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. 2014 Oct 11;35(2):283–292. doi: 10.1007/s10571-014-0123-0

Knockdown of STIM1 Improves Neuronal Survival After Traumatic Neuronal Injury Through Regulating mGluR1-Dependent Ca2+ Signaling in Mouse Cortical Neurons

Peng-Fei Hou 1,2, Zhan-Hui Liu 2, Nan Li 1,3, Wen-Jia Cheng 4, Shi-Wen Guo 1,✉
PMCID: PMC11486307  PMID: 25304289

Abstract

Activation of glutamate receptors and followed increase of intracellular calcium concentration is a key pathological mechanism involved in secondary neuronal injury after traumatic brain injury (TBI). Stromal interaction molecule (STIM) proteins are considered to be important players in regulating neuronal Ca2+ homeostasis under normal aging and pathological conditions. Here, we investigated the role of STIM1 in regulating metabotropic glutamate receptor 1 (mGluR1)-related Ca2+ signaling and neuronal survival by using an in vitro traumatic neuronal injury (TNI) model. The expression of STIM1 was significantly increased at both mRNA and protein levels after TNI. Down-regulation of STIM1 by specific small interfere RNA significantly preserved neuronal viability, decreased lactate dehydrogenase release, and inhibited apoptotic cell death after traumatic injury. Moreover, knockdown of STIM1 significantly alleviated the mGluR1-related increase of cytoplasmic Ca2+ levels after TNI. By analyzing Ca2+ imaging in Ca2+-free conditions, we demonstrated that the mGluR1-dependent inositol trisphosphate receptor and/or ryanodine receptor-mediated Ca2+ release from the endoplasmic reticulum after TNI is strongly attenuated in the absence of STIM1. Together, our results demonstrate that in the mammalian nervous system, STIM1 is a key regulator of mGluR1-dependent Ca2+ signaling and knockdown of STIM1 might be an effective intervention target in TBI.

Keywords: Traumatic brain injury, STIM1, mGluR1, Endoplasmic reticulum

Introduction

Traumatic brain injury (TBI) is defined as an injury caused by a blow or jolt to the head or a penetrating head injury that disrupts the normal function of the brain (Ghajar 2000). It is a leading cause of death and disability throughout the world, especially in children and young adults (Chen et al. 2011). Brain damage after TBI can be classified into the primary injury, the damage that occurs at the moment of trauma, and the secondary damage, which is initiated immediately after trauma and persists over hours, days, and even months after injury due to biochemical and physiological derangements (Algattas and Huang 2014). At present, the major known mechanisms of secondary injury after TBI include excessive release of excitatory neurotransmitters, influx of calcium and sodium ions, activation of pro-apoptotic signaling, overproduction of free radicals, inflammatory responses, and mitochondrial dysfunction (Chen et al. 2012a, c; Stoica and Faden 2010; Raghupathi 2004). However, the exact molecular mechanisms of neuronal damage following TBI are still poorly understood, and no effective neuroprotective drugs for TBI have emerged to date.

Glutamate is the most abundant excitatory neurotransmitter in neuronal and glial cells of the central nervous system (CNS) (Abramov and Duchen 2008; O’Shea 2002). Acute increases in extracellular glutamate levels have been observed in both experimental TBI models and in human patients (Arundine and Tymianski 2004). These elevated levels of glutamate induced by an imbalance between glutamate release and uptake can lead to glutamate excitotoxicity, which plays an important role in the development of secondary injury and contributes to the expansion of infarct volume in TBI patients (Arundine and Tymianski 2004; Belousov 2012; Abramov and Duchen 2008). Glutamate can induce Ca2+ overload and many downstream neurotoxic cascades through over-stimulation of ionotropic glutamate receptors (iGluRs), especially via N-methyl-d-aspartate (NMDA) receptors (Smith et al. 2000). Recently, accumulating studies have demonstrated the involvement of metabotropic glutamate receptors (mGluRs) in glutamate-mediated pathological increases in intracellular Ca2+ (Chen et al. 2012b; Cucchiaroni et al. 2010; Stone and Addae 2002). Moreover, mGluR1 was shown to mediate glutamate-induced excitotoxicity through mitochondrial dysfunction and Ca2+ release from endoplasmic reticulum (ER) stores (Chen et al. 2012b). Therefore, regulation of mGluRs-mediated Ca2+ signaling cascades might be an ideal therapeutic target for TBI treatment.

Cytosolic Ca2+ overload is derived mainly from two sources, Ca2+ release from the ER stores and Ca2+ influx from the extracellular spaces (Baba and Kurosaki 2009). A main calcium entry pathway linking these two sources is represented by the so-called store-operated calcium channels (SOCs), which is implicated in maintaining the Ca2+ homeostasis in neurons (Schindl et al. 2009). Human stromal interaction molecule 1 (STIM1) is an ER-resident Ca2+ binding protein that regulates SOCs and allows an expanding molecular definition of SOCs (Worley et al. 2007). It is known that STIM1 can directly bridge the ER to the plasma membrane at specialized junctions via its Ca2+-binding EF-hand motifs and a single sterile α-motif (SAM) domain, and then open SOCs to mediate store-operated calcium entry (SOCE) to ensure optimal refilling of the ER or lead to a prolonged increase in cytosolic Ca2+ (Fahrner et al. 2009; Schindl et al. 2009). These Ca2+ influx through STIM1-mediated SOCE is a key component of the receptor-evoked Ca2+ signal, including mGluR1-dependent Ca2+ signaling (Ng et al. 2011; Hartmann et al. 2014). Due to increasing evidence that STIM1-mediated SOCE plays important roles in neuronal injury (Li et al. 2014; Chen et al. 2013a, b), it is necessary to elucidate the relationship between STIM1 and mGluRs post TBI. Thus, in the present study, we investigate the effects of STIM1 knockdown on mGluR1-dependent Ca2+ signaling and neuroprotection in an in vitro TNI model.

Experimental Procedures

Primary Culture of Cortical Neurons

Cortical neurons were cultured from C57BL/6 J mice using a modified method reported by Chen et al. (2012b). Briefly, cerebral cortices were removed from embryos at 13–15 days, stripped of meninges and blood vessels, and minced. Tissues were dissociated by 0.25 % trypsin digestion for 15 min at 37 °C and gentle trituration. Neurons were re-suspended in neurobasal medium containing 2 % B27 supplement and 0.5 mM l-glutamine and plated at a density of 3 × 105 cells/cm2. Before seeding, culture vessels, consisting of 96-well plates, 24-well plates, 1.5-cm glass slides or 6-cm dishes were pre-coated with poly-l-lysine (PLL, 50 μg/ml) overnight. Neurons were maintained at 37 °C in a humidified 5 % CO2 incubator, and half of the culture medium was changed every other day. The cultured neurons were used for studies on in vitro days 8–10 (DIV 8–10).

Traumatic Injury Model

Traumatic neuronal injury was performed according to the previous published method (Huang et al. 2005) with modifications. In brief, a traumatic injury was performed on cultured cortical neurons by using a punch device that consisted of 28 stainless steel blades joined together and produced 28 parallel cuts. As a result, the injury produced parallel cuts at 0.5 mm intervals that were uniformly distributed through the cell layer. These cuts caused immediate neuronal death under the blade. The appearance of secondary insults at a distance from the cuts can be observed by an electron microscope, beginning at 6 h after the mechanical injury. This model was shown to be highly reproducible and to induce severe traumatic neuronal injury.

RNA Interference (RNAi) Transfection

STIM1 siRNA (Si-STIM1, sc-76590, Santa Cruz, CA, USA) and control siRNA (Si-control, sc-37007, Santa Cruz, CA, USA) were dissolved separately in Optimem I (Invitrogen, CA, USA). After 10 min of equilibration at room temperature, each RNA solution was combined with the respective volume of the Lipofectamine 2000 solution (Invitrogen, CA, USA), mixed gently, and allowed to form siRNA liposomes for 20 min. Primary cortical neurons were transfected with Si-STIM1 or Si-control according to the manufacturer’s protocol (Invitrogen, CA, USA). Optimal transfection efficiency and conditions were determined by using GFP-labeled non-specific siRNA. The transfection efficiency was approximately 80 % (data not shown), and the mRNA and protein expression levels were measured 72 h after transfection.

Neuronal Viability

Neuronal viability assay was performed using the Cell proliferation Reagent WST-1 (Roche, Basel, CH) 24 h after traumatic injury following the manufactory protocol. Cortical neurons were cultured in 24-well plates with a final volume of 500 μl/well culture medium. After various treatments, 50 μl WST-1 was added into each well and incubated for 4 h at 37 °C, and then shaken thoroughly for 1 min on a shaker. One hundred and ten microliters of supernatant from each well was collected in a 96-well microplate. The absorbance of the samples was measured by using a microplate reader (Bio-Rad Laboratories).

Lactate Dehydrogenase (LDH) Assay

Neuronal cytotoxicity was determined by the release of LDH into the culture medium using a diagnostic kit following the manufacturer’s instructions (Jiancheng Bioengineering Institute, Nanjing, China). Briefly, 50 µl of the supernatant from each well was collected to assay the LDH release. The samples were incubated with the reduced form of nicotinamide adenine dinucleotide (NADH) and pyruvate for 15 min at 37 °C, and the reaction was stopped by adding 0.4 M NaOH. The activity of LDH was calculated from the absorbance at 440 nm, and the background absorbance from the culture medium that was not used for any cell cultures was subtracted from all of the absorbance measurements.

TUNEL Staining

Apoptotic neuronal death after traumatic injury was detected by TUNEL staining. Briefly, the cortical neurons on 1.5-cm glass slides were fixed by immersing slides in freshly prepared 4 % methanol-free formaldehyde solution in phosphate buffered saline (PBS) for 20 min and permeabilized with 0.2 % Triton X-100 for 5 min. Neurons were labeled with fluorescein TUNEL reagent mixture for 60 min at 37 °C according to the manufacturer’s suggested protocol (Roche, Basel, CH). After that, slides were examined by fluorescence microscopy and the number of TUNEL-positive (apoptotic) cells was counted. PI (50 μg/ml) was used to stain nucleus.

Measurement of Caspase-3 Activity

The activity of caspase-3 was measured using a colorimetric assay kit according to the manufacturer’s protocols (Cell Signaling, MA, USA). Briefly, after being harvested and lysed, 106 neurons were mixed with 32 μl of assay buffer and 2 μl of 10 mM Ac-DEVD-pNA substrate. Absorbance at 405 nm was measured after incubation at 37 °C for 4 h. Absorbance of each sample was determined by the subtraction of the mean absorbance of the blank and corrected by the protein concentration of the cell lysate. The results were described as relative activity to that of control group.

Real-Time RT-PCR

Total RNA was isolated from primary cortical neurons using Trizol reagent (Invitrogen, CA, USA). After the equalization of the RNA quantity in each group, the mRNA levels of STIM1 were quantitated using a Bio-Rad iQ5 Gradient Real-Time PCR system (Bio-Rad Laboratories), and GAPDH was used as an endogenous control. Primers for all Real-Time PCR experiments were listed as follows: STIM1: forward: 5′-GGCCAGAGTCTCAGCCATAG-3′, reverse: 5′-TCCACATCCACATCACCATT-3′; GAPDH: forward: 5′-AAGGTGAAGGTCGGAGTCAA-3′, reverse: 5′-AATGAAGGGGTCATTGATGG-3′. Samples were tested in triplicates, and data from five independent experiments were used for analysis.

Western Blot Analysis

Equivalent amounts of protein were loaded and separated by 10 % SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5 % nonfat milk solution in tris-buffered saline with 0.1 % Triton X-100 (TBST) for 1 h, and then incubated overnight at 4 °C with the primary STIM1 antibody (1:1,000) or β-actin antibody (1:600) dilutions in TBST. Next, the membranes were washed and incubated with a secondary antibody for 1 h at room temperature. The immunoreactivity was detected with Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). The analysis software Image J (Scion Corporation) was used to quantify the optical density of each band.

Calcium Imaging

Intracellular Ca2+ levels in cortical neurons were recorded using the ratiometric Ca2+ indicator dye Fura-2AM, and imaging was performed as described previously (Gruszczynska-Biegala et al. 2011). The neurons grown on coverslips were loaded with 2 μM acetoxymethyl (AM) ester of Fura-2 for 30 min at 37 °C. After rinsing, the ratio of the two emission intensities for excitation at 340 and 380 nm was detected, and the intracellular Ca2+ levels in individual neurons were calculated after subtracting background fluorescence. The ratio of emissions at 510 nm was recorded every 5 min (Fig. 3) or 60 s (Fig. 4). Ca2+-free solution instead of CaCl2 contained 0.5 mM EGTA.

Fig. 3.

Fig. 3

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Knockdown of STIM1 attenuates mGluR1-dependent intracellular Ca2+ overload after traumatic neuronal injury. a Mouse cortical neurons were pretreated with DHPG (100 μM) or BAY 36-7620 (BAY, 50 μM) for 10 min before traumatic injury, and intracellular Ca2+ was measured up to 120 min. b Mouse cortical neurons were transfected with STIM1-specific targeted siRNA (Si-STIM1) or control siRNA (Si-control) for 72 h before traumatic injury, and intracellular Ca2+ was measured up to 120 min. c, d Mouse cortical neurons were transfected with siRNA and pretreated with DHPG (100 μM) for 10 min before traumatic injury, and intracellular Ca2+ was measured up to 120 min. Each trace represents the average response of 15–20 cells measured on a single coverslip from three independent experiments. Fluorescence values beginning just before the traumatic injury were normalized to the same values

Fig. 4.

Fig. 4

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Knockdown of STIM1 inhibits mGluR1-dependent ER Ca2+ release after traumatic neuronal injury. a Mouse cortical neurons were treated with Tg (2 μM) or traumatic injury, and intracellular Ca2+ was measured up to 30 min. b Mouse cortical neurons were pretreated with DHPG (100 μM) or BAY 36-7620 (BAY, 50 μM) for 10 min before traumatic injury, and intracellular Ca2+ was measured up to 30 min. c Mouse cortical neurons were transfected with STIM1-specific targeted siRNA (Si-STIM1) or control siRNA (Si-control) for 72 h before traumatic injury, and intracellular Ca2+ was measured up to 30 min. d–f After transfection with siRNA and traumatic injury, intracellular Ca2+ in mouse cortical neurons was measured up to 30 min, and the neurons were treated with 100 μM DHPG, photolytic release of IP3, or 80 mM caffeine at 5 min after Ca2+ measurement, respectively. All the experiments were performed in Ca2+-free culture medium. Each trace represents the average response of 15–20 cells measured on a single coverslip from three independent experiments. Fluorescence values beginning just before the traumatic injury were normalized to the same values

IP3 Uncaging

For photolytic uncaging experiments, the internal saline was supplemented with NPE-IP3 (400 mM; Invitrogen). Uncaging of IP3 was produced by directing the output of a diode laser (Coherent Cube; 375 nm, 15 mW at the laser head) onto the surface of the cultures with the use of a tapered lensed optical fiber (Hartmann et al. 2014).

Statistical Analysis

Statistical analysis was performed using SPSS 16.0, a statistical software package. Statistical evaluation of the data was performed by one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons or unpaired t test (two groups). A value of p < 0.05 was considered statistically significant.

Results

Knockdown of STIM1 Improves Neuronal Survival After Traumatic Neuronal Injury

To investigate the effect of traumatic injury on STIM1 expression, the expression levels of STIM1 mRNA and protein were assayed at different time points (control, 3, 6, 12, and 24 h). The expression levels of STIM1 mRNA and protein were significantly increased within 24 h after injury, and peaked at 6 h or 12 h, respectively (Fig. 1a, b). To investigate the biological functions of STIM1 in traumatic neuronal injury, mouse cortical neurons were transfected with STIM1-targeted siRNA (Si-STIM1) or control siRNA (Si-control). The results showed that STIM1 expression was significantly reduced in neurons at both mRNA (Fig. 1c) and protein (Fig. 1d) levels after their transfection with Si-STIM1. Si-STIM1 transfection did not alter the expression of STIM2 mRNA (Fig. 1e) and protein (Fig. 1f) compared with Si-control group. After transfection and traumatic injury, the viability of the neurons transfected with Si-STIM1 was higher than that of the neurons transfected with Si-control (Fig. 1g). In contrast, knockdown of STIM1 significantly decreased the LDH release induced by traumatic injury (Fig. 1h). As compared to control group, STIM1 knockdown only partially rescued neuronal viability and LDH release after injury.

Fig. 1.

Fig. 1

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Knockdown of STIM1 improves neuronal survival after traumatic neuronal injury. a, b Traumatic injury was induced in primary cultured mouse cortical neurons, and the expression of STIM1 was measured by Real-Time RT-PCR and Western blot analyses. c–f Mouse cortical neurons were transfected with STIM1-specific targeted siRNA (Si-STIM1) or control siRNA (Si-control) for 72 h, and the expression of STIM1 and STIM2 was measured by Real-Time RT-PCR and Western blot analyses. g, h After transfection with siRNA and traumatic injury, the neuronal viability was measured by the WST assay, and the cytotoxicity was measured by the LDH release assay. The data are shown as mean ± SD of five experiments. # p < 0.05 versus control. *p < 0.05 versus Si-control

Knockdown of STIM1 Reduces Neuronal Apoptosis After Traumatic Neuronal Injury

To evaluate the effects of STIM1 on neuronal apoptosis, the main cell death type of the secondary insults of neurons after TNI, TUNEL staining was performed at 24 h after traumatic injury (Fig. 2a). There were no obvious TUNEL-positive neurons in the control group, whereas the number of TUNEL-positive cells increased after traumatic injury, and this effect was significantly decreased by Si-STIM1 transfection compared with that in Si-control-transfected neurons (Fig. 2b). As shown in Fig. 2c, down-regulation of STIM1 expression also inhibited the traumatic injury-induced activation of caspase-3 compared with neurons in Si-control-transfected group. These data indicated that down-regulation of STIM1-induced neuroprotection in our in vitro TNI model was mediated via anti-apoptotic activity.

Fig. 2.

Fig. 2

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Knockdown of STIM1 reduces neuronal apoptosis after traumatic neuronal injury. Mouse cortical neurons were transfected with STIM1-specific targeted siRNA (Si-STIM1) or control siRNA (Si-control) for 72 h before traumatic injury. a The neuronal apoptosis was detected by TUNEL staining, and the total neuron number was detected by propidium iodide (PI). b The apoptotic rate was calculated. c The activity of caspase-3 was measured at 24 h after traumatic injury. Scale bars = 100 μm. The data are either representative of three similar experiments or shown as mean ± SD of five experiments. # p < 0.05 versus control. *p < 0.05 versus Si-control

Knockdown of STIM1 Attenuates mGluR1-Dependent Intracellular Ca2+ Overload After Traumatic Neuronal Injury

To examine the relationship between elevation of cytosolic Ca2+ levels and STIM1 knockdown-induced neuroprotection, Ca2+ imaging was performed up to 120 min after traumatic injury (Fig. 3). First, we tested the involvement of mGluR1 in the Ca2+ overload after traumatic injury. The results showed that the traumatic injury-induced increase of cytosolic Ca2+ levels was significantly decreased by the mGluR1 antagonist BAY 36-7620, but further increased by the mGluR1 agonist DHPG pretreatment (Fig. 3a). Down-regulation of STIM1 significantly attenuated the elevation of cytosolic Ca2+ levels induced by traumatic injury compared with Si-control-transfected neurons (Fig. 3b). In addition, the mGluR1 agonist DHPG significantly increased the cytosolic Ca2+ levels in Si-control-transfected neurons after traumatic injury (Fig. 3c), but had no effects on the cytosolic Ca2+ levels in Si-STIM1-transfected neurons (Fig. 3d).

Knockdown of STIM1 Inhibits mGluR1-Dependent ER Ca2+ Release After Traumatic Neuronal Injury

To determine whether STIM1 knockdown affects mGluR1-dependent Ca2+ release from the ER, we performed Ca2+ imaging in Ca2+-free culture medium (Fig. 4). Tg yielded an early but transient (within 10 min) elevation of cytosolic Ca2+ levels in cortical neurons, whereas traumatic injury-induced Ca2+ increase was slower and more modest (Fig. 4a), indicating the presence of ER Ca2+ release after traumatic neuronal injury. The ER Ca2+ release after traumatic injury was significantly increased by DHPG, decreased by BAY 36-7620 (Fig. 4b), and almost abolished by STIM1 knockdown compared with that in Si-control-transfected neurons (Fig. 4c). Activation of mGluR1 with DHPG at 5 min after injury significantly increased ER Ca2+ release in Si-control-transfected cells, but not in STIM1-down-regulated neurons (Fig. 4d). As shown in Fig. 4e and f, the similar results with application of RyR agonist caffeine and IP3 were also observed, suggesting that the RyR and IP3R-mediated ER Ca2+ release after traumatic neuronal injury were both prevented by STIM1 knockdown.

Discussion

In this study, we first investigated the expression of STIM1 in primary cultured mouse cortical neurons by establishing an in vitro model of TNI, and explored the dynamic changes of STIM1 expression after TNI. Although STIM1-mediated SOCE is considered to be ubiquitous in non-excitable cells, there are studies demonstrating that it is also present in excitable cells such as smooth muscle cells and neuronal cells (Potier et al. 2009; Ng et al. 2011; Klejman et al. 2009). Previous studies have shown that STIM1 was present in Pukinje neurons, in primary dendrites of pyramidal neurons of the hippocampus, and in pyramidal neurons of layer V of the cerebral cortex (Dziadek and Johnstone 2007), which was also confirmed in our study in mouse cortical neurons. Depletion of the ER Ca2+ store in cultured cortical neurons induced a change in the localization of STIM1 from disperse to puncta-like in thapsigargin-treated cells (Klejman et al. 2009). In the present study, we found that STIM1 expression changed after TNI as indicated by western blot, showing a dramatic increase of STIM1 occurring as early as 6 h after TNI, and peaked at 12 h post-injury. As further demonstrated by Real-Time RT-PCR, STIM1 mRNA expression increased as early as 3 h post-injury, and peaked at 6 h after TNI. These results are consistent with a recent study showing that the expression of STIM1 was increased in the early stages of diffuse axonal injury (Li et al. 2013). Thus, our current study confirmed that STIM1 is highly expressed at both mRNA and protein levels in cortical neurons after TBI.

The important roles of mGluRs in neuronal cell death and survival have been demonstrated in several neurodegenerative diseases (Goudet et al. 2009). Recently, the relationship between mGluRs (especially group I mGluRs) and TBI is highlighted by both in vitro and in vivo studies. A previous study showed that the expression of group I mGluRs and the number of mGluR1α-positive neurons significantly increased after TBI (Fei et al. 2005). Using selective mGluRs agonists and antagonists, further studies have suggested that the activation of group I mGluRs subtype mGluR1, but not mGluR5, contributes to neuronal injury after TBI (Gong et al. 1995; Faden et al. 2001; Chen et al. 2012a, c). Thus, selective blockade of mGluR1 by specific antagonists represents a potential pharmaceutical approach in the treatment of TBI, and the underlying mechanisms are thought to be associated with downstream protein kinases signaling, such as protein kinase C (PKC), Akt, and mitogen-activated protein kinases (MAPKs) (Fei et al. 2006). After brain injury, sustained elevation of extracellular glutamate leads to intracellular Ca2+ overload, which is crucial to secondary neuronal damage through triggering downstream lethal cascades. Stimulation of mGluR1 was shown to increase intracellular concentration of IP3, which enhances the release of Ca2+ form ER stores via stimulating IP3R (Lau and Tymianski 2010). In the present study, we found that the activation of mGluR1 by DHPG increased ER Ca2+ release, whereas blocking mGluR1 by BAY 36-7620 attenuated Ca2+ response in Ca2+-free conditions after TNI. These results are accompanied with similar changes of cytosolic Ca2+ concentration using DHPG and BAY 36-7620, which were consistent with some of the other TBI studies (Faden et al. 2001; Chen et al. 2012b). Therefore, mGluR1 might participate in neuronal calcium overload and be an important regulator of ER Ca2+ release after TNI. Overall, our study confirmed the involvement of mGluR1 activation in traumatic neuronal injury and introduced a Ca2+ signaling-associated mechanism in mGluR1-mediated neuronal damage.

Although the molecular mechanisms underlying TBI-induced neuronal injury are not fully understood, intracellular Ca2+ overload is considered to be a key factor in apoptotic neuronal death after TBI (Cheng et al. 2012; Feyen et al. 2012). It is well known that high STIM1 expression inside cells will increase Ca2+ influx by altering SOCE (Worley et al. 2007). However, the role of STIM1-mediated SOCE in TBI-induced neuronal injury is still not known, and there is little information in the literature about the role of STIM1 knockdown in Ca2+ homeostasis after TBI. Chen et al. demonstrated that blocking STIM1-mediated SOCE through non-selective antagonist SKF-96365 or via down-regulating the postsynaptic scaffold protein Homer1 attenuated the neuron toxin-induced neuronal injury (Chen et al. 2013a, b). Notably, Li et al. recently showed that down-regulation of STIM1 reduced 6-hydroxydopamine (6-OHDA)-induced intracellular Ca2+ overload through inhibiting orai1 and L-type channel-dependent SOCE in PC12 cells (Li et al. 2014). Thus, STIM1 might also play an important role in the regulation of Ca2+ homeostasis after TBI. Our data here demonstrated that knockdown of STIM1 using specific siRNA significantly attenuated the increase of intracellular Ca2+ concentration induced by traumatic neuronal injury. STIM1-associated SOCE can be mediated by several calcium channels in the membrane, such as TRPC channels and Orai1 and L-type Ca2+ channels (Harraz and Altier 2014). Under cellular stress conditions, STIM-induced activation of these channels not only ensures optimal refilling of the intracellular Ca2+ stores, but also leads to a prolonged increase in cytosolic Ca2+ (Parekh and Putney 2005). Therefore, we speculate that after STIM1 is down-regulated, its role as a calcium sensor is blocked and the activities of TRPC channels and Orai1 and/or L-type Ca2+ channels would be subsequently reduced. Then, the Ca2+ influx through these membrane Ca2+ channels and followed Ca2+ overload after TNI would be inhibited, and the neuronal death might be sequentially attenuated.

In central mammalian neurons, activation of mGluR1 evokes a complex synaptic Ca2+ response, but it is unknown how mGluR1 is linked to its downstream effectors. The underlying mechanisms are mediated by two major intracellular signaling pathways: IP3R or RyR-dependent rapid Ca2+ release from the ER stores and a slow Ca2+ influx from extracellular spaces through SOC channels (Batchelor et al. 1994; Finch and Augustine 1998). The mGluR1-mediated ER Ca2+ release after brain injury can be detrimental not only through aggravating intracellular Ca2+ overload, but also via activating ER stress-related apoptotic pathways (Urra et al. 2013). By coupling to Gq protein, mGluR1 activates phospholipase C and thus induces the generation of IP3, which opens IP3R channels and thereby leads to the Ca2+ release from the ER (Stone and Addae 2002). Recent studies have shown that this process was mediated by several postsynaptic proteins, such as Homer and Shank (Hayashi et al. 2009). Our data showed that in the absence of STIM1, ER stores become largely devoid of Ca2+ ions and in these conditions, mGluR1-mediated IP3 signaling and RyR signaling fail to induce a Ca2+ release signal from the ER stores (Fig. 4). The underlying mechanism might be that decreased STIM1 expression might result in the mGluR complexes separating at the synapse, decreasing the second messenger IP3 transduction, and consequently decreasing the Ca2+ release from intracellular calcium pool. Such a Ca2+ signaling mechanism is in agreement with what has been previously found and is well established in neuronal cells (Hartmann et al. 2014). Therefore, STIM1 might participate in neuronal calcium release and be an important regulator of the mGluR1-IP3R/RyR-Ca2+ signal transduction pathway.

The in vitro TNI model used in our study was performed according to the Faden’s model, which also found the important role of mGluR receptors after traumatic neuronal injury (Faden et al. 2001). This model mimics neuronal injury after TBI by lacerating neurons, which represents a different type of injury as compared with stretching injury used in many previous TBI studies. Although these two different type of injuries can both be observed in TBI animal models, some more experiments using stretching model or in vivo TBI models will be helpful for confirming the mechanism detected in our in vitro model.

In conclusion, our findings showed that down-regulation of STIM1 decreased intracellular Ca2+ concentration and mGluR1-mediated Ca2+ release from ER stores, which may cause improved neuronal viability. Our results identify STIM1 as one of the intracellular links between mGluR1 and its downstream effectors, which is essential for the regulation of calcium homeostasis after TBI.

Acknowledgments

Conflict of interest

The authors declare that there are no conflicts of interest.

Footnotes

Peng-Fei Hou and Zhan-Hui Liu have contributed equally to this work.

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