Abstract
Traumatic brain injury (TBI) is a global risk factor that leads to long-term cognitive impairments. To date, the disease remains without effective therapeutics because of the multifactorial nature of the disease. Here, we demonstrated that activation of the c-Jun N-terminal kinase (JNK) is involved in multiple pathological features of TBI. Therefore, we investigated the disease-modifying therapeutic potential of JNK-specific inhibitor (SP600125) in TBI mice. Treating 2 different models of TBI mice with SP600125 for 7 days dramatically inhibited activated JNK, resulting in marked reductions of amyloid precursor protein (APP) expression level and in amyloid beta production and hyperphosphorylated tau and regulation of the abnormal expression of secretases. Furthermore, SP600125 strongly inhibited inflammatory responses, blood–brain barrier breakdown, apoptotic neurodegeneration, and synaptic protein loss, regulated prosurvival processes and improved motor function and behavioral outcomes in TBI mice. More interestingly, we found that SP600125 treatment ameliorated amyloidogenic APP processing and promoted the nonamyloidogenic pathway in TBI mouse brains. Our findings strongly suggest that active JNK is critically involved in disease development after TBI and that inhibition of JNK with SP600125 is highly efficient for slowing disease progression by reducing multiple pathological features in TBI mouse brains and regulating cognitive dysfunction.
Introduction
Traumatic brain injury (TBI) is an environmental risk factor that triggers a variety of events and causes devastating chronic neurodegenerative disease including Alzheimer’s disease (AD), Parkinson’s disease, and amyotrophic lateral sclerosis. Post-TBI consequences include altered expression of amyloid precursor protein (APP), increased amyloid beta (Aβ) generation and tau proteins phosphorylation, which is believed to be involved in the pathogenesis of AD (Ikonomovic et al. 2004; Yu et al. 2012; Wright et al. 2016). Elevated levels of Aβ42 in the cerebrospinal fluid of head trauma patients and postmortem studies revealed Aβ deposition in TBI patients (Olsson et al. 2004; Uryu et al. 2007). Necessary APP enzymes that generate Aβ, the β-APP cleaving enzyme-1 (BACE1), and Presenilin-1 were found increased after TBI, and the deposition of Aβ may occurs less than a day after head injury (Chen et al. 2004; Nadler et al. 2008). Several lines of evidence have reported Aβ-induced neuronal insult and inflammatory and proapoptotic pathway activation (Chang et al. 2015). A previous study reported that overproduction and deposition of Aβ induce microglial and astrocyte activation and oxidative stress in an AD model. Furthermore, in a rodent model of brain injury, active astrocytes and microglial were reported in the injured brain area (López-Rodríguez et al. 2011, 2015; Abeti and Duchen 2012). To date, no disease-modifying therapeutic agent is currently approved for treatment of TBI because of the multifactorial nature of the disorder.
The c-Jun N-terminal kinase (JNK) plays critical roles in cell regulation including cell proliferation, gene expression and apoptosis (Repici and Borsello 2006). A growing body of evidence suggests that the JNK is involved in numerous pathophysiological mechanisms of AD (Mehan et al. 2011), with increased expression in AD patients and in animal models of AD as well as consistently surrounding Aβ plaques, suggesting the possible association of JNK with Aβ deposition in the brain (Shoji et al. 2000; Zhu et al. 2001; Thakur et al. 2007). Several reports have demonstrated that active JNK is involved in the phosphorylation of tau proteins (Vogel et al. 2009; Ploia et al. 2011). More recently, a study reported that inhibition of active JNK ameliorated neuroinflammatory responses and alleviated synaptic loss in an AD mouse model (Zhou et al. 2015). The above-mentioned evidence clearly demonstrates that active JNK is potentially involved in APP processing, Aβ generation, hyperphosphorylation of tau proteins, neuroinflammation, and synaptic loss. Based on all these observations, we hypothesized that JNK activation may be a potential therapeutic target against TBI-induced AD-like pathological hallmarks in the brain. However, it remains unclear whether inhibition of active JNK via a specific inhibitor has disease-modifying therapeutic effects in TBI-induced neurodegeneration.
Among the available JNK inhibitors, SP600125 (anthra [1,9] pyrazol-6(2H)-one) is a well-reported selective JNK inhibitor that can substantially pass through the blood–brain barrier (BBB) and inhibit active JNK in animal brains (Gao et al. 2005; Chen et al. 2012a). Zhou et al. reported that inhibition of active JNK via SP600125 could alleviate multiple neuropathologies in APP/PS1 transgenic mice that were correlated with AD (Zhou et al. 2015). In the present study, we developed a TBI mouse model that exhibits pathological characteristics of AD to investigate the potential disease-modifying therapeutic properties of SP600125, a specific JNK inhibitor. In the present study, using a SP600125, we showed that systematic administration of SP600125 possibly inhibited active JNK, resulting in significant reductions in Aβ generation, altered APP processing, tau phosphorylation, neurodegeneration, and neuroinflammatory response, synaptic loss and improved memory in TBI mice. Taken together, our data show for the first time that specific inhibition of active JNK by the specific inhibitor SP600125 significantly abrogated neuropathologies associated with TBI in mouse brains, suggesting the potential efficacy of JNK inhibition by SP600125 treatment as a disease-modifying therapy for TBI-induced neurological dysfunction.
Materials and Methods
Animals and Treatment
Male C57BL/6N mice, 8 weeks of age (25–30 g), were obtained from Samtako Bio, Korea and were maintained in the animal care center of Gyeongsang National University, South Korea. The animals were provided with water and ad libitum and kept under a 12/12 light cycle at 23 °C, 60 ± 10% humidity. After 1 week of acclimatization, the mice were randomly divided into 4 groups (n = 13). The animals were carefully handled according to the animal ethics committee (IACUC) of the Division of Life Science, Department of Biology, Gyeongsang National University, Republic of South Korea. The experimental techniques were approved (Approval ID: 125) by the animal ethics committee (IACUC) of the Division of Life Science, Department of Biology, Gyeongsang National University, Republic of South Korea.
TBI Models
Feeney’s Weight Drop Model
In the present study, an in vivo model of TBI was induced as previously described (Feeney et al. 1981; Tian et al. 2012) with slight modifications. Briefly, the mice subjected to TBI were anesthetized with Zoletil and Rompun and were placed in a stereotaxic apparatus followed by making a mid-longitudinal incision and exposing the skull. A circular craniotomy, 5 mm in diameter (1 mm posterior to the bregma and 2 mm lateral to the midline), was made with a dental drill. The intact dura was left undisturbed. A 50-g steel rod with a flat end diameter of 4 mm was allowed to fall on the piston placed on the dura. The height was set to 20 cm to generate standardized parietal contusions. The piston was adjusted to compress the tissue a maximum of 4 mm with a brain deformation depth of 2 mm. The cranium was sealed using bone wax, and the skin was tightly closed using silk suture. The mice of the control group were subjected to the same procedure without any injury to the head. The animals were maintained under continuous heating with a heat lamp until recovering from anesthesia. The animals were visually monitored every 15–20 min until safe recovery was verified.
Repetitive Mild Traumatic Brain Injury
Eight- to 10-week-old mice were randomly divided into 2 groups to undergo repetitive mild traumatic brain injury (rmTBI) or shame injury as previously described with modification (Zhang et al. 2015). Briefly, mice were anesthetized with Zoletil and Rompun and were carefully placed on stereotaxic frame. Following anesthesia the skull was exposed by midline skin incision. The anesthetized mice were placed on a delicate task wiper (Kimwipe, Kimberly Clark) and manually positioned under a hollow guided perforated tube. rmTBI was induced by dropping a 58-g weight from a 28-cm height along the guided tube on the right frontal side of the head. The mice were subjected to rmTBI (3 times/4 days). Following impact, the skin was sutured and mice were allowed at regular heating until fully recovered. The recovered mice were carefully returned to their home cages. A second injury was performed next day of the first injury. After second injury the mice were allowed for 1 day rest and the third injury was performed at day 4 of the first injury. The sham-injured mice were exposed to anesthesia without head injury. The experimental treatment was performed under continuous heating with a heat lamp. The mice were exposed to fresh air in the experimental room and visually monitored until they were fully recovered.
For the in vitro model of TBI, transaction model was established (Zhao et al. 2012). Briefly, SH-SY5Y cells were grown in 6-well plates containing Dulbecco’s modified Eagle medium (DMEM) cell media and were manually scratched with a sterile pipette tip in a 9 × 9 square grid (4 mm spacing between the lines). After scratching, the cells were incubated at 37 °C. The cells were divided into 3 groups as follows: cells without injury (con), cells with scratch injury (Scr), and cells with injury plus SP600125. After treatment the cells were collected at different time intervals (8, 24, and 36 h) for immunoblot analysis.
Brain endothelial cells line (bEnd.3 cell line (ATCC)) was grown in 6-well plates containing DMEM and was manually scratched with same procedure as in SH-SY5Y. After scratching, the cells were treated with SP600125 and were incubated at 37 °C for 8, 24, and 36 h. The cells were collected at different time intervals for immunoblot analysis.
SP600125 Treatment of Mice
Mice undergoing TBI were divided into the following 2 groups: the craniotomy-weight drop model (Feeney’s weight drop model) and rmTBI. For craniotomy-weight drop model the treatment groups were as follows: (1) saline-treated control group; (2) TBI group; (3) TBI+SP600125 (20 mg/kg/i.p./daily) group; and (4) Sham treated group. For rmTBI, the mice were divided into 4 groups: (1) saline-treated control group; (2) rmTBI group; (3) rmTBI + SP600125 (10 mg/kg/i.p./daily) group; and (4) rmTBI + SP600125 (20 mg/kg/i.p./daily) group. For in vitro cell treatment (SH-SY5Y and bEnd.3 cell lines), the cells were treated with 20 μM and SH-SY5Y at different concentrations of SP600125 (10, 20, or 30 μM).
Evaluation of BBB Integrity by Evans Blue
BBB permeability in the TBI mouse brains was evaluated by measuring Evans blue (EB) extravasation with some modification (Okuma et al. 2014). EB was prepared in 0.9% NaCl solution and a volume of 3 mL/kg was injected intravenously into the tails of the mice over 1 min (40 mg/kg) at 24 h after TBI. After 2 h circulation, the EB content was measure at 620 nm. The data are expressed as EB ng/g wet brain weight.
Brain Water Content
Beam Walking Test
Beam walking test (BWT) was performed by mice after TBI to assess motor coordination and function (Chen et al. 2012a, 2012b). The BWT is a method used to discriminate fine motor coordination differences between injured animals and sham-injured animals. The animals tended to escape the open area by walking on a narrow beam to enter a black box on the opposite end of the beam. The latency to reach the black box was recorded. The animals were allowed to complete 3 trials 2 h before TBI and at 1, 3, and 7 days post-TBI.
Morris Water Maze Test
Inhibitory effects of SP600125 treatment on the cognitive ability of TBI-injured mice were analyzed via the Morris water maze (MWM) test as previously described with modifications (Ahmad et al. 2017). The MWM apparatus was a circular tank filled with water and made opaque with white ink. A platform (10 cm in diameter) was hidden 1 cm below the water surface in 1 quadrant of the tank during the entire course of the experiment. In each trial, the mice were allowed 60 s to find the hidden platform; if they failed to locate the platform within 60 s, the mice were manually guided to the hidden platform and were allowed to remain there for 30 s. The animals received 4 training trials per day for 4 consecutive days. For each trial, escape latencies and the swim speed of the animals were calculated. The next day, the probe test was performed while removing the hidden platform to test spatial memory. For the probe trial, the latency to the platform location, the number of crossings, the swimming speed, and the time spent in the target quadrant were calculated. The data were recorded using a video tracking system (SMART, Panlab Harvard Apparatus, Bioscience Company).
Protein Extraction
After treatment, all animals were anesthetized and decapitated. The brains were carefully removed, and the ipsilateral cortex and hippocampus regions were dissected with care and were frozen at −80 °C. Tissues of the hippocampal and cortical regions were homogenized in pro-prep extraction solution (iNtRON Biotechnology) followed by centrifugation. Proteins were stored at −80 °C and were processed for immunoblotting.
Aβ1–42 ELISA Analysis
Aβ1–42 levels were analyzed in the cortex and hippocampus homogenates of all 4 groups at days 3 and 5 using a sandwich ELISA kit (Cat#: KHB3442; Thermo Fisher Scientific). Aβ1–42 levels were analyzed according to the manufacturer’s protocol.
Measurement of Inflammatory Markers Using ELISA
Inflammatory markers in the cortex homogenates were measured in all 4 groups of experimental animals at day 3 and day 7 post-TBI. The levels of total NF-KB and TNF-α were analyzed using commercially available kits (Cat #: KHO037; Thermo Fisher Scientific, USA, and Cat #: DY410; R&D Systems, Minneapolis, MN, respectively).
Western Blot Analysis
Western blot analysis was performed to detect the protein expression levels, as previously described (Badshah et al. 2016). Briefly, protein concentration in the ipsilateral cortex and hippocampus regions were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories) according to the manufacturer’s instructions. Equal volumes of the proteins were separated by 10% SDS polyacrylamide gel electrophoresis, transferred to PVDF membranes followed by incubation in 5% skim milk. The membranes were incubated with primary antibodies (overnight, 4 °C) which are tabulated in Table 1. All the blots were normalized to antibeta actin. After incubation, the blots were developed using horseradish peroxidase-conjugated secondary antibodies and an ECL chemiluminescence system (Atto Corporation Tokyo).
List of antibodies used for immunoblot and immunofluorescence analysis
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List of antibodies used for immunoblot and immunofluorescence analysis
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Tissue Collection and Sample Preparation
For brain section staining, the male mice (n = 5) were anesthetized followed by transcardially perfusion with saline to wash away the blood and then fixed with paraformaldehyde (4%). The brains were separated, postfixed in 20% sucrose solution for 48 h, frozen in OCT compound (A.O USA) and then cut in 14-μm sections using a vibratome (Leica, Germany). The sections were thaw-mounted on probe-on plus charged slides (Fisher) and stored at −80 °C.
Assessment of Brain Lesion Volume
The slides containing the brain tissues were selected and stained with cresyl violet (Desai et al. 2014). The digital photograph of the injured mice brain and that of SP600125 treated was taken and were analyzed using ImageJ software. The injured area of the TBI and TBI plus SP600125 was carefully traced; calculated and obtained the lesion volume by multiplying the sum of the ipsilateral hemispheres area by distance between the sections.
Immunofluorescence Staining
The slides containing hippocampus and cortex tissues were selected for immunofluorescence staining. The slides were dried overnight at room temperature and washed twice with PBS (0.01 mM) for 8–10 min. The slides were incubated with proteinase K for 5 min, rinsed with PBS (0.01 mM) and blocked with 2% normal serum (goat/rabbit) in PBS containing 0.1% Triton X-100. The slides were incubated with primary antibodies overnight at 4 °C including anti-p-JNK, anti-Aβ (B-4), anti-p-Tau (Ser413), anticaspase-3, and anti-SNAP23 from Santa Cruz Biotechnology and anti-APP (Millipore). The slides were then incubated with tetramethylerhodamine isothiocyante–fluorescein isothiocyante (FITC)-labeled secondary antibodies (antigoat, antirabbit and antimouse) at room temperature for 90 min. For double immunofluorescence, the primary and secondary antibodies were applied on the following day. The slides were covered with glass coverslips using mounting medium. Images were taken using a confocal laser-scanning microscope (FluoView FV 1000 MPE).
Measurement of Intracellular ROS
ROS generation in the mouse cortices was measured by a 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) assay as previously described with some modification (Rehman et al. 2016).
Fluoro-Jade B Staining
Fluoro-Jade B (FJB) (cat# AG310, Millipore) Staining was performed according to the manufacturer’s instructions. The slides were dried at room temperature, immersed in 1% sodium hydroxide solution and 80% ethanol for 4–5 min, washed with 70% ethanol and then rinsed with distilled water (DW) for 2–3 min. Next, the slides were kept in 10% MgCl2 solution in the dark for 10 min, rinsed with DW and transferred to a solution containing acetic acid (0.1%) and FJB (0.01%) for 20 min. Xylene was used for 5 min to clear the slides, and the slide were covered with glass coverslips using DPX mounting medium. Images were captured using a confocal laser-scanning microscope (Flouview FV 1000 MPE).
Statistical Analysis
The obtained immunoblot bands were scanned and analyzed through densitometry using Sigma gel software (SPSS Inc.). The immunohistological analysis was performed using ImageJ software. The values were calculated as the mean ± S.E.M. The data were analyzed using one-way ANOVA followed by Student’s t-test. Statistical analysis was done using GraphPad Prism 5 software. Significant differences were determined at P < 0.05. The symbols *P < 0.05, **P < 0.01, and ***P < 0.01 represent significant differences between control and TBI; #P < 0.05, ##P < 0.01, and ###P < 0.01 represent significance differences between TBI and TBI plus SP600125.
Results
SP600125 Inhibited JNK Phosphorylation in the Hippocampus and Cortex of TBI Mice
We first investigated active JNK in the mouse brain homogenates using western blot in 2 models of TBI. Our findings showed increased levels of phosphorylated JNK (p-JNK) in the ipsileteral cortex and hippocampus of the craniotomy-weight drop model of TBI (Fig. 1a) and in rmTBI mice (Fig. 1b) compared with the saline-treated control mice (P < 0.01). However, treatment with SP600125 resulted in a ∼31% reduction of JNK protein at a dose of 10 mg/kg and ∼52% reduction at dose of 20 mg/kg in the brain regions of the TBI groups. Interestingly, SP600125 treatment alone showed no toxic effects in the mouse brains (Fig. 1a). Next, the immunofluorescence results showed increased amounts of p-JNK in the hippocampal region of the mice with brain injury, which was significantly reversed by SP600125 treatment in the hippocampal region of the TBI mouse brains (Fig. 1c). Our results are consistent with a previous study that SP600125 treatment inhibited active JNK in ischemia/reperfusion (I/R) injury (Kim et al. 2016). Our findings suggest that SP600125 strongly inhibited JNK phosphorylation in the TBI mouse brains.
SP600125 ameliorates JNK activation in TBI mouse brains. (a) Representative western blot and histogram analysis of active JNK in the ipsilateral cortex and hippocampus from mice that underwent TBI and TBI plus SP600125 treatment for 7 days post-TBI (n = 8). The same immunoblots were probed using β-actin antibody as a loading control. Sigma gel software was used for quantification of the protein bands. (b) Representative western blot and histograms of active JNK, APP and Aβ in the ipsilateral cortex and hippocampus of mice brain following rmTBI and rmTBI plus SP600125 (10 and 20 mg/kg/daily) treated groups at day 7. (c) Confocal image showing active JNK in the mouse hippocampus. Confocal images were analyzed via ImageJ software. Magnifications X10. The values represent the mean ± SEM. Statistical analysis was done via one-way ANOVA followed by Student’s t-tests. *P < 0.05, **P < 0.01 versus TBI, #P < 0.05, ##P < 0.01 versus TBI+SP600125.
SP600125 Treatment Reduces APP Processing and Aβ Generation in the Cortex and Hippocampus of TBI Mice
Previous studies reported altered APP processing and Aβ deposition in TBI mouse brains and in human brains (Kawai et al. 2013; Mouzon et al. 2014; Yang et al. 2015). We investigated whether JNK inhibition reduced the levels of APP and Aβ in the ipsilateral cortex and hippocampus of TBI mouse brains. Using western blot, we observed increased expression of APP and Aβ accumulation in the ipsilateral cortex and hippocampus of the craniotomy-weight drop model of TBI mice (Fig. 2a) and in rmTBI groups (Fig. 1a) as compared to the saline-treated mice. However, SP600125 treatment reduced the increased expression of APP and Aβ to the baseline in the TBI plus SP600125 group in 2 types of TBI models. The level of Aβ1–42, which is more toxic than Aβ1–40, was measured using ELISA. We evaluated Aβ1–42 at 2 different time points following TBI with increased levels of Aβ1–42 in the ipsilateral cortex and hippocampus regions of TBI mice. However, SP600125 treatment reduced the level of Aβ1–42 on day 3 and 7 in the cortex and hippocampus of the TBI plus SP600125 treatment group compared to the TBI mice (Fig. 3a,b). Consistent with our western blot and ELISA results, the immunofluorescence results also showed increased accumulation of Aβ (FITC-labeled) in the ipsilateral cortex and hippocampus region of TBI mouse brains compared to the saline-treated control mouse group. However, SP600125 treatment significantly reduced the immunoreactivity of Aβ in the cortex and hippocampal regions of the treated mouse group (Fig. 2c). We observed no significant difference between the saline-treated control group and the SP600125-treated group. Furthermore, we examined whether there was any direct interaction of JNK and APP in the TBI mouse brains on day 7 (Ahn et al. 2016). Cortical tissues were immunostained with antibodies that detect p-JNK and APP. The results showed no colocalization of JNK and APP in the TBI cortices on day 7 (Fig. 2b).
SP600125 reduced the expression of BACE1, APP, and Aβ in TBI mouse brains. (a) Representative western blot and histogram analysis of BACE1, APP, and Aβ in the ipsilateral cortex and hippocampus homogenates from mice that underwent TBI and TBI plus SP600125 treatment (n = 8). In each case, β-actin was used as a loading control. Proteins were quantified using Sigma gel software. (b) Confocal microscopy images of the double immunofluorescence of p-JNK (green) and APP (red) in the ipsilateral cortical regions of the 3 groups. (c) Confocal images of Aβ in the cortex and hippocampus of mice that underwent TBI and TBI plus SP600125 treatment. The values are taken from 3 independent experiments. ImageJ software was used for immunohistological analysis. Values represent the mean ± SEM. Statistical analysis was done via one-way ANOVA followed by Student’s t-test. *P < 0.05, **P < 0.01 versus TBI, #P< 0.05, ##P < 0.01 versus TBI+SP600125.
SP600125 treatment reduced Aβ level and regulated the expression of secretases in TBI cortices. (a, b) Aβ1-42 levels in the ipsilateral cortex and hippocampus of TBI and TBI plus SP600125 groups at 2 different time points were measured via enzyme-linked immunosorbent assay. (c) Representative immunoblots and histograms of ADAM17, Neprilysin, and IDE protein expression in the ipsilateral cortical regions of the 4 groups (n = 8). The same immunoblots were probed using β-actin antibody as a loading control. Protein bands were quantified using Sigma gel software. Data are represented as the mean ± SEM. *P < 0.05 versus TBI, #P < 0.05 versus TBI+SP600125.
SP600125 Treatment Corrects Secretase Expression Abnormalities in TBI Mouse Brains
Previous studies reported abnormal expression of several secretases in TBI mouse brains (Blasko et al. 2004; Loane et al. 2009). To further investigate the deregulated expression of various secretases and enzymes involved in AD and the possible underlying mechanism of the inhibitory effects of SP600125, TBI-induced deregulated β-secretase (BACE1) and α-secretase (ADAM17) were analyzed using western blot. Our results clearly revealed increased expression of BACE1 (Fig. 2a) and decreased expression of ADAM17 (Fig. 3c), which were significantly restored by SP600125 treatment in the TBI mouse brains, suggesting that SP600125 reduces Aβ deposition via regulating secretase expression. Furthermore, we investigated 2 of the major enzymes involved in Aβ degradation, insulin degrading enzyme (IDE) and NEP, using western blot analysis. No significant difference was found among the 4 groups (Fig. 3c), suggesting that TBI results in Aβ generation via the amyloidogenic pathway and suppression of the nonamyloidogenic pathway without any effects on Aβ degrading enzymes. Treatment with SP600125 resulted in reduced Aβ via promoting the nonamyloidogenic pathway and suppressing the amyloidogenic pathway in the TBI mouse cortices.
SP600125 Treatment Ameliorates Tau Pathology in TBI Mouse Brains
We investigated the state of tau phosphorylation at Ser413 and Ser404 sites in the TBI mouse cortices using western blot analysis. Consistent with available literature from both animal and human studies (Begum et al. 2014; Olivera et al. 2015; Ojo et al. 2016), we observed increased tau phosphorylation in the TBI mouse brains compared to the saline-treated control mouse brains. Phosphorylated tau was examined using specific antibodies against phosphorylation at the Ser413 and Ser404 sites using western blot (Fig. 4a). Interestingly, SP600125 treatment significantly reversed tau phosphorylation at specific sites in the cortices of the TBI plus SP600125-treated mice. Next, we determined the expression levels of the major kinase glycogen synthase kinase-3b (p-GSK3β) and p-CDK5, which are involved in tau phosphorylation. Our western blot results clearly showed increased expression of p-GSK3β in the TBI mouse cortices, which was significantly reversed by SP600125 treatment in the cortical region. However, we observed no change in p-CDK5 expression. Furthermore, using immunofluorescence, we observed a marked increase in tau phosphorylation at Ser413 in the TBI mouse cortices compared to the saline-treated control mice. Treatment with SP600125 significantly reduced the level of p-tau at Ser413, suggesting that SP6001245 treatment suppresses the amyloidogenic signaling pathway and p-tau phosphorylation in TBI mouse brains (Fig. 4b).
SP600125 treatment reduced phosphorylation of tau protein in TBI mouse cortices. (a) Representative Immunoblots of p-GSK-3β, p-CDK5, p-Tau 413, p-Tau 404, and total tau in the cortex of the 4 groups (n = 8). The same immunoblots were probed using β-actin antibody as a loading control. Protein bands were quantified using Sigma gel software. (b) Quantitative analysis of p-tau confocal microscopy images in gray scale of the p-tau 413 in the cortical regions of the 3 groups. Arrow head represents dapi and arrow indicates expression of p-tau 413. ImageJ software was used for immunohistological analysis. Values represent the mean ± SEM. Statistical analysis was done via one-way ANOVA followed by Student’s t-test. *P < 0.05 **P < 0.01 versus TBI, #P < 0.05, ##P < 0.01 versus TBI+SP600125.
SP600125 Inhibited the Amyloidogenic Pathway in SH-SY5Y Cells
We examined the increased expression of p-JNK, APP and Aβ post-8, 24 and 36-h scratch injury in SH-SY5Y cells. The results showed increased expression of p-JNK, APP, and Aβ in injured cells at 24 h. However, the effects were reversed by treatment with SP600125 (20 μM), suggesting that neuronal injury induces stress that activates JNK and amyloidogenic pathway, which were significantly reversed by SP600125 treatment (Fig. 5a, b). Furthermore, we examined inhibition of active JNK at 24 h by different concentrations (10, 20, and 30 μM) of SP600125. Our results showed significant inhibition of JNK at concentration of 20 μM and 30 μM of SP600125 (Fig. 5c).
SP600125 treatment reduced scratches-induced p-JNK, APP, and Aβ levels in the SH-SY5Y cell line. (a) The protein expression levels of p-JNK. (b) APP and Aβ were assessed via western blot analysis in the SH-SY5Y cell line at 3 different time intervals (8, 24, and 36 h) and SP600125 treatment (n = 3). (c) Protein expression level of activated JNK (24 h) postscratch and treatment of SP600125 (10, 20, and 30 μM). The same immunoblots were probed using β-actin antibody as a loading control. Protein bands were quantified using Sigma gel software. Values represent the mean ± SEM. Statistical analysis was done via one-way ANOVA followed by Student’s t-test. *P < 0.05 versus scratch injury, #P < 0.05 versus scratch injury+SP600125.
SP600125 Treatment Restores BBB Breakdown and Reduced Lesion Volume in TBI Mouse Cortices
Several studies have demonstrated the impact of TBI on BBB breakdown, an important indicator of brain injury (Puvenna et al. 2014). EB content was significantly increased in the TBI mouse brains. However, SP600125 treatment significantly reversed the effects of BBB leakage, as revealed by EB content (Fig. 6a). Lesion volume was quantified in cresyl violet-stained brain sections following TBI at day 7. SP600125 treatment reduced the lesion volume in TBI plus SP600125 group as compare to TBI group (Fig. 6b). Next, we examined the BBB breakdown in the TBI mouse cortices using specific antibodies including claudin-5 and ZO-1 in western blot. The results indicated decreased expression of claudin-5 and ZO-1, which was significantly reversed by SP600125 treatment (Fig. 6d). Our results are consistent with previous results that inhibition of JNK with a specific inhibitor prevented BBB breakdown (Chen et al. 2012a, 2012b). These findings suggested that SP600125 treatment prevents breakdown of the BBB and restored the transcriptional changes and inflammatory cytokine infiltration that lead to neuronal degeneration. Furthermore, we analyzed whether neuronal injury has any direct effects on BBB break down, using transaction in vitro model of injury in brain endothelial cells (bEnd.3 cells). our western blot results showed no changed in the expression level of claudin-5 and ZO-1 at different time intervals (Fig. 6e), suggesting that BBB breakdown may be due to TBI-induced neuroinflammation in the mouse brains, as suppression of neuroinflammation can ameliorates BBB injury (Han et al. 2016). From these observations, we concluded that reversal of BBB breakdown in TBI mouse brains may be due to inhibition of neuroinflammation via JNK dependent mechanism.
Effect of SP600125 treatment on BBB permeability and lesion volume in TBI mouse brains. (a) Mice received i.p. injections of SP600125 (20 mg/kg/i.p.) 30 min after and 1 h before EB injection. The permeability of the brain was examined by i.v. injection of EB (40 mg/kg) at 24 h after injury. The EB was measured at 2 h after circulation. The amount of EB was determined in the TBI and TBI plus SP600125 groups. (b) Representative images of cresyl violet-stained coronal brain sections from TBI and TBI+SP600125 at day 7 post-TBI. (c) A significant increase in brain water content was detected in the TBI mouse brains compared with the control saline-treated mouse brains. SP600125 treatment reduced water content in the ipsilateral side of the mouse brains compared with the TBI mouse brains. (d) Representative western blots and a relative density histogram of claudin-5, ZO-1, and β-actin in the cortex. Homogenates from the TBI and TBI plus SP600125 groups at day 7 post-TBI. β-Actin antibody was used as a loading control. Protein bands were quantified using Sigma gel software. (e) Representative western blots and a relative density histogram of cloudin-5, ZO-1, and β-actin in bEnd-3 cell line using in vitro transaction model of TBI and TBI+SP600125 (10, 20, and 30 μM). Values represent the mean ± SEM. Statistical analysis was done via one-way ANOVA followed by Student’s t-test. *P < 0.05 versus TBI, #P < 0.05 versus TBI+SP600125.
Effect of SP600125 on Posttraumatic Brain Edema Formation
Brain edema formation was analyzed 24 h post-TBI. The results demonstrated that SP600125 treatment significantly reduced the level of brain water content compared to the TBI mouse brains. We observed no significance difference between control and SP600125-only-treated mouse brains (Fig. 6c).
SP600125 Treatment Inhibited Apoptotic Neurodegeneration
We examined neuronal apoptosis in the TBI cortices, as TBI leads to neuronal apoptosis in the brain (Chen et al. 2012a, 2012b). The western blot results showed increased expression of apoptotic markers including cleaved caspase-3, Bax, PAPR1 (DNA damage marker) and reduced expression of Bcl2 in the TBI mouse cortices. However, SP600125 treatment inhibited the apoptotic markers in the TBI plus SP600125-treated group (Fig. 7a). Next, our immunofluorescence results showed increased expression of capsase-3 in the TBI cortices, which was significantly reduced by SP600125 treatment (Fig. 7b). Furthermore, we observed increased FJB-positive cells in the TBI cortices. However, the number of FJB-positive neurons was significantly reduced in the TBI mouse cortices that were treated with SP600125. These findings suggest that SP600125 is significantly involved in reducing TBI-induced neuronal degeneration in TBI brains (Fig. 7c).
SP600125 treatment attenuated TBI-induced neuronal apoptosis in the mouse cortices. (a) Representative Immunoblots and a relative density histogram of PARP-1, cleaved caspase-3 Bax and Bcl2 in the cortices of the TBI and TBI plus SP600125 groups (n = 8). The same immunoblots were probed using β-actin antibody as a loading control. Protein bands were quantified using sigma gel. (b) Confocal microscopy images of caspase-3 in the cortical regions of the 3 groups. (c) Confocal microscopy images of FJB staining in the cortical regions of the 3 groups. ImageJ software was used for immunohistological analysis (n = 3). (d) SP600125 treatment for 7 days reduced ROS level in the ipsileteral cortex of the TBI mice brain. Values represent the mean ± SEM. Statistical analysis was done via one-way ANOVA followed by Student’s t-test. *P < 0.05, **P < 0.01 versus TBI, #P < 0.05, ##P < 0.01 versus TBI+SP600125.
SP600125 Treatment Reduced Oxidative Stress in the TBI Cortices
Oxidative stress is the main hallmark of neurodegeneration resulting from reactive oxygen species (ROS) following TBI (Bains and Hall 2012). We therefore assessed ROS generation in the TBI cortices on day 7. The results clearly demonstrated elevated ROS level in the TBI cortices, which was significantly reversed by SP600125 treatment, suggesting that JNK inhibition might be protective against ROS-induced neurodegeneration in TBI brains (Fig. 7d).
SP600125 Treatment Inhibited Inflammatory Mediators in the TBI Mouse Cortices
Several studies have reported inflammatory responses in the cortex and hippocampus regions of TBI mouse brains (Lloyd et al. 2008; Bachstetter et al. 2015; Webster et al. 2015). It is also believed that Aβ induces inflammatory responses via activation of JNK (Wang et al. 2011). We therefore assessed the inflammatory response in TBI mouse cortices via western blot analysis. Our data clearly showed marked increases in inflammatory markers including p-NF-KB, TNF-α, IL-1β, and inducible nitric oxide synthase (iNOS) in the TBI mouse cortices compared to the saline-treated mice, which were significantly reversed by SP600125 treatment (Fig. 8a). We further confirmed the inflammatory response by measuring inflammatory mediators using Elisa that include NF-KB 65 and TNF-α in the ipsilateral cortices of the TBI mice, which were significantly increased in the TBI cortices. However, the levels were significantly reversed by SP600125 treatment (Fig. 8b,c). Furthermore, our confocal microscopy results also indicated increased expression of IL-1β in the TBI mouse cortices, which was significantly inhibited by SP600125 treatment (Fig. 8d). These findings indicate that treatment with SP600125 is largely effective against the TBI-induced inflammatory response that occurs in injured mouse cortices.
SP600125 treatment inhibited TBI-induced neuroinflammation in the mouse cortices. (a) Representative Immunoblots and a relative density histogram of p-NF-KB, IL-1β, and TNF-α in the ipsilateral cortices of the TBI and TBI plus SP600125 groups (n = 8). The same immunoblots were probed using β-actin antibody as a loading control. Protein bands were quantified using Sigma gel software. (b,c) NF-KB and TNF-α level in the ipsilateral cortex of TBI and TBI plus SP600125 groups at 2 different time points were measured via enzyme-linked immunosorbent assay. SP600125 inhibited the levels of NF-KB and TNF-α in the ipsilateral cortex on 7-day postinjury. (d) Confocal microscopy images in gray scale of IL-1β in the cortical regions of the 3 groups. Arrow head represents dapi and arrow indicates expression of IL-1β. ImageJ software was used for immunohistological analysis. ImageJ software was used for immunohistological analysis. Values represent the mean ± SEM. Statistical analysis was done via one-way ANOVA followed by Student’s t-test. *P < 0.05, **P < 0.01 versus TBI, #P < 0.05, ##P < 0.01 versus TBI+SP600125.
SP600125 Treatment Regulates P-p38, p-Akt, and p-Erk1/2 Levels In Vivo and In Vitro
To further elucidate the role of SP600125 on p38, ERK and AKT phosphorylation, we examined the effects in TBI mouse cortices and in SH-SY5Y neuronal cells. P-p38 a member of mitogen-activated protein kinase (MAPK) is activated in response to several forms of stress (Reynolds et al. 2000). Our immunoblot results clearly demonstrated the increase expression levels of P-p38 in the ipsilateral cortices of the TBI mice with significant reversal in SP600125-treated mice group. Similarly, we investigated the expression levels of p-ERK1/2 and p-AKT (ser413) following TBI. Consistent with a previous observation (Cohen-Yeshurun et al. 2011), we observed a significant decline in the levels of p-PERK1/2 and p-AKT in the epsilateral cortices. Interestingly, treatment of SP600125 reversed the effects and regulated the expression levels of these proteins. From our observation it is interesting that SP600125 regulate other kinases either directly or indirectly in TBI mice brain (Fig. 9a).
SP600125 regulates the expression levels of P-p38, p-Akt, and p-Erk1/2 levels in vivo and in vitro. (a) Immunoblots and histogram represents P-p38, p-Akt (Ser413), and p-Erk1/2 expression levels in the ipsilateral cortices of TBI and TBI plus SP600125 group (n = 8). (b) Representative Immunoblots and histogram showing the expression level of P-p38, p-Akt (Ser413), and p-Erk1/2 in SH-SY5Y cell line using in vitro transaction model of TBI and TBI+SP600125 (20 μM) at 3 different time intervals. The results were obtained from 3 independent experiments (n = 3). All the blots were probed with β-actin antibody as a loading control. The immunoblots of P-p38, p-Akt (Ser413), and p-Erk1/2 were quantified with Sigma gel software. Values represent the mean ± SEM. Statistical analysis was done via one-way ANOVA followed by Student’s t-test. *P < 0.05, **P < 0.01 versus TBI, #P < 0.05, ##P < 0.01 versus TBI+SP600125.
Furthermore, we investigated the effect of SP600125 in SH-SY5Y neuronal cells using scratch injury model. We examined the expression level of P-p38, p-AKT (ser413), and p-ERK1/2 post-8, 24 and 36-h scratch injury in SH-SY5Y cells. We observed highest expression levels of P-p38, post-24, and 36-h scratch injury that was significantly reversed by SP600125 treatment at a dose of 20μM. Next, we examined a significant decline of p-AKT (Ser413) post-24-h scratch injury in SH-SY5Y cells and these effects were reversed by SP600125 treatment post-24-h. However, we observed no significant difference post-8 and 36-h scratch injury. Furthermore, we examined the effect of SP600125 treatment on p-ERK1/2. The levels of p-ERK1/2 were significantly declined post 8-h and 24-h injury. SP600125 treatment showed no significant elevation of p-ERK1/2 post 8-h scratch injury. However, a dramatic increase of p-ERK1/2 was observed post-24-h scratch injury in the SP600125-treated group (Fig. 9b), suggesting that SP600125 treatment inhibits the activation of active JNK as well as regulates activity of other kinases and regulates the prosurvival processes in the injured neuronal cells.
SP600125 Treatment Reversed Synaptic Protein Loss in the TBI Mouse Cortices
Several studies have reported that TBI contributes to severe synaptic protein loss in animal brains (Ansari et al. 2008; Merlo et al. 2014). To investigate the synaptic marker loss in TBI mouse cortices, we examined synaptic markers via western blot analysis and confocal microscopy. Our results showed that synaptic proteins including SNAP23, SNAP-25, and synaptophysin were significantly downregulated in the TBI mouse cortices compared to the saline-treated control mice. Interestingly SP600125 significantly reversed these effects and restored the lower expressions of these synaptic proteins (Fig. 10a). The immunofluorescence results indicated up-regulated SNAP23 protein levels in the TBI plus SP600125-treated mice group. These findings suggest that SP600125 treatment reverses synaptic loss via regulation of oxidative stress or neuroinflammation in the mouse cortex (Fig. 10b).
SP600125 Treatment Improved Motor Function and Rescued Memory Impairment in the TBI Mouse Brains
Previous studies reported that TBI mice exhibit motor function abnormalities and severe abnormalities in spatial learning and memory in the MWM test (Zhao et al. 2013; Rubovitch et al. 2015). Mice were tested on a beam walk before injury and postinjury on days 1, 3, and 7. Our results indicated significant differences between the saline-treated and TBI groups on days 1, 3, and 7. Notably, SP600125 treatment improved motor function abnormalities after TBI with significant difference between the TBI and TBI plus SP600125-treated groups (Fig. 11g).
SP600125 reversed TBI-induced synaptic dysfunction in the mouse cortices. (a) Representative immunoblots and a relative histogram of PSD95, SNAP23, and synaptophysin in the ipsilateral cortices of the TBI and TBI plus SP600125 groups (n = 8). The same immunoblots were probed using β-actin antibody as a loading control. Protein bands were quantified using Sigma gel. (b) Confocal microscopy images in gray scale of SNAP23 in the cortical regions of the 3 groups Arrow head represents dapi and arrow indicates expression of SNAP23. ImageJ software was used for immunohistological analysis. Values represent the mean ± SEM. Statistical analysis was done via one-way ANOVA followed by Student’s t-test. *P < 0.05, **P < 0.01 versus TBI, #P < 0.05, ##P < 0.01 versus TBI+SP600125.
In the MWM test, our results revealed that the TBI mice showed spatial learning deficits as indicated by longer escape latencies during the training days compared to the saline-treated control mice. However, SP600125 treatment reversed these abnormalities and improved the spatial learning performance of mice in the TBI plus SP600125 group, as indicated by shorter latencies (Fig. 11a). Figure 11c shows the increased latencies of the TBI mice during the probe trial on day 5 compared to the saline-treated mice, which was significantly reversed by SP600125 treatment. The representative histograms showing the short time spent in the target quadrant and the fewer number of platform crossings by the TBI mice during the probe trial on day 5 compared with the saline-treated mice. However, SP600125 treatment reversed these effects and improved the memory performance, as indicated by the increased number of platform crossings (Fig. 11e) and the longer time spent (Fig. 11f) in the target quadrant in the TBI plus SP600125-treated group. Furthermore, we examined the mean swim speeds during training days and in the probe trial. We confirmed the reduced swimming speeds in the TBI mice group compared to the saline-treated group, suggesting that TBI may induce motor problems in mice. However, SP600125 treatment reversed the abnormalities and enhanced the mean swim speed (Fig. 11b) in training trial as well as in the probe trail (Fig. 11d). Interestingly, we observed no significant differences in escape latencies, the time spent in the target quadrant or the number of platform crossings between the saline-treated group and the SP600125-treated group, suggesting that SP600125 has no direct effect on learning and memory performance. Overall, these data indicated that, in traumatic brain injuries, inhibition of active JNK via its inhibitor SP600125 can significantly improve cognitive deficits in TBI mice without any major side effects (Fig. 11).
Treatment with SP600125 reversed cognitive deficits in TBI mice. Results of the hidden platform tests performed for 4 consecutive days in the MWM. (a) The mice with TBI had longer escape latencies compared to the saline-treated mice, whereas SP600125-treated TBI mice showed reduced escape latencies compared to the TBI mice (P < 0.05). (b) Histogram of the comparison of the swim speeds during the training days. (c) Comparison of the latency (s) on day 5 during the probe trial (P < 0.05). (d) Histogram showing the comparison of the swim speeds during the probe trial (P < 0.05). (e) The number of crossings over the hidden platform location on day 5 (P < 0.05). (f) The time spent in the target quadrant among the 4 groups (P < 0.05). (g) Represents the number of foot faults. The data are shown as the mean ± SEM (n = 8). *P < 0.05 versus TBI, #P < 0.05 versus TBI+SP600125.
Discussion
The search for an effective treatment of TBI has been extensively investigated, as TBI has been recognized as a multifactorial disorder. Therefore, multifunctional approaches are likely to be effective for TBI patients. In the present study, we unveiled the critical role of active JNK and its activation of the intracellular signaling cascade that leads to synaptic dysfunction and neurodegeneration in TBI mouse brains. The major observations of the present study are that inhibition of active JNK by SP600125 markedly reduced several pathological events in TBI mouse brains that included APP expression, Aβ production, Aβ-synthesizing enzymes, phosphorylation of tau proteins, neuroinflammation, BBB breakdown, neurodegeneration, and synaptic loss. These findings clearly establish that activated JNK plays a key role in several pathological events in TBI mouse brains and its inhibition may have disease-modifying potential for treating TBI.
Activated JNK has been reported in various neurodegenerative disorders including AD and PD.
Several studies have reported aberrant JNK activation in AD patients and in AD transgenic mouse brains and inhibition of active JNK with specific JNK inhibitors alleviated JNK mediated neuronal degeneration (Shoji et al. 2000; Zhu et al. 2001; Braithwaite et al. 2010; Chambers et al. 2013). However, whether inhibition of active JNK can prevent the development of different pathological events in TBI mouse brains remains to be fully elucidated. The current study revealed increased activation of JNK in the ipsilateral cortex and hippocampus of 2 different types of TBI mice compared with saline-treated mice. However, administration of SP600125 in TBI mice for 7 days strongly inhibited active JNK in the above mentioned regions of the TBI brains. Previous studies reported strong inhibition of active JNK via SP600125 in AD transgenic mouse hippocampi and cortices (Zhou et al. 2015). Inhibition of active JNK via SP600125 markedly reduced the level of Aβ in the ipsilateral cortex and hippocampus of TBI mice, as indicated by biochemical and histological analysis. Biochemical analysis indicated increased levels of Aβ in the TBI mouse brains. However, SP600125 treatment reduced the level of Aβ in the mouse brains. Immunohistological results also showed increased immunoreactivity of Aβ in both ipsilateral regions of the TBI brains that was significantly reduced by SP600125 in both regions of the TBI brains. We then examined the direct interaction of JNK and APP in TBI mouse cortices on day 7, as JNK might be a major kinase for APP phosphorylation (Ahn et al. 2016). We found no colocalization of JNK and APP in the TBI mouse cortices on day 7, implicating that JNK regulates APP expression via BACE1 expression regulation, and previous studies have demonstrated the role of JNK in the expression of BACE1 (Tamagno et al. 2005; Guglielmotto et al. 2011). We found increased expression of BACE1 in the TBI mouse cortices and hippocampi, which was markedly inhibited by SP600125 treatment. More interestingly, we found decreased levels of ADAM17 in the TBI mouse cortices, and potentially restored levels of ADAM17 in the SP600125-treated TBI cortices, suggesting that SP600125 reduces Aβ deposition by regulating secretase expression. However, the unchanged expression levels of the 2 Aβ degrading enzymes, IDE and NEP, indicate that the observed Aβ reduction was not due to Aβ clearance in the TBI cortices; however, there was a slight increase in IDE with no significance difference in the TBI mouse cortices compared to the control saline-treated mice, presumably as a neuroprotective mechanism in the TBI mouse cortices. Overall, these findings showed that the remarkable effect of SP600125 treatment on Aβ reduction in TBI mouse brains may be due to suppression of the amyloidogenic pathway and promotion of the nonamyloidogenic pathway without the clearance of Aβ.
Hyperphosphorylation of tau protein is a major pathological factor involved in AD (Querfurth and LaFerla 2010; Ando et al. 2016), and previous studies reported hyperphosphorylated tau protein in TBI mouse brains (Zhang et al. 2015). We explored TBI-induced tau phosphorylation and its possible reversal via SP600125 in TBI mouse cortices. Our immunoblot results showed significant increases in the phosphorylation of specific tau epitopes in the ipsilateral cortex of TBI mice, which was significantly reduced by SP600125 treatment. Previous studies reported that TBI accelerated tau phosphorylation in a 3xTg-AD model, which was significantly reduced by D-JNKi1, a specific JNK inhibitor peptide (Tran et al. 2012). To elucidate the molecular mechanism of reduced tau phosphorylation via SP600125, we assessed CDK5 and GSK3β, the main kinases responsible for tau phosphorylation. We found no change in p-CDK5; however, p-GSK3β was significantly increased in the TBI mouse cortices and was significantly normalized to baseline upon treatment with SP600125 in the TBI mouse cortices. Our findings are similar to those previously reported (Dash et al. 2011; Zhang et al. 2015). Logically, we can hypothesize from the observation that reduced tau phosphorylation may be attributable to its suppression of JNK activation, as some Aβ species may be involved in tau phosphorylation via the JNK pathway (Ma et al. 2009). These findings may provide novel insight related to the active JNK pathway that may involve tau phosphorylation in an animal model of TBI, as tau phosphorylation is a critical player in the progression of TBI-induced neurodegeneration (Yang et al. 2016).
The BBB is a semipermeable membrane that is crucial for the maintenance of brain homeostasis. Tight junction proteins including claudin, occludin and zonula occludin (ZO-1) are important in the maintenance of BBB integrity. However, BBB breakdown leads to cytokine infiltration, which enhances neuronal vulnerability and plays a critical role in the progression of TBI-induced neurodegeneration (Shlosberg et al. 2010; Bachstetter et al. 2015; Webster et al. 2015). In the present study, we examined BBB breakdown, elevated Brian water content, enlarged lesion volume and the increased expression levels of inflammatory mediators in TBI cortices, and these effects were significantly inhibited by treatment with SP600125. Previous studies reported that SP600125 treatment restores tight junctions, attenuates BBB breakdown and induces anti-inflammatory activity, as JNK triggers glial cells and stimulates inflammatory cytokines (Waetzig et al. 2005; Chen et al. 2012a, 2012b; Graczyk 2013). Herin, we observed no change in the expression levels of the tight junction proteins using an in vitro trasection model of injury in brain endothelial cells (bEnd.3 cells). From these observations we concluded that reversal of the BBB breakdown may be due to inhibition of neuroinflammation via JNK dependent mechanism in TBI mice brain. Thus, the inhibitory effect of SP600125 on inflammatory responses in TBI mouse brains could be mainly attributed to its direct anti-inflammatory effects through inhibition of active JNK and or through inhibition of BBB breakdown.
Increased level of neuroinflammation is critical in the progression of TBI-induced neurodegeneration (Lloyd et al. 2008; Bachstetter et al. 2015). In the present study, using biochemical and histological measurements, we observed increased inflammatory responses in the ipsilateral cortices of TBI mice. Importantly, the effects were strongly inhibited by SP600125 treatment, as indicated by the reduced levels of p-NF-KB, IL-1β, TNF-α and iNOS in the ipsilateral cortices of the TBI mouse brains.
Emerging evidence has demonstrated neuronal apoptosis in TBI brains (Chen et al. 2014). Oxidative stress is the main hallmark of neuronal apoptosis. Our results clearly revealed the TBI-induced increased oxidative stress, increased expression of apoptotic markers (cleaved caspase-3, Bax and cleaved PARP-1), and reduced expression level of Bcl2, ERK and AKT that are involved in several prosurvival and antiapoptotic processes which were significantly attenuated by SP600125 treatment. Furthermore, the FJB-positive cells represent dead neuronal cells in the ipsilateral cortices of the TBI mice, which were significantly reversed by SP600125 treatment.
Increased synaptic loss is another major hallmark of TBI that is associated with cognitive deficits (Ansari et al. 2008). We therefore examined synaptic markers and memory impairment in the TBI mice. Our results showed decreased expression of synaptic proteins including PSD95, SNAP23, and synaptophysin in the TBI mouse cortices. Interestingly, the decreased synaptic proteins were significantly restored to basal levels in the saline-treated mice. Furthermore, progressive cognitive and motor dysfunctions are associated with TBI, which are a major cause of disability and poor quality of life. We analyzed cognitive dysfunction using the MWM test and the BWT for motor dysfunction. Our results revealed that TBI induces cognitive dysfunction, as evident in the MWM test. However, treatment with SP600125 reversed the effects and regulated cognitive dysfunction. We assessed motor dysfunction after TBI for up to 7 days using the BWT and confirmed that TBI-induced motor function deficits compared to saline treatment. Importantly, SP600125 significantly reversed the motor function deficits up to 7 days post-TBI. A growing body of evidence has shown that Aβ, phosphorylated tau and neuroinflammation are strongly associated with cognitive dysfunction in TBI mouse brains (Kabadi et al. 2015; Kondo et al. 2015). Our results are similar to previous reports that inhibition of JNK is involved in the restoration of cognitive dysfunction induced by Aβ1–42 or in APP/PS1 transgenic mice (Ramin et al. 2011; Zhou et al. 2015).
In conclusion, this study offers momentous preclinical evidence that inhibition of active JNK by treatment with a SP600125 could efficiently reverse the pathological effects following TBI. Treatment with SP600125 can effectively restore cognitive deficits, reduces Aβ, p-tau protein accumulation and restored the multipathological conditions in TBI-induced mouse cortices and hippocampi. Our findings clearly demonstrated that inhibition of JNK via SP600125 would be a new disease-modifying therapeutic strategy to treat TBI-induced neuronal dysfunction as well as AD and PD associated neurodegeneration. Further preclinical research is needed for the development and specificity of SP600125 towards a broad range of kinases before initiating the clinical trials of JNK inhibition in human subjects with neurodegenerative consequences related to TBI, AD and PD. In addition, combating these devastating neurodegenerative diseases, the other pivotal kinases such as ERK, CDK5, GSK3β, and p38 MAPK needs to be considered.
Funding
This research work was supported by the Brain Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (2016M3C7A1904391).
Notes
We thank Dr Shahid Ali Shah, Dr Haroon Badshah and Tahir Ali, for their valuable technical assistance, Sohail Khan for animal caring and support for behavioral study. Conflict of Interest: None declared.
