Up‐regulation of CHMP4B alleviates microglial necroptosis induced by traumatic brain injury

Abstract Microglial cells are key component of central nervous system (CNS) and mediate the immune response of the brain under physiological or pathological conditions. It tends to activate into a pro‐inflammatory M1 phenotype after traumatic brain injury (TBI) and promote secondary brain damage. Recently, necroptosis was found to promote microglial activation and neuroinflammation after TBI. However, the mechanism and specific interventions of microglial necroptosis after TBI remain poorly investigated. Here, we reported that overexpress the charged multivesicular body protein 4b (CHMP4B) which is a core member of the endosomal sorting required for transport complex III (ESCRT‐III) significantly decreased the level of necroptosis in microglia, improved neurological function recovery and protected against cell death after TBI. Further investigation showed that forkhead transcription factor O1 (FOXO1) was a crucial transcription factor that increased CHMP4B transcription by binding to the promoter region, thereby inhibiting necroptosis in microglia. Collectively, our findings demonstrated that CHMP4B relieved microglial necroptosis and neuroinflammation after TBI, and promote the recovery of nerve function. FOXO1 is an important factor in promoting CHMP4B expression. This study provides the novel viewpoint for TBI prevention and treatment.

cell death. In contrast, necrosis is thought to be an unregulated form of cell death. 6,7 However, with the progress of research, increasing evidence has shown that necrosis can also be regulated, similar to apoptosis, which is now called necroptosis. 7 Necroptosis is initiated by activation of tumour necrosis factor alpha (TNFα) and/ or Fas, which differentiates it from caspase-dependent apoptosis. 8 Necroptosis was recently observed in many nervous system pathologies, including brain trauma and cerebral ischaemia. 9,10 Long after the discovery of necroptosis, phosphorylated mixed lineage kinase domain-like pseudokinase (p-MLKL)-labelled cell membranes were considered to indicate an irreversible cell death process that was not regulated by other factors. 11,12 However, current research has demonstrated that MLKL localizes to sites of broken membrane bubbles, where it recruits ESCRT-III components, including CHMP4B, which in turn reduces cell membrane damage caused by p-MLKL. 13,14 Some studies have proved that necroptotic cells induce a robust inflammatory response, in contrast to apoptotic cells, which are generally not associated with inflammation. [15][16][17] Microglia are intrinsic immune effector cells in the central nervous system (CNS) that play important roles in both physiological and pathological processes.
Studies have shown that microglia undergo necroptosis in the course of various nervous system injuries, such as intracerebral haemorrhage, 18 spinal cord injury 19 and ischaemic stroke. 20 Therefore, inhibiting microglial necroptosis may alleviate neuroinflammation and improve neuronal survival after traumatic brain injury.
Forkhead transcription factor O1 (FOXO1) is a member of the forkhead box-containing transcription factor O family that participates in various cell physiological and pathological processes, 21 including glycogenolysis and gluconeogenesis. 22,23 Except in glucose metabolism, FOXO1 has been shown to have a protective effect in organ damage. Some studies have reported that FOXO1 can alleviate cell damage, lower reactive oxygen species (ROS) levels and inhibit cell apoptosis in various pathological condition. 24,25 However, the role of FOXO1 in TBI and the underlying cellular and molecular mechanisms remain unclear.
In this study, we investigated the role of CHMP4B in brain injury. Interestingly, CHMP4B is involved in inhibiting necroptosis in microglia, in turn alleviating neuroinflammation and improving neuronal survival. Mechanistically, we found that FOXO1 regulates the expression of CHMP4B, in which knockdown of endogenous FOXO1 significantly reduces the transcription of CHMP4B. Thus, FOXO1 participates in the anti-necroptotic effect of CHMP4B.

| Patients
The Department of Neurosurgery at the First Affiliated Hospital of Nanjing Medical University provided human brain tissue (Table 1), which was approved by the Institutional Review Board. Brain tissues resected from patients with severe TBI were snap-frozen and stored in liquid nitrogen until assay. Brain tissues resected from epilepsy patients were used as control. The Ethics Committee of Nanjing Medical University approved the use of human brain tissue, and all procedures were conducted in accordance with approved guidelines. The participant's explicit permission was obtained, and the patient provided informed consent.

| Animals
Experimental Animal Central of Nanjing Medical University provided adult male C57BL/6J mice (25 ± 2 g). We arranged all mice five per cage under humid environment and at regulated temperature

| Experimental TBI model
As described previously, the TBI groups underwent surgery to produce a controlled cortical impingement model. 26 Mice underwent anaesthesia with 4% isoflurane in 70% N 2 O and 30% O 2 . Anaesthesia was maintained with 2%-3% isoflurane. Then, in the left parietotemporal cortex (the relative coordinates centre of craniotomy to bregma: 1.5 mm posterior and 2.5 mm lateral), we performed a 5 mm craniotomy with an electric drill. For the sham groups, only the dura mater was exposed. In the TBI groups, the exposed dura mater was struck by a 3-mm metal impounder at 6 m/s velocity with 1 mm set depth and 100-ms impacting period. We closed the scalp by suturing, and then mice were caged. cortex around the lesion was collected and stored in 4% paraformaldehyde or at −80°C for the following study.

| Primary cortical neuronal culture
The SPF grade one-day-old C57BL/6J mice were used for this study. The mice were decollated immediately after sterilizing the skin with 75% alcohol, and the intact brain was immersed in pre-cooled DMEM/F12 medium. The meninges and blood vessels were removed from a thin layer of cerebral cortex and placed in a fresh pre-cooled DMEM/F12 medium, and then minced into small pieces with ophthalmic scissors.

| Adeno-associated virus (AAV) intracerebroventricular injection
According to the process described in the previous study. 27 We an-

| BV2 cell culture, transfection and in vitro injury model
The BV2 cell line was purchased from the Chinese Academy of Sciences Cell Bank and was cultured in Dulbecco's modified Eagle's medium (DMEM). All of the medium were mixed with penicillin, streptomycin and 10% foetal bovine serum (FBS) (Gibco).
The cultivation environment was maintained at 37°C and 5% CO 2 .
Generally, the BV2 cells were used for the following experiments when the growth density reaches 70%-80%. The BV2 cells were transfected with the indicated plasmids using Lipofectamine 3000 (Invitrogen). After 24 hours, we treated BV2 cells with 100 μm glutamate (Glu) for inducing cellular injury for 24 hours according to the study protocol.

| Western blotting
Cytosolic, cytomembrane and overall extracts of protein were requested following the previous description. 28,29 Proteins at the site of trauma and on the contralateral side were harvested at different time and extracted using radioimmunoprecipitation assay (RIPA) buffer on ice (Sigma) and then centrifuged for 15 minutes 1000 g 4°C. The proteins underwent separation using 12% SDS-PAGE gel and placed to polyvinylidene fluoride (PVDF) membranes (Millipore).
Membranes underwent incubation with 5% (w/v) non-fat dried milk for 2 hours at ambient temperatures and then incubated using Abcam) and FOXO1 (1:1000, ab39670; Abcam). Using an enhanced chemiluminescence detection system (GE Healthcare), we ascertained bound antibodies. Applying ImageJ software (National Institutes of Health), we studied the achieved bands' optical density.

| TUNEL assay
Apoptosis in 8-μm frozen brain sections was examined by TUNEL

| Brain water content
After sacrifice, the mice brain underwent immediate removal and weighing process. Subsequently, it underwent drying process at 70°C for 72 hours to achieve the dry weight. The brain water content was obtained based on water content (%) = [(wet weight − dry weight)/wet weight] × 100%. 30

| Morris water maze
Each experimental animal was taken to a behavioural room kept at 25°C (housing area during training period). After a two-day adaptation period, the training lasted seven days. The water was made opaque by the addition of milk. In brief, the mice were given 90 seconds to find the platform, and if the mouse could not reach the platform within 90 seconds, the researcher placed it on the platform for 15 seconds for spatial learning and orientation memory training. The first 5 days were used for training, and four trials were conducted, and the mice were released from the four different quadrants for each trial. On day 6, the time to the platform from each quadrant was recorded. If the platform was not found, a time of 90 seconds was recorded. On day 7, the platform was removed, and the time spent in the target quadrant and the number of times the mouse crossed the target platform location was recorded as indexes of learning and memory. In the study, a researcher with no knowledge of each mouse's experimental groups performed the behavioural tests.

| Rotarod test
The mice were allowed to walk for three days on a rotating rod at 10 revolutions every min (RPM). On the 4th day, we assessed the ability of each groups of mice to walk on rotarod. Rotarod was accelerated from 4 rpm to 40 RPM for 5 minutes, and we recorded the time for each mouse to fall off the rod to be the retention time.
Every mouse was tested three times, and the data were statistically analysed.

| Cellular fractionation
According to the manufacturer's instructions, nuclear/plasma separation kits (Norgen Biotek Corp.) were used for nuclear/plasma separation. The efficiency of cellular fractionation was tested by cytoplasmic and nuclear markers TH and UBF, respectively.

| RNA extraction and qRT-PCR
Total RNA was extracted from brain and BV2 cell line using TRIZOL reagent (Invitrogen). cDNA was produced using the PrimeScript-RT Reagent Kit (Takara). qRT-PCR was performed using the SYBR Green PCR kit

| Dual-luciferase gene reporter assay
In order to analyse the transcriptional activity of CHMP4B, we gen-

| Chromatin immunoprecipitation (ChIP)
Purification of sonicated nuclear lysates and immunoprecipitation were performed using the Simple Chip Enzymatic Chromatin IP kit (Cell Signaling Technology) according to manufacturer's instructions.

| Flow cytometric analysis
Nerve cells were cultured in 6-well plates and subjected to different treatments. Cells were trypsinized with 0.25% trypsin (without EDTA), centrifuged at 160 g for 5 minutes and resuspended in 300 μL of binding buffer. Subsequently, the cell suspension was stained with 3 μL Annexin V-APC and 3 μL 7-AAD (KGA1023, KeyGEN biotech). After 20 minutes incubation, cells were analysed using a flow cytometer.

| Statistical analysis
Results of images and data are reported to be means ± SEM from more than three separated experimental processes. Grey levels were detected with ImageJ. Variance underwent two-way ANOVA analysis for determining the diversification between treating processes, and non-parametric t test was adopted for comparing the diversifications of the two groups. P< .05 was considered statistically significant

| Necroptosis after clinical and experimental TBI
At present, it is generally accepted that receptor-interacting protein kinase-3 (RIPK3) and phosphorylated mixed lineage kinase domainlike pseudokinase (p-MLKL) constitute the core of the necroptosis machinery termed as the necrosome. [26][27][28] We hypothesized that the severity of CNS damage after TBI is related to the levels of RIP3 and p-MLKL.
We used Western blot analysis to compare the levels of RIP3 and p-MLKL in severe trauma patients with intractable intracranial pressure and control patients. Obviously, increased levels of RIP3 and p-MLKL were observed in trauma patients compared with controls ( Figure 1A,B). Furthermore, immunohistochemistry showed that RIP3 and p-MLKL levels were significantly higher in trauma patients than controls ( Figure 1C).
In TBI, necroptosis has been found to be involved in the process of CNS tissue damage. However, the temporal peak of necroptosis may vary because of differences in the mouse models used. Here, we found that the level of necroptosis varied over time in our experimental TBI model. Western blot analysis showed that RIP3 and p-MLKL protein contents in the traumatized cortex were markedly increased at 6 hours after TBI compared with the sham groups.
However, 24 hours after TBI, RIP3 and p-MLKL protein levels decreased markedly compared with 6 hours ( Figure 1D,E). These data show that necroptosis peaks at 6 hours in the traumatized cortical area after TBI.
In view of the above results, the CHMP4B protein level was detected at 6 hours in the traumatized cortex. Western blotting and immunohistochemistry revealed that CHMP4B protein levels were almost threefold higher than in sham mice ( Figure 1F-H).

| CHMP4B exerts protective effects after TBI
To explore the role of CHMP4B in TBI, we injected mice with adeno- To examine memory retention, Morris water maze tests were performed to assess cognitive deficits at 3 weeks after injury. In the first 2 days, all the groups treated with TBI performed poorly compared with the sham groups. Over the next 4 days, the TBI + AAV-CHMP4B group performed significantly better. Contrast, the TBI and TBI + AAV-vector groups continued to perform poorly and the improvement is not obvious than TBI + AAV-CHMP4B group ( Figure 2A). On the last day, compared with TBI and TBI + AAVvector groups, the swimming distance of TBI + AAV-CHMP4B group was significantly ameliorated ( Figure 2B,C). Next, we removed the platform and observed the performance on day 7. The TBI + AAV-CHMP4B group crossed the platform more often and spent more time in the target quadrant than the TBI groups and TBI + AAVvector groups ( Figure 2D,E). The rotarod test, which tests the motor ability, revealed that TBI + AAV-CHMP4B groups remained longer on the rotarod than the TBI and TBI + AAV-vector groups ( Figure 2F).
Brain water content was significantly decreased in the TBI + AAV-CHMP4B group compared with the TBI and TBI + AAVvector groups ( Figure 2G). Moreover, the cortical lesion volume and oedema area were significantly decreased in the TBI + AAV-CHMP4B group compared with the TBI and TBI + AAV-vector groups after 3 weeks ( Figure 2J).
We continue to study the role of CHMP4B on necroptosis, and the results of Western blotting and immunohistochemistry reflected that TBI + AAV-CHMP4B group had lower level of necroptosis compared with the TBI group ( Figure 3A-C). Finally, we used transmission electron microscopy (TEM) to observe cells' morphology.
TBI + AAV-CHMP4B group showed a denser cytoplasm, more intact cell membrane and less mitochondria swelling than TBI group ( Figure 3D).

F I G U R E 2 CHMP4B improves memory and motor ability and reduces the number of cell death and the size of oedema area after TBI.
There are four treatment groups take part in the experiments: sham, TBI, TBI + vector and TBI + CHMP4B (#P: TBI groups compared with sham groups; *P: TBI + CHMP4B groups compared with the TBI + vector groups). A, Mean latency to the platform of each group over 6 d (n = 10 mice; data are presented as the means ± SEM). B and C, Mean swimming speed (B) and distance (C) of each group on the day 6 (n = 10 mice; data are presented as the means ± SEM). D and E, Time (s) spent in the target quadrant (D) and the number of times the mouse crossed the target platform location (E) during the probe trials on day 7 (n = 10 mice; data are presented as the means ± SEM). F, The time mice can stay on the rotarod (n = 10 mice; data are presented as the means ± SEM). G, Brain water content after TBI (n = 3 mice; data are presented as the means ± SEM). H, Representative images of TUNEL staining after TBI in each group (Scale bar = 100 μm). I, Statistical analysis of the positive cell shown in G (n = 5 mice; data are presented as the means ± SEM). J, Mouse brain tissue MRI T2W1 images in each group. *P < .05; #P < .05, ##P < .01 These results demonstrate that up-regulation of CHMP4B enhances recovery of motor and memory functions, reduces cell death and alleviates the necroptosis after TBI in mice.

| CHMP4B plays a protective role by alleviating the necroptosis in microglia
Previous studies have shown that activated microglia increase inflammation in the CNS and reduce the survival of neurons after injury. 32 To clarify the molecular mechanisms underlying the Next, three independent plasmids encoding short hairpin RNAs targeting CHMP4B were constructed to silence CHMP4B expression in microglia. qRT-PCR and Western blot analyses indicated that sh-CHMP4B #1 had the highest interference efficiency and was therefore chosen for the following experiments ( Figure S2D-F).
Western blotting showed theGlu + sh-CHMP4B group had higher levels of the necroptosis marker proteins than the Glu and Glu + vector groups. (Figure 4E,F) Then, we performed the results by immunofluorescence. Again, the Glu + sh-CHMP4B group had a higher level of the necroptosis markers than the Glu and Glu + vector groups ( Figure 4G,H).
These results suggest that CHMP4B decreases necroptosis in injured BV2 cells.

| CHMP4B suppresses activated microgliamediated injury to neurons
Given the involvement of CHMP4B in the inhibition of necroptosis,  Figure 5H). Furthermore the Glu group showed the more fold of released LDH, whereas theGlu + CHMP4B group showed a comparatively lower release of LDH ( Figure 5I).
Thereafter, we examined the growth of neurons. After stimulation, neurons displayed axonal shortening and aggregation. When cultured with MCM from microglia treated with Glu and overexpressing CHMP4B, neuronal appearance remained normal ( Figure 5J).
Based on these results, CHMP4B can reduce a variety of harmful secretions from activated microglia.

| FOXO1 regulates the mRNA and protein levels of CHMP4B after TBI
Many studies have confirmed that changes in protein expression can be attributed to upstream transcriptional inducers when tissues or cells are stimulated. 35 According to the JASPAR database prediction, Dxl3, Nr2e1, Foxo1 and Gata4 had the highest prediction scores.
Then, we sought to validated this database prediction by luciferase assay and found that FOXO1 had the strongest luciferase inducing The immunofluorescence assay showed a higher fluorescence intensity of FOXO1 in nuclei after stimulation ( Figure 6B,C). And using qPCR and Western blotting, we found that the level of Glu stimulation ( Figure S4).

| FOXO1 regulates CHMP4B at the transcription level by binding to the promoter region
To further explore the regulatory interaction between FOXO1 and CHMP4B, we explored the promoter region of CHMP4B with the JASPER database, and three potential binding sites with the highest prediction scores were selected ( Figure 7A). The ChIP assay revealed that FOXO1 bounds to CHMP4B only at Site 1, the −958 to −968 bp region ( Figure 7B). Finally, we verified the binding site of FOXO1 on the CHMP4B promoter by generating mutations. As expected, luciferase assay showed that the transcriptional activity of the CHMP4B promoter is significantly increased, when the CHMP4B promoter binding by FOXO1 ( Figure 7C). These results indicate that FOXO1 directly enhances CHMP4B transcription by binding to a specific region on the CHMP4B promoter.

| D ISCUSS I ON
Traumatic brain injury is common in neurosurgery, and not only results in high mortality and disability, but is also considered to contribute to chronic diseases such as Alzheimer's disease, chronic traumatic encephalopathy and Parkinson's disease. 29,30 However, there is still no treatment that has been proven to be effective in improving prognosis of TBI and reducing complications. 29 In the pathophysiological process of TBI, microglia are activated first, in turn promoting neuroinflammation and secondary brain injury. 34 Therefore, a better understanding of the microglial activation mechanism is urgently needed for better treatment of TBI. Based on accumulating evidence, necroptosis occurs in the acute phase after TBI and can cause massive loss of neurons, which is a major neuropathological event. Traditionally, necroptosis is triggered by the TNF signalling pathway, which has been reported to be involved in neurobehavioural and histological outcomes after TBI. 36 can inhibit necroptosis and promote CNS recovery.
In subsequent in vitro experiments, we treated BV2 cells with Glu to induce cell injury. Our results confirmed that overexpression of CHMP4B inhibited necroptosis after microglial injury and reduced the pro-inflammatory effect of microglia, thereby alleviating neuronal damage. As previously reported, the expression of proteins is regulated by upstream transcription factors. 35 By a database prediction, we obtained a number of potential candidate regulators. Among these, FOXO1, a classical transcription factor, had the strongest luciferase inducting ability. We found that FOXO1 increased CHMP4B transcription by binding to the promoter region (located at −1499 to −1489 bp) in microglia.
Stable knockdown of FOXO1 can reduce the expression of CHMP4B, thereby increasing the level of necroptosis after microglia damage.
Previous studies have shown that the endosomal sorting complexes required for transport III (ESCRT-III) are involved in many physiological and pathological processes including the multivesicular body (MVB) pathway, cytokinesis, viral budding, plasma membrane repair and apoptosis. [43][44][45] Although it has been confirmed that CHMP4B affects neuronal growth, differentiation and apoptosis, its role in TBI had remained unknown. Here, we found that in TBI, CHMP4B inhibits necroptosis and reduces cell death, and its protective effect is greater than its adverse effect.
There are several limitations to this study. First, we used a TBI model employing juvenile mice, whereas the clinical samples were obtained from adult patients. Differences in age and species may affect the generalizability of our findings. Second, our microglial injury model was induced by Glu. Although it is a widely used model, the physiological and pathological processes may differ from those of traumatic injury. Third, for the clinical samples, epileptic patients were used as controls and were therefore not true healthy controls. However, because it is unethical to remove brain tissue from a normal healthy control, they represent the best available source of control samples. In addition, the clinical TBI tissues were mainly from the frontal lobe, whereas those in the control groups were all from the temporal lobe. Furthermore, the mean age of the control patients was lower than that of the TBI patients. Nevertheless, they represent the best control simples available for this study.

| CON CLUS ION
In summary, we found that CHMP4B effectively reduces necroptosis after TBI. And FOXO1 functions as a transcriptional activator that binds CHMP4B at its promoter region to induce expression.
Inhibition of TBI-induced necroptosis improves motor and memory functional recovery in mice. Our findings are important to better understand the mechanisms underlying secondary injury after TBI and may lead to the discovery of new necroptotic inhibitors as potential new therapies for TBI and other acute CNS injuries.

ACK N OWLED G EM ENTS
This study was supported by the funding from National Natural Science funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed in the current study are included either in this article or in the additional files.