led to a decreased UPR and inhibited the expression of CHOP (Fig. 7A, B). We also detected the expression of the IRE1-JNK pathway. The results indicated a change in p-IRE1, but no change in the expression of p-JNK (Fig. 7C, D).

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3.8. IP3R inhibition relieved endoplasmic reticulum stress and improved hemorrhagic brain injury

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To confirm that IP3Rs influenced the function of TRPV4 after ICH, we administrated 10 μM

2-APB (an antagonist of IP3R) through the lateral ventricle at 1 h before ICH. GSK1016790A

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(1 μM) or HC-067047 (3 μM) was injected into the lateral ventricle after operation, and then

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the ipsilateral brain tissues were extracted for immunoblotting at 24 h after ICH. The results

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showed that application of 2-APB alone could inhibit the activation of CHOP, but combined

4. Discussion

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reticulum stress (Fig. 8).

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application of HC-067047 and 2-APB exerted a better effect on relieving endoplasmic

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TRPV4 channels are widely distributed in the central nervous system (Kanju and Liedtke, 2016). It has been reported that activated TRPV4 channels participate in multiple

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pathophysiological processes, such as Aβ40-induced hippocampal cell death (Bai and Lipsi, 2014), cerebral hypoxia/ischemia (Butenko et al., 2012), and the apoptosis of mouse retinal

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ganglion cells (Ryskamp et al., 2011). Our investigations demonstrated that the expression of TRPV4 protein was upregulated after ICH by immunoblotting and immunofluorescence. Consistent with our study, other research has suggested that TRPV4 proteins are increased after traumatic brain injury (Lu et al., 2017),

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in a rat middle cerebral artery occlusion model (Jie et al., 2015), and in cortical lesions of patients with focal cortical dysplasia (Chen et al., 2016). Furthermore, TRPV4 protein was found to be increased in aged rats compared with young rats, which may be associated with neurodegenerative diseases (Lee and Choe, 2014). This indicates that the TRPV4 channel, as

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an osmotic sensor, mechanical stimuli sensor, and chemical sensor (Liedtke et al., 2000; Liedtke and Friedman, 2003; Liedtke et al., 2003; Liedtke and Kim, 2005; Moore et al., 2018;

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Ciura et al., 2018; Mizuno et al., 2003; Suzuki et al., 2013; Ryskamp et al., 2014), can sense

changes in the internal environment and adjust its expression. Our results also showed that

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inhibition of TRPV4 channels contributed to the alleviation of ICH-induced brain edema, the

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suppression of neuronal apoptosis, and the improvement of mouse neurological function.

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Hence, TRPV4 inhibition may constitute a novel therapeutic target for ICH. We also noticed

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that the blockage of TRPV4 was invalid when ICH happened more than 12 h. Since the

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period from 12h to 24h after ICH is a period in which pathophysiological changes become aggravated, the results suggest that inhibition of TRPV4 as soon as possible after ICH can

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exert a better effect.

It has been demonstrated that cell swelling, eicosanoid metabolites, and mechanical

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stimulation can activate TRPV4 channels (Virens et al., 2004; Ryskampet al., 2014). As is known, intracerebral hemorrhage can cause brain edema and cell swelling. Thus, cell swelling

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may be an activator of TRPV4. The other endogenous activator of TRPV4 may be anandamide. The endocannabinoid anandamide activates TRPV4 via the formation of 5,6-EET (Watanabe, et al., 2003). It has been shown that anandamide content is increased after stroke (Muthian, et al., 2004). Therefore, we believe the above two factors may activate

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the TRPV4 channels after ICH. Some researchers found that activated TRPV4 channels mainly mediated the influx of Ca2+ ions, and formed calcium oscillation in the cells (Dunn, et al., 2013; Liedtke et al., 2000; Suzuki et al., 2013; Ryskamp, et al., 2011). Our research also confirmed that GSK1016790A，

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an agonist of TRPV4 channels, could evoke TRPV4 channel-dependent Ca2+oscillations in neurons, and these oscillations appeared in the form of propagating Ca2+ waves with a high

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amplitude. The Ca2+ oscillations in the mouse neurons appeared in two variants: a sustained

high level of Ca2+ with low amplitude oscillation, and a “finger”-shaped Ca2+ oscillation (Fig.

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5A), which was supported by other groups’ research (Ryskamp et al., 2014; Liedtke et al.,

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2000). We also found that the Ca2+ oscillation disappeared in the Ca2+-free extracellular fluid.

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When Ca2+ ions were added again, [Ca2+]i was elevated immediately. If the ER Ca2+ store was

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depleted with thapsigargin, and Ca2+ replenishment was prevented with SKF 96365 in

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advance, the GSK1016790A-induced Ca2+ oscillations disappeared. These results not only implied that TRPV4 mediates the extracellular Ca2+ influx that promotes neuronal Ca2+ oscillations, but also hinted that Ca2+ oscillations were associated with Ca2+ release from the

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endoplasmic reticulum (ER). The process is called Ca2+ -induced Ca2+ release (CICR).

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The endoplasmic reticulum is a reservoir for intracellular Ca2+. Usually, to maintain Ca2+ homeostasis, the flow of Ca2+ into and out of the endoplasmic reticulum has to be precisely

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regulated to be in balance (Krebs et al., 2015). Ca2+ ions are released through inositol-1,4,5-trisphosphate receptors (IP3Rs) located on the membrane of the ER and recycled by Ca2+ pumps of the ER, which use ATP as an energy source to pump Ca2+ against a steep ion gradient across the membrane. We believe that the factors that follow may result in

18

the disruption of ER Ca2+ homeostasis after ICH. 1) Upregulated expression of TRPV4 and many adverse factors (such as cell swelling and eicosanoid metabolites) can cause the overactivation of TRPV4 channels, promoting the chronic depletion of the ER Ca2+ store. 2) Loss of nutrients/energy leads to unusual Ca2+ recycling by Ca2+ pumps after ICH. 3) We

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speculate that there is a positive regulation mechanism that results in sustained TRPV4 activation after ICH. It has been found that TRPV4 has a site binding to the IP3Rs within the

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second ANK domain at the C-tail (aa 812-831), and IP3Rs are positive modulators of TRPV4 channel activity (Fernandes et al., 2008; Garcia-Elias et al., 2008). In the present study, we that

ICH

recruits

IP3Rs

into

the

TRPV4

protein

complex

by

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demonstrated

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coimmunoprecipitation, which may cause the sustained activation of TRPV4 channels, and

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result in an enhanced CICR response, finally give rising to chronically prolonged depletion of

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ER Ca2+ ions.

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Depletion of Ca2+ store leads to a rapid accumulation of misfolded proteins and promotes dissociation of GRP78 from PERK, IRE1 and ATF6, thereby activating the UPR pathway

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(Krebs et al., 2015). The UPR prevents further accumulation of newly synthesized proteins in the ER for the sake of reducing further burden to the ER. However, prolonged UPR activation

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occurs when normal ER function fails to be restored, which then causes ER stress and cell apoptosis (Ryu et al., 2002). PERK is a key protein responsible for ER stress. The

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phosphorylation of PERK induces the apoptosis-relevant transcriptional factor CHOP and then activates the mitochondrial apoptosis pathway (McCullough et al., 2001; Marciniak et al., 2004). IRE1 is another transmembrane protein that is located in the membrane of the ER. Prolonged ER stress also stimulates the phosphorylation of IRE1, which causes downstream

19

activation of stress kinase Jun-N-terminal kinase (JNK), which promote apoptosis (Urano et al., 2000; Nishitoh et al., 2002). In the present study, we observed that ICH increased PERK activation by increasing cellular levels of phosphorylated PERK, which in turn upregulated CHOP expression and inhibited the expression of Bcl-2. These results are consistent with

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previous studies indicating that UPR triggered cell apoptosis through the PERK-CHOP-Bcl-2 pathway (McCullough et al., 2001; Marciniak et al., 2004). We found that inhibition of IP3Rs

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by 2-APB could relieve ER stress. This implies that IP3Rs regulate the activation of TRPV4 channels after ICH. However, it was interesting that phosphorylation of IRE1 increased but

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phosphorylation of JNK did not change after ICH. This may be caused by activation of IRE1

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involving activation of p38 MAPK, which in turn activates CHOP (Wang and Ron, 1996).

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Other evidence also supports our results. For example, TRPC3, a member of the TRP

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superfamily, mediates the Ca2+ influx that is necessary for ER stress-induced apoptosis

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(Ampem et al., 2016). Furthermore, activation of transient receptor potential vanilloid 1 results in ER stress and cell death in human lung cells (Tomas et al., 2007).

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In conclusion, our results suggest that ICH causes upregulation of TRPV4 channels and recruitment of IP3Rs, which results in an enhanced CICR response, and finally leads to the

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depletion of ER Ca2+ ions. Disruption of ER Ca2+ homeostasis activates the PERK-CHOP-Bcl-2 pathway and induces neuronal apoptosis. Therefore, the present study

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highlights the mechanisms of TRPV4 in ICH-induced neuronal apoptosis and supplies a novel target of neuroprotection with ICH. In future work, we will use TRPV4 knockout mice or TRPV4 gene silencing mice to verify our experimental results. Furthermore, GSK2193874, an orally active and selective blocker of TRPV4, has been shown to resolve pulmonary edema

20

induced by heart failure (Thorneloe et al., 2012). We will test the effectiveness of GSK2193874 in a mouse model of intracerebral hemorrhage so as to promote the clinical application of TRPV4 channel blockers.

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Conflicts of Interest None

This work was supported

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Acknowledgements

in part by the National Nature Science Foundation of China

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(81401234, 2015), the Chongqing Municipal Education Committee, 2015, and the Ba-yu

A

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Overseas Planning Project (2015-47, 2015).

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Figures and Legends

Fig. 1. The expression of TRPV4 in the brain tissues was upregulated after ICH. (A)

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Immunoblots of the extracts from the perihematoma brain tissues using the indicated

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antibodies. β-actin served as a loading control. (B) Quantification of the TRPV4 protein levels

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(n = 4). * P < 0.05, ** P < 0.01 vs. sham. (C) Representative images of ipsilateral brain

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tissues at 24 h after ICH double-staining with the indicated antibodies. Magnification 400×,

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scale bar: 50 μm. (D) Quantification of the optical density for TRPV4 and the number of NeuN-positive cells (n = 5 per group，N = 10 mouse). **P < 0.01 vs. sham. Data are

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presented as mean ± SD.

Fig. 2. Dose-dependent effects of GSK1016790A or HC-067047 on neurological deficits and

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brain injury at 24 h after ICH. (A) Neurological deficits. (B) Rope grip test. (n = 6~8 per group); *P < 0.05, **P < 0.01 vs. vehicle. Data are presented as mean ± SD. (C) The effectiveness of HC-067047 in delayed treatment after ICH. n =6 per group; **P < 0.01 vs. vehicle.

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Fig. 3. Inhibition of TRPV4 channels improved brain edema. (A) Representative images of brain morphological changes in the perihematoma brain tissues were obtained at 24 h after ICH. Magnification 400×, scale bar: 50 μm. Green arrows indicate the cells with typical

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characteristics. Black arrows indicate the changes in the gap around the cells. (B) Quantification of brain water between vehicle vs. sham, GSK1016790A-treated or

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HC-067047-treated group (n = 8 per group, N = 32 mouse). *P < 0.05, **P < 0.01 vs. vehicle.

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Data are presented as mean ± SD.

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Fig. 4. Blockage of TRPV4 channels improved the neuronal apoptosis after ICH. (A)

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Representative images of the peri-hematoma brain section obtained at 24 hours after ICH

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stained with Nissl staining, scale bar: 50 μm. Red arrows indicate the cells with typical

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characteristics; yellow arrows indicate the cells with decreased Nissl bodies; green arrows indicate the cells with vacuolar changes. (B) Quantification of the Nissl staining mean gray

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value at vehicle vs. sham, GSK1016790A (1 μM)/HC-067047 (3 μM)–treatedgroup (n = 6, N = 24 mouse). (C) Representative images of the perihematoma brain tissues at 24 h after ICH

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double staining with NeuN and TUNEL labeling. Scale bar: 50 μm. (D) Quantification of the

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numbers of TUNEL-positive neurons in the perihematoma brain tissues of the vehicle vs. sham, GSK1016790A (1 μM)/HC-067047 (3 μM)-treated group (n = 6~8 per group, N = 26 mouse). **P < 0.01 vs. vehicle. Data are presented as mean ± SD.

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Fig. 5. GSK1016790A induced CICR activity in the neurons. (A) Representative images of the neurons loaded with the Ca2+ indicator fluo-3 AM. Scale bar: 10 μm. (B) The representative trace for intracellular Ca2+ fluorescence intensity when individual cells responding to GSK1016790A. (C) Quantification of the mean intensity in the intracellular

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fluorescence. n = 36 cells, **P < 0.01 vs. control. Data are presented as mean ± SD. (D) Representative images of the neurons after depleting internal Ca2+ stores with 10 μM

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thapsigargin (Tg) and blocking potentiation channels with SKF96365 (20 μM). The intracellular fluo-3 AM fluorescence dose not increase when exposed to GSK1016790A.

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Scale bar: 100 μm. (E) The representative trace for intracellular Ca2+ fluorescence intensity

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after adding Tg and SKF96365. (F) Quantification of the mean intensity in the intracellular

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fluorescence. N = 36 cells. Data are presented as mean ± SD. (G) Representative images of

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the neurons in extracellular fluid of free Ca2+ exposed to GSK1016790A (1 μM); no change

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occured in the intracellular fluo-3 AM fluorescence. When 2 mM Ca2+ was added to the medium, however, the intracellular Ca2+ concentration promptly rose. Scale bar: 100 μm. (H) The representative trace for intracellular Ca2+ fluorescence intensity when individual cells

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responding to GSK1016790A with /without Ca2+ in the extracellular fluid. (I) The statistical

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analysis results for the change of the mean intensity in the intracellular fluorescence with/without Ca2+ in the extracellular fluid. Data are presented as mean ± SD. **P < 0.01. n

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= 20 cells.

Fig. 6. ICH recruits TRPV4 into IP3R complex. (A) The extracts (1 mg) from the ipsilateral brain tissues of mice 24 h after sham (lane 1) or ICH (lane 2) were precipitated with 30

nonspecific IgG (rIgG) or anti-TRPV4 and probed with anti-IP3R. Input: 50 μg of protein was loaded in each lane. Right panels: Quantification of TRPV4 protein levels between sham and vehicle (co-ip). n = 3 per group. *P < 0.05. (B) The extracts (1 mg) from the ipsilateral brain tissues of mice 24 h after sham (lane 1) or ICH (lane 2) were precipitated with nonspecific

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IgG (mIgG) or anti-IP3R and probed with anti-TRPV4. Input: 50μg of protein was loaded in each lane. Right panels: Quantification of IP3R protein levels between sham and vehicle

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(co-ip). n = 3 per group. *P < 0.05.

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Fig. 7. TRPV4 channels induced ER stress through the PERK-CHOP-Bcl-2 pathway after

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ICH. (A) Immunoblots of the extracts from the ipsilateral brain tissues at 24 h after ICH using

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the indicated antibodies. β-actin served as a loading control. (B) Quantification of the GRP78, p-PERK, CHOP, and Bcl-2 protein levels in sham, vehicle, GSK1016790A-treated,

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HC-067047-treated. (n = 4 mouse per time point). *P < 0.05, **P < 0.01 vs. vehicle. (C) Immunoblots of the extracts from the ipsilateral brain tissues at 24 h after ICH using the

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indicated antibodies. β-actin served as a loading control. (D) quantification of the GRP78,

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p-IRE1, p-JNK protein levels in sham, vehicle, GSK1016790A-treated, HC-067047-treated.

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(n = 4 mouse per time point). *P < 0.05, **P < 0.01 vs. vehicle.

Fig. 8. IP3R inhibition relieved endoplasmic reticulum stress and improved hemorrhagic brain injury. (A) Immunoblots of the extracts from the ipsilateral brain tissues at 24 h after ICH using the CHOP and Bcl-2 antibodies. β-actin served as a loading control. (B)

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Quantification of the CHOP and Bcl-2 protein levels in the corresponding group (n = 3 mouse

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