Traumatic Brain Injury: Crosstalk Between ER and Mitochondria Contributes to Oligodendrocyte Cell Death and Demyelination

Traumatic Brain Injury: Crosstalk Between ER and Mitochondria Contributes to Oligodendrocyte Cell Death and Demyelination | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Traumatic Brain Injury: Crosstalk Between ER and Mitochondria Contributes to Oligodendrocyte Cell Death and Demyelination Rahul Shankar Rao Rayilla, Prakash Babu Phanithi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1128634/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective Demyelination is one of the manifestations of traumatic brain injury (TBI). Mechanisms underlying oligodendrocyte cell death in white matter lesions have not been fully studied. In the present study our focus is to investigate how the pro-inflammation contributes to the ER stress, mitochondrial protein alteration leading to oligodendrocyte cell death that aggravates demyelination in TBI. Methods Weight drop method was used to induce the TBI. Immunohistochemical studies were done to understand the role of pro-inflammation in activating ER-stress leading to cell death and demyelination. Proteomic analysis of mitochondria was done using 2D-gel electrophoresis. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to identify the mitochondrial functional protein changes and their role in cell death causing demyelination. Remyelination was assessed by treating with teriflunomide (TF). Results Histopathology studies revealed the infiltration of activated macrophages leading to cytokines secretion which initiates ATF6 activation by releasing BiP, the endoplasmic reticulum (ER) chaperone binding protein and unfolded proteins at the site of ER lumen. Activated ER stress alters the mitochondrial functional proteins leading to oligodendrocyte cell death causing white matter lesions and demyelination. Treatment with TF inactivates the macrophages and reduces the cytokines secretion at the site of injury promoting recovery and remyelination. Conclusion Our findings confirm pro-inflammation activates the ER stress sensors. The cross-talk between ER and mitochondria leads to cell death followed by demyelination. TF acts as an anti-inflammatory drug promoting oligodendroglial precursor cells maturation for recovery and secretion of myelin. Neurology Immunology White matter injury demyelination ER stress pro-inflammation Teriflunomide therapy remyelination Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Traumatic brain injury (TBI) usually results from a violent blow to the head caused by concussive head injury (external mechanical force) or diffuse injury (crash injuries, athletic head injuries)[ 1-3 ]. TBI causes the pathological alterations in the neuronal cells within the grey matter. But, according to recent studies the pathological changes depend on the impact or intensity of damage occurring to the brain and have highlighted the equal importance of white matter integrity which has axons[ 4-6 ]. White matter atrophy has a low probability of repair, due to the loss of neuron cell bodies in the corresponding regions[ 7-9 ]. Demyelination is the term used in white matter injuries and is characterized by loss of the myelin sheath and oligodendrocytic cell death. In the injured brain activated macrophages trigger cytokines causing inflammation at the injured site inducing ER stress sensors[ 10 ]. ER mediates the unfolded protein response (UPR) signaling pathway and is classified into three domain cascade sensors, protein kinase R (PKR)-like ER R kinase (PERK), activating transcription factor6 (ATF6), and inositol requiring enzyme 1 (IRE1)[ 11 ]. BiP which is a protein chaperone, maintains the physical interactions between the ER stress sensors and unfolded proteins in the ER lumen to prevent their activation[ 12 ]. During ER stress the abnormal accumulation of unfolded proteins results in the release of BiP from the ER stress sensors and activates ATF6 which translocate into the cytosol[ 13 ]. Proteases S1P and S2P at golgi apparatus cleaves the translocated ATF6 sensors into fragments which moves into the nucleus that causes more inflammation in several neurodegenerative diseases[ 14 , 15 ]. Mitochondria play an important role in cell survival and cell death mechanisms. The outer membrane is composed of integral proteins and voltage-dependent anion channels (VDAC) which is the most common pathway in exchanging the ions from the cytosol like Ca 2+ , K + , and Na + through OM by ER-mitochondrial junction called mitochondrial associated membrane (MAM). The inner membrane is composed of many compartmentalized functional proteins and molecules that require special membrane transporters to enter and exit the mitochondrial matrix. IM proteins involved in oxidative phosphorylation, ATP synthase, transporter proteins which regulate enter and exit metabolites exchanges in the mitochondrial matrix, import machinery, mitochondrial fusion, and fission. Whereas the respiratory chain protein complexes are accumulated in the cristae matrix[ 16 , 17 ]. TF is an immunomodulatory, anti-inflammatory and multi-functional drug for multiple sclerosis (MS) patients[ 18 , 19 ]. Treatment with TF inactivates the immune cells and reduces the cytokine secretion from macrophages. In addition, TF promotes oligodendroglia precursor cells (OPCs) maturation which results in recovery and remyelination[ 20-22 ]. In this study, we elucidated the role of cytokines secretion from the activated macrophages followed by ER stress activation. Further, we evaluated the impact of ER stress on mitochondrial damage and observed the ER-mitochondria cross-talk promotes oligodendroglia cell death and aggravates demyelination. In addition, we have evaluated the remyelination and recovery by treatment with TF. Methods Animal selection Sixty Sprague Dawley (SD) rats (3 months old, 220 – 250gms male) were purchased from the National Institute of Nutrition (NIN), Hyderabad. As per the experimental requirement, animals were acclimatized in the Animal house facility, University of Hyderabad before 10 days of the experiments, (Reg number: 151/1999/CPCSEA). Experiments were approved by the institutional animal ethical committee (UH/IAEC/PBP/2019 – I/03). The animals were housed in cages with an ambient temperature of 24°C, constant and standard air humidity and natural day/night cycles, quality food and water ad libitum. TBI animal model of brain axonal damage (BAD) SD rats were anesthetized and BAD were induced by a well-established weight-drop method. In Sham group (n=6) animals were placed on the weight drop platform without inducing BAD, post-experimental 48-hour point (n=6), post-experimental 1-week point (n=6), Vehicle treated (n=6) [carboxymethylcellulose made up to 0.06% (w/v) in water, to which Tween 80 was added to reach a final concentration of 0.5% (v/v)] administrated orally for two weeks after post BAD. One group of rats were allowed for two weeks for self-recovery (n=6) and Teriflunomide therapy (n=6). Before performing the weight drop experiment, all the experimental instruments were sterilized using 70% ethyl alcohol. Animals were weighed and anesthetized through intraperitoneal administration of anesthesia: Avertin (2,2,2 - Tribromoethanol 1.25gms, 2 - Methyl 2 - Butanol - 2.5ml per 100ml) Dose: 150mg/kg or 12.4ml/kg body weight of the animal model. Site: Intraperitoneal, Volumes: 3ml/250gms of selected SD rats. 200gms of stainless steel iron ball was dropped on the anesthetized rat head from a height of 60cms to induce BAD. BAD-induced rats were shifted to the recovery chamber which maintains the temperature 37°C followed by accommodating in cages with food and water in different day points 48 hours, 1 week, 2 weeks self-recovery, and Teriflunomide therapy for a 2-week point post BAD. TF administration to the BAD induced rats After inducing BAD, a batch of n=6 rats was maintained for the TF treatment. TF was dissolved in vehicle: carboxymethylcellulose made up to 0.06% (w/v) in water, to which Tween 80 was added to reach a final concentration of 0.5% (v/v). TF was administrated at a concentration of 10mg/ml orally using rat oral gavage at a volume of 1.0ml/kg body weight for two weeks every alternate day after post-48-hour injury. Euthanasia followed by animal perfusion fixation After BAD, rats were euthanized by an overdose of sodium pentobarbital (100mg/kg) injected intraperitoneally at different time points. Rats were then perfused and the brain samples were excised for further studies. Perfusion fixation Chemical composition: i) Stock solution: 8% paraformaldehyde: Add 80gm paraformaldehyde to 1000ml distilled water. Stir the solution while heating (not exceed more than 60- 65°c). Reduce heat and add 2-3ml of 1.0M NaOH with a dropper. Filter and store at 4°c or up to 1 month. ii) Prepare 0.2 M Sodium Phosphate Buffer, pH 7.4: Add 27.8gm of NaH 2 PO 4 to 1litre distilled water for sodium phosphate monobasic stock; add 28.4gm of Na 2 HPO 4 to 1litre of distilled water for sodium phosphate dibasic stock. The final stock of sodium phosphate buffer was prepared by adding 810ml of the monobasic stock to 190ml of the dibasic stock and pH adjusted at 7.4 iii) 4% Paraformaldehyde Fixative preparation: Add equal parts 1:1 ratio of 8% paraformaldehyde stock to 0.2M Sodium Phosphate Buffer. 4% paraformaldehyde was prepared freshly before 72hour experimentation plans. iv) Phosphate buffered saline preparation, pH 7.4: Add NaCl-9gms, KH 2 PO 4 -144mg, Na 2 HPO 4 -795mg to the 1litre of distilled water and pH adjusted to 7.4. Procedure Euthanized animals were placed on the shallow tray filled with crushed ice. Beneath the rib cage lateral incisions of 5-6 cm were made through the integument and abdominal wall carefully, to separate the liver from the diaphragm and followed by another small incision in the diaphragm using the curved, blunt scissors which continued till the entire rib cage along with the pleural cavity. Lung displacement was done using curved, blunt scissors and a cut was made through the rib cage up to the collarbone followed by a similar cut on the contralateral side. The tip of the sternum was clamped with the haemostat and placed over the head. A small incision to the posterior end of the left ventricle using iris scissors was made and a 15-gauge blunt- or olive-tipped perfusion needle had inserted through the cut ventricle into the ascending aorta. The heart was clamped using a haemostat to secure the needle and prevents leakage. Finally, an incision was made to the animal's right atrium using iris scissors to create as large an outlet as possible without damaging the descending aorta. Outlet port perfusion equipment was attached to the needle base by avoiding air bubbles and 80mm Hg pressure of the manometer bulb was maintained throughout the buffer infusion period with a proper needle angle adjustment to achieve the maximum flow rate. Fixation was almost pumped to the animal and the clear running of the fluid was monitored. The clearance of the liver was observed which is indicative of proper perfusion. Fixation tremors had been observed within seconds; which can be considered as the true time of fixation indicator. Further, the pressure was gradually increased up to a maximum of 130mm Hg 2 to maintain a steady flow rate. The outlet valve was closed soon after the completion of the fixation process followed by the ending time recordings and the stiffness of the animal was observed. Paraffin-embedded tissue sections Freshly collected brain samples were sliced into 3mm slices and fixed with 10% paraformaldehyde or formalin for 48 hr at room temperature. After fixation samples were washed under running tap water for 1 hrfollowed by dehydration steps using 70%, 80%, 95% alcohol changes 30min each and 3 changes of 100% alcohol for 1hour each. Brain samples were cleared in 1 change of xylene for 5min and another step of xylene + melted paraffin 1: 1 ratio for 5mins followed by immersing the samples in 3 changes of paraffin (Paraplast® - polyisobutylene mixture, catalogue no P3558 – SIGMA – ALDRICH®) 1hour each and the brain slices were embedded in a paraffin block. Brain slice blocks were fixed to the microtome (LEICA RM2145) and 10µm sections were done and floated in a 40°c maintained water bath containing clear distilled water. Tissue sections were transferred carefully to the glass slides for further neuropathological studies. Histopathological studies Hematoxylin and eosin staining (H & E) Formalin-fixed paraffin-embedded FFPE brain sections were firstly deparaffinized on the heating pad or heating plate and followed by tissue clearing for 2min each in two xylene changes. Slides containing FFPE sections were rehydrated accordingly in two changes of 100% alcohol for 3min each, thereafter 95%, 80%, and 70% alcohol for 3min and followed by a 10min water wash. Processing slides were then dipped in nuclear stain (Haematoxylin) containing coupling jars for 3 – 5min and shifted to the stain (eosin) for extracellular matrix and cytoplasmic staining for 2min followed by a tap water wash for 6min. After the staining procedures, sections were transferred to 30%, 50% alcohol for 6min each, 70% alcohol for 10 min, 95% alcohol, and 2 changes of 100% alcohol 6min each for dehydration and followed by tissue clearing with 2 changes of xylene, 3min each and DPX mounting followed by microscopic evaluations. Immunohistochemistry Brain sections were deparaffinized using a heating pad and placed or dipped in 2 changes of xylene containing coupling jars for 2min and quickly transferred to a 1:1 ratio of xylene: 100% ethanol for 3min. Tissue sections were further rehydrated in 2 changes of 100%, 95%, 70%, 50%, and 30% ethanol for 3min and rinsed in tap water for 5min. The tissue sections were transferred to antigen retrieval buffer (trypsin 0.05% in 100 ml of PBS) for 15min at 37°c and permeabilized (triton x – 100 0.2% in PBS) for 7 – 10min followed by 1 change of wash in PBS for 5min. Tissue section slides were transferred into blocking solution (BSA 1%, NGS 5% in PBS) and left for 1hour at room temperature. Approximately 100µl of diluted primary antibodies myelin basic protein MBP (AB clonal Catalogue No: A1664, 86 Cummings Park Dr, Woburn, MA 01801, United States), CNPase -2, 3-cyclic nucleotide 3-phosphodiesterase, GenScript, Catalogue no: A01308-40, 860 Centennial Ave., Piscataway, NJ 08854, USA), S100 Beta EP 32 (PathnSitu Catalog No: CR070 – 0.1 ML Concentrated, USA-Registered Office 538, Selby Ln, Livermore, CA-94551 USA) as myelin and glial markers. GRP 78 (76-E6): Catalogue no: Sc-13539 and ATF-6α Antibody (H-280): Catalogue no: sc-22799 from Santa Cruz Biotechnology, Inc. 10410 Finnell Street, Dalla, Texas 75220, U.S.A, as ER stress markers. CD68 KP 1 (PathnSitu Catalogue no: PM113 – 0.1 ML concentrated, USA-Registered Office 538, Selby Ln, Livermore, CA-94551 USA, and TNFα antibody-NBP1-19532, Novus Biologicals, LLC-10730 E. Briarwood Avenue, Building IV Centennial, CO 80112, USA. (dilution 1:200 PBS with 0.02% sodium azide, 50% glycerol, Ph 7.3) were applied to the tissue sections and incubated in a humidified chamber at room temperature for 30min. Sections were transferred for washing with PBS for 2 changes of 5min. Approximately 100µl of diluted biotinylated secondary antibody was applied (using the antibody dilution buffer) to the sections on the slides and incubated in a humidified chamber at room temperature for 30min (protected from sunlight) followed by washing with 2 changes of PBS for 5min. DAB substrate solution (freshly prepared before use: 0.05% DAB, 0.015% H 2 O 2 in PBS) was applied to the sections on the slides to reveal the color of antibody staining. The color development was done for less than 5min until the desired color intensity was obtained followed by washing in 3 changes of PBS for 2min each. For counterstaining slides were immersed in haematoxylin for 2 – 3min as per the thickness of tissue sections followed by water wash for 15 – 20min. Tissue slides were dehydrated in graded alcohol solutions of 2 changes 95% and 2 changes 100% alcohol 5min. Tissue sections were mounted by using DPX mounting solution and covered with coverslips. Mitochondria isolation Rat brains were collected and finely minced by adding around 5 ml of buffer: 1 – 200 μl of 500 mM EGTA for a final concentration of 10 mM, 0.392 g of D-mannitol for a final concentration of 215 mM, 1.25 ml of 8X mitochondria buffer (10.28 g of sucrose for a final concentration of 0.6 M, 400 mg of free-fatty acid bovine serum albumin (BSA) for a final concentration of 0.8%, 2.08 g of HEPES for a final concentration of 160 mM, pH to 7.4 and made up to 50 ml with distilled water) pH to 7.4 and made-up to 10 ml with distilled water. Minced brain with the solution was then transferred to the polytron homogenizer. Homogenized tissue samples were collected into pre-chilled microcentrifuge tubes and centrifuged at 700 g for 10 minutes at 4°c. Supernatant was transferred to new pre-chilled microcentrifuge tubes and pellets were discarded. The supernatant was centrifuged at 10,500 g for 10 minutes at 4°c and the pellet was resuspended in 500 μl of (buffer: 2 - Add 60 μl of 500 mM EGTA for a final concentration of 3 mM, 0.392 g of D-mannitol for a final concentration of 215 mM, 1.25 ml of 8X mitochondria buffer, pH to 7.4 and makeup to 10 ml with distilled water) and centrifuged at 10,500 g for 10 minutes at 4°c. Final mitochondrial pellet was suspended in 100 μl of Buffer: 2 followed by quick-spin for final mitochondrion for a few seconds. Supplement Lysis Buffer: 1 and Buffer: 2, 1/100 volume of Protease Inhibitor Solution (100x) (i.e., if using 2 ml Disruption Buffer, add 20 μl Protease Inhibitor Solution. Determine protein concentration using the Bradford assay. 2D – gel electrophoresis Rehydration of IPG strips and First dimension separation IEF (Isoelectric focusing) The first – dimension separation of 2Dgel is IEF, in this proteins are separated based on differences in their isoelectric point (pI)[ 23 ]. Samples were added to the rehydration tray and cover removed from the selected IPG strip pH 4-10 and gel was allowed to slide down onto the rehydration buffer in the tray. The tray was overlaid with the mineral oil to prevent the evaporation and precipitation of urea during rehydration and left for 20 hours for complete rehydration. The rehydrated IPG strips were transferred to the IEF tray by gel side up using two notches and IPG strips were overlaid with mineral oil and run the IEF accordingly to the protocol (GE Healthcare). IPG strip equilibration and running second dimension by SDS - PAGE After IEF the strips were transferred to the new tray gel side up and filled with the recommended volume of equilibration buffer 1 followed by incubation for 10 min. Buffer 1 was removed and equilibration buffer 2 was added for 10 min incubation. After equilibration, IPG strips were removed and followed by a rinse with SDS – PAGE running buffer, and the strips were placed on the prepared 12% SDS – PAGE gel and sealed with 0.5% of agarose solution, and the electrophoresis unit was run. Comparative studies of the gels for the up-regulation and down-regulation for the detection of the protein was analyzed by IMR software version7 GE Healthcare (all the above experimental protocols and buffers are from GE Healthcare ) In-gel digestion and mass spectrometric analysis MS An automated spot cutter was used to collect the spots of interest. For in-gel digestion, the gel plugs were washed with water twice for 5 min and incubated with 100% acetonitrile for 10 min. Then, the gels were dried completely for at least 30 min. The protein in the gel was digested by treatment with TPCK – treated trypsin in 50 mM ammonium bicarbonate at 37°c overnight with gentle agitation. After digestion, peptides were extracted with 50 µl of 50% acetonitrile, 0.1% Trifluoroacetic acid twice, concentrated, and extensively treated with ZipTip. Then trypsin digests were mixed with an equal volume of matrix solution, comprising of saturated dihydroxy benzoic acid in 50% acetonitrile/0.1% TFA. The samples were spotted on a MALDI target plate and subjected to mass spectrometric analysis. Mass spectrometric analysis of trypsin digestion was performed using a Q-STAR Pulser-i equipped with a MALDI ion source. Raw data to mzml file conversion was done using Flex analysis 3.2 software and followed by mascot online search engine tool for the protein identification and peptide sequencing (Fig B). Results Cytokines secretion from activated macrophages in traumatic brain Cellular damage triggered the macrophage's activation and secretion of cytokines at the site of injury when compared to the normal brain. Immunostaining with macrophage marker CD68 and cytokine marker TNFα demonstrated early expressions, 48 hours post BAD (Fig1). CD68 and TNFα were highly expressed in the late stages, around one-week, post-BAD. Treatment using TF for two weeks on alternate days resulted in the decreased expression levels of CD68 and TNFα respectively as shown in Fig4. ER stress sensors activation via BiP ER chaperone binding protein In the traumatic brain, secreted cytokines from the activated macrophages translocate ATF6 sensor from ER lumen to the cytosol by releasing BiP, ER chaperone binding protein (Fig2). BiP expression was observed soon after 48 hours post BAD and further increased after one-week post BAD (Fig2). ATF6 expression was observed at 48 hours and slightly increased after one-week post BAD (Fig2). However, BiP and ATF6 expressions decreased after TF treatment (Fig5). Mitochondrial dysfunction under stress condition We confirmed mitochondrial damage induces apoptotic cell death upon ER stress. We observed differential expression of mitochondrial proteins in the traumatic brain which were restored upon treatment with teriflunomide. Proteins were analysed using 2D-gel electrophoresis and identified by MALDI MS. Peptide sequences were identified as COX7B - Cytochrome c oxidase subunit 7B, PRDX3 - Thioredoxin-dependent peroxide reductase, SPYA - Serine--pyruvate aminotransferase, ACPM - Acyl carrier protein, M2OM - mitochondrial 2-oxoglutarate/malate carrier protein, MTCH1 - Mitochondrial carrier homolog 1, TTC19 - Tetratricopeptide repeat protein 19, AATM - Aspartate aminotransferase, IPYR2 - Inorganic pyrophosphatase 2, GCSH -glycine cleavage system H protein, CH10, Hspe1 - 10 kDa Heat shock protein, and Mitochondrial matrix proteins as ACADL - long-chain specific acyl-CoA dehydrogenase, RM55 - 39S ribosomal protein L55 (Fig7, 8&9). Demyelination and remyelination after Teriflunomide treatment Immunostaining with myelin markers, myelin basic protein (MBP), CNPase, and Neuroglia S100 detected high expression in white matter tracts, in the sham group (Fig3) and the vehicle group (Fig6). Early myelin loss was observed in the post 48-hour BAD group and moderate in the one-week post BAD group (Fig3). Remyelination or recovery was observed after TF treatment by the activation, recruitment, and differentiation of resident OPCs as shown in Fig6. Discussion The brain consists of grey and white matter regions which majorly consist of glial cells. Grey matter contains neuronal cell bodies which serve as an information processor for the CNS[ 1 ]. On the other hand, white matter is composed of myelin-producing oligodendrocytes, and other glial cells which maintains myelin, signal transmission, and communication[ 7 ]. Oligodendrocytes are the primary cells that are richly present in white matter and are responsible for maintaining the myelin sheath under normal conditions and for remyelination after axonal damage[ 24 ]. Oligodendrocyte cell death causes the myelin loss and is known to be a significant factor underlying demyelination after brain injury. Increased demyelination is a major pathological condition of white matter injury-causing BADs which contributes to significant long-term sensorimotor and cognitive deficits[ 24 , 25 ]. To date, many TBI animal models are in use to induce neurodegeneration and help in studying the mechanism of cell death[ 26 ]. However, to the best of our knowledge, there are no reports representing demyelination in TBI during ER stress induced mitochondrial protein alterations. Here in the present study, we used the weight-drop method as a tool to understand the mechanism of cell death leading to demyelination. Further, we attempted to reveal role of inflammation in aggravating the demyelination. We demonstrated the early pro-inflammation by CD68 and TNFα at forty-eight hour and moderate at one-week injured rat brain white matter lesions. In similar studies increased expression levels of CD68 and TNFα markers were found to detect the activated macrophages and secreted cytokines[ 27-29 ]. ATF6 and BiP markers detected the initial expression at forty eight hour and increased expression at one week TBI. Pro-inflammatory cytokines mediates ER stress sensor ATF6 activation, initiates the release of BiP chaperone binding protein which commences the cell death[ 14 , 30 ] , [ 12 , 31 ]. MBP, CNPase (2',3'-Cyclic-nucleotide 3'-phosphodiesterase) and S100 markers showed significant early myelin loss at forty eight hours which increased at one week intervals after inducing TBI. Comparably, myelin loss or demyelination was assessed by using MBP, CNPase and S100 markers in many neurological diseases[ 32-35 ]. To our knowledge oligodendrocyte cell death under ER-stress promoting demyelination is not reported so far. Further we demonstrated the decreased expression of CD68 and TNFα markers after TF treatment; this influenced the expression of ER stress sensors ATF6 and BiP markers. Additionally, our results showed the increased expression of myelin markers MBP, CNPase, and S100 after treatment with TF. TF is an anti-inflammatory drug which helps in the inactivation of macrophages and reduces the cytokines secretion at the injured site[ 18 , 19 ]. Similar reports indicates that TF promotes oligodendrocyte precursor cell differentiation which causes remyelination in white matter lesions[ 20 ]. Though the mitochondrial cell death has been reported in various pathological models but the involvement of specific proteins is spars. Mitochondrial dysfunction maybe considered to be one of the early events that can cause cell death in traumatic brain[ 36 ]. Therefore, in the present study mitochondria has been isolated from the control, traumatic brain and treated brain samples. Proteins were subjected to 2D- gel electrophoresis and identified using MALDI-MS. In proteomic studies, we observed significant changes in the mitochondrial functional proteins. Mitochondrial carrier homolog 1 (MTCH1) acts as the receptor for truncated BID (tBID) which involves in apoptosis[ 37 ]. Herein we found MTCH1 down-regulated in trauma brain group and total protein degraded and disappeared in the treatment. In TBI, MTCH1 down-regulation results in signalling the tBID (pro-apoptotic protein) on the surface of mitochondria. tBID triggers the release of cytochrome c by causing oligomerization of BAX/BAK (apoptotic regulators) and results in outer membrane permeabilization. On the other hand MTCH1 down-regulation increases the opening of voltage dependent anion channels (VDAC) leading to the membrane potential loss and release of cytochrome c. Mitochondrial 2-oxoglutarate/malate carrier protein (M2OM) involves in the regulation of apoptosis[ 38 , 39 ]. This was down-regulated after TBI and slightly noticeable up-regulation was observed after treatment. Reduced expression of M2OM mitochondrial inner membrane protein in traumatic brain can transport the glutathione (GSH) from the cytosol into the mitochondrial matrix promotes apoptosis. Additionally, over accumulation of the GSH in the mitochondrial matrix promotes the BAX/BAK activation (apoptotic regulars) and cytochrome c release. Restoration of M20M after treatment can help in normalising the transportation of GSH from cytosol to mitochondrial matrix. Cytochrome c oxidase subunit 7B (COX7B) functionally drives oxidative phosphorylation and plays an important role in proper central nervous system development in vertebrates. We demonstrated COX7B down-regulation after TBI and slight noticeable up-regulation was observed after treatment, the reduced expression of COX7B in the mitochondria indicates that this must have released into the cytosol and forms apoptosis protease activating factor (APAF1) complex thereby activating caspase cascade promoting the apoptosis. Whereas restored COX7B after treatment in the mitochondria reduces the activation of apoptotic pathway. Thioredoxin-dependent peroxide reductase (PRDX3) functionally protects the cell against oxidative stress by detoxifying peroxides[ 40 ]. In the present study PRDX3 down-regulated in traumatic brain samples and slightly up-regulated after treatment. Decreased expression of PRDX3 led to the increased oxidative stress in the mitochondria, this might have responsible for the cell death in our model. However, upon TF treatment PRDX3 expression restored, this appeared to have a protective role against traumatic brain mitochondrial damage. Serine pyruvate amino transferase (SPYA) involves in gluconeogenesis and glyoxylate detoxification[ 41 ]. We noticed SPYA down-regulated in TBI and up-regulated after treatment. Down-regulation of SPYA influences in the breakdown of metabolic processes and must have aggravated the cell death leading to demyelination in TBI whereas recovery was observed after the treatment. Long chain specific acyl-CoA dehydrogenase (ACADL) catalyses the first step of mitochondrial fatty acid beta-oxidation, an aerobic process[ 42 ]. In our study ACADL was down-regulated in the trauma brain and up-regulated after treatment. After using TF, we showed the restoration of ACADL expression can promote cell cycle arrest and inhibit cell proliferation and growth. Acyl carrier protein (ACPM) involves in the transfers of electrons from NADH to the respiratory chain[ 43 ]. ACPM down-regulation was noticed in TBI and slight up-regulation after treatment. Down-regulated ACPM imbalances the functions of electron transport chain. Whereas functioning of the electron transport chain and maintenance of normal mitochondrial membrane potential can be restored after TF treatment. Tetratricopeptide repeat protein (TTC19) plays a key role in the preservation of structural and functional integrity of mitochondrial respiratory complex III[ 44 ]. TTC19 disappeared in the trauma brain group and up-regulated after treatment. Disappearance of TTC19 can lead to mitochondrial respiratory complex chain III deficiency. However, after TF treatment, reappearance of the TTC19 can restore the complex III deficiencies, progressive neurological and metabolic decline. 39S ribosomal protein (RM55) involves in the protein synthesis within the mitochondria[ 45 ]. We found RM55 totally absent or degraded in the trauma brain group but increased expressions were found in the treated. In traumatic brain RM55 fails in protein synthesis and causes adenosine tri-phosphate (ATP) deficiency within the mitochondria. TF treatment can help in mitochondrial protein synthesis recovery and ATP production. 10 kDa heat shock protein (CH10, Hspe1) prevents misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix[ 46 ]. CH10 Hspe1 up-regulated in the traumatic brain group and increased expression was observed in the treated. Up-regulated CH10, Hspe1 represents the prevention of protein denaturation and aggregation by refolding. After treatment with TF, normalisation of CH10, Hspe1 was noted. Aspartate aminotransferase (AATM) exchanges metabolites between mitochondria and cytosol, including amino acid metabolism. We observed the degradation or absence of AATM in traumatic brain and up-regulation after treatment. In similar studies AATM levels were decreased soon after post blast exposure TBI[ 47 ]. Inorganic pyrophosphatase 2 (IPYR2) regulates mitochondrial membrane potential, and mitochondrial organization and function[ 48 ]. We demonstrated down-regulation of IPYR2 in traumatic brain and up-regulation after treatment. Down-regulation of IPYR2 influences in the functional loss of mitochondria and restores after the treatment with TF. Proteomics of mitochondrial expression level in sham, traumatic brain and treated groups reveal the prominent changes of proteins which are mainly involved in activating the caspase cascade, cytochrome c release, induction of the apoptosis, detoxification, respiratory chain, exchange of metabolite and restoration or refolding of denatured proteins in the mitochondria. Earlier research predicts ER releases more metabolites under stress conditions and mitochondria uptakes the excess Ca 2+ through VDAC at the MAM junction[ 49 ]. Calcium overload in mitochondria induces permeability transition pore which promotes apoptotic cell death at the site of injury[ 50 ]. After treatment with TF significant changes were noticed, which suggests restoration of mitochondrial functional proteins. Conclusion Overall, our findings explain the involvement of activated macrophages in ER stress sensors activation. Proteomics studies demonstrated the cross-talk between ER and mitochondria through mitochondrial dysfunction followed by oligodendrocyte cell death in white matter lesions causing demyelination. Treatment with TF reduced the inflammation by immunomodulation and promoted recovery or remyelination and can be used as a remyelination therapy for TBI patients. Abbreviations CNS: Central nervous system TBI or TBIs: Traumatic brain injury or Injuries BAD: Brain axonal damage ER: Endoplasmic reticulum BiP: Immunoglobulin-heavy-chain-binding protein ATF6: Activating transcription factor 6 TNFα: Tumour necrosis factor alpha MAM: Mitochondrial associated membrane VDAC: Voltage dependent anion channels PTP: Permeability transition pores CD68: Cluster of differentiation 68 MBP: Myelin basic protein TF: Teriflunomide UPR: Unfolded protein response PKR: Protein kinase R PERK: (PKR) – Like endoplasmic reticulum kinase IRE1: Inositol requiring enzyme1 SD: Sprague Dawley FFPE: Formalin fixed paraffin embedded OPCs: Oligodendroglia precursor cells Declarations Acknowledgments PPB and RRSR thank NATCO Pharma for Teriflunomide. The authors acknowledge the help of Dr. Ranjani Chakravarthy, MD (Pathology), FRCPath (UK), Managing Director & Consultant Histopathologist, Cpathlabs (India). Tirumala chari, Histopathologists, Cpathlabs & PathnSitu for their help in Neuropathological studies. Funding Financial assistance from the Department of Science and Technology (DST- India) (Grant no: SR/CSRI/196/2016), Department of Biotechnology (DBT-India) (Grant No. BT/PR18168/MED/29/1064/2016). RRSR is thankful to DST – Inspire Fellowship under the Ref.no: DST/INSPIRE/03/2015/003047 Availability of data Data generated and analysed during this study are available from the authors on reasonable request Author information Affiliation Department of Biotechnology and Bioinformatics, School of Life sciences, University of Hyderabad, Hyderabad-500046 (T.S), INDIA; Contact details: Ph. No: 7993379999, 040-23134584/4684; Email id: [email protected] ; [email protected] ; Rahul Shankar Rao Rayilla and Prakash Babu Phanithi Authors Contribution RRSR performed animal experiments; PPB and RRSR analyzed and interpreted the data. RRSR drafted the work, PPB revised it critically and both the authors have approved the submission. Corresponding Author Prakash Babu Phanithi Ethical approval for animal experiments Experiments were approved by the Institutional animal ethical committee IAEC (UH/IAEC/PBP/2019 – I/03). As per the experimental requirement, animals were acclimatized in the Animal house facility, University of Hyderabad before 10 days of the experiments, (Reg number: 151/1999/CPCSEA). Consent for publication The authors provide consent for publication. Competing interests The authors declare that they have no conflict of interests References Shi H, Hu X, Leak RK, Shi Y, An C, Suenaga J, Chen J, Gao Y: Demyelination as a rational therapeutic target for ischemic or traumatic brain injury. Exp Neurol 2015, 272: 17-25. Cernak I: Understanding blast-induced neurotrauma: how far have we come? Concussion 2017, 2: 2017-0006. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1128634","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":67649024,"identity":"4b760f62-6421-4407-97dc-1c9b9b19636d","order_by":0,"name":"Rahul Shankar Rao Rayilla","email":"","orcid":"","institution":"University of Hyderabad","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rahul","middleName":"Shankar Rao","lastName":"Rayilla","suffix":""},{"id":67649026,"identity":"71d2b665-5c36-45a7-a974-77411eca2fef","order_by":1,"name":"Prakash Babu Phanithi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYHACZiA+IMfAwANGED4xWoxJ15LYANdCCJhLH35s8OPPnfQNx3uPSbxhsJNnYOc9gFeLZV+acWJv27PcDWfOpUnOYUg2bGDmS8CrxeAMg/EB3obDuRtu5JhJ8zAwJzAw8xgQ0ML++eCfP4fTDSBa6onRwmOczMN2OAGq5TBhLZY9PMXGsm2HDWeeOWNsOcfguGEbIS3mPOybJd/8OSzPd7zH8Mabimp5fv4zBByGxGaRAHHZ8KpH08L8gZDqUTAKRsEoGJkAAP8eP+EjqDTLAAAAAElFTkSuQmCC","orcid":"","institution":"University of Hyderabad","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Prakash","middleName":"Babu","lastName":"Phanithi","suffix":""}],"badges":[],"createdAt":"2021-11-30 16:44:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1128634/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1128634/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":16211454,"identity":"cfb9e820-f609-4cdc-893e-de549ccf101c","added_by":"auto","created_at":"2021-12-06 15:21:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1686147,"visible":true,"origin":"","legend":"Secretion of TNFα by activated macrophages in traumatic brain\nMicroscopic evaluations of FFPE rat brain sections in detecting the activated macrophages by CD68 and cytokines with TNFα markers (Images captured at 20X and 100 X magnifications Olympus BX51). a Sham group, n=6, mean-56.182, SEM-1.944, b, 48-hour post BAD group, n=6, mean-74.533, SEM-3.819, and c, one week post BAD group, n=6, mean-88.854, SEM-3.095 reveals the spontaneous activation of macrophages in b, and severe in c, one week post BAD FFPE sections with immunostain CD68 marker. In d, e, and f TNFα cytokines marker FFPE sections, increased expression found in e, 48-hour BAD group, n=6, mean-76.212, SEM-3.999 and f, one-week BAD group, n=6, mean-80.892, SEM-2.111 comparing with Sham group, n=6, mean-54.492, SEM-1.922 (Statistical analysis are calculated using SigmaPlot 11.0 and the values are graphically represented using GraphPad Prism 8).","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1128634/v1/c4f5f69f9902e8ba9435b35f.png"},{"id":16211111,"identity":"9893d764-8a81-48b9-bb40-89e64567574a","added_by":"auto","created_at":"2021-12-06 15:18:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1826330,"visible":true,"origin":"","legend":"Expression of BiP, ER chaperone binding protein in the activation of ATF6 ER stress sensors\nImmunostaining of rat brain FFPE sections to detect the activation of ER stress sensors via BiP ER Chaperone binding protein. a, sham group, n=6, mean-167.037, SEM-1.786; b, 48-hour post BAD group, n=6, mean-172.475, SEM-2.145; c, one-week post BAD group, n=6, mean-183.027, SEM-2.695 revealed the early expression levels in b and increased expression in c of BiP by comparing with sham group a; d, sham group, n=6, mean-159.998, SEM-2.474; e, 48-hour post BAD group, n=6, mean-167.860; f, one-week post BAD group, n=6, mean-176.691, SEM-3.319 represents FFPE sections immunostained with ATF6 marker and identified early expression in e and increased expression in f comparing with d.\n","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1128634/v1/9767b2cb1ca1638667af878a.png"},{"id":16211117,"identity":"6e218265-de65-408f-897b-e1ff71f96297","added_by":"auto","created_at":"2021-12-06 15:18:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3529009,"visible":true,"origin":"","legend":"Expression of myelin markers in white matter lesions at different time points \nImmunostaining to detect demyelination in FFPE rat brain sections. a, b, c hematoxylin, and eosin H\u0026E staining images revealed the alteration of cell morphology in the white matter lesions of rat brain. a, Sham group, n=6; b, 48 hours post BAD damage group, n=6; c, one week post BAD group, n=6 represents the alteration of the cell morphology in b and more in c; d, Sham group, n=6, mean – 131.120, SEM – 1.69; e, 48-hour post BAD group, n=6, mean – 123.371, SEM, 1.988; f, one-week post BAD group, n=6, mean – 112.168, SEM – 3.451.4 myelin basic protein (MBP) marker immunostaining revealed the decreased expressions in e and highly reduced in f comparing with d; Images g, h, and i are stained with CNPase marker, g, sham group, n=6, mean-77.651, SEM-2.143; h, 48-hour post BAD group, n=6, mean-54.908, SEM-3.499, n=6; I, one week post BAD group, n=6, mean-54.620, SEM-2.941 represents decreased expression in h and highly decreased in I comparing with g; j, sham group, n=6, mean-179.992, SEM-1.583; k, 48-hour post BAD group, n=6, mean-152.392, SEM-3.178; l, one week post BAD group, n=6, mean-138.398, SEM-4.450, n=6 immunostained with S100 marker and revealed the reduced expressions in k and highly reduced expressions in l in comparison with j. \n","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-1128634/v1/f1cf7129ca0bad9f18741b08.png"},{"id":16211455,"identity":"cb4f4a2b-c9b2-439f-befc-8949716a7d25","added_by":"auto","created_at":"2021-12-06 15:21:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1707727,"visible":true,"origin":"","legend":"This figure shows the inactivation of the macrophages and reduced cytokines secretion after Teriflunomide treatment using CD68 and TNFα markers \nImmunostaining and microscopic evaluations of CD68 and TNFα markers. a, vehicle group, n=6, mean-54.492, SEM-1.922; b, one-week BAD group, n=6, mean-80.892, SEM-2.111; c, self-recovery group, mean-82.107, SEM-2.500; and d, drug therapy group, n=6, mean-65.169, SEM-3.487 represents immunostaining of CD68 marker show decreased expression in d, no self-recovery in c comparing with a, and b. Immunostaining with TNFα marker e, sham, n=6, mean-54.492, SEM-1.922; f, one week BAD group, n=6, mean-80.892, SEM-2.111; g, self-recovery group, n=6, mean-82.107, SEM-2.500; h, drug therapy group, n=6, mean-65.169, SEM-3.487; identified decreased expression in h, no self-recovery in g comparing with e, and f. \n","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-1128634/v1/ae508587068e8de5c20da68b.png"},{"id":16211113,"identity":"2c9f6ccd-5e99-45e7-9683-4a4f555edd4a","added_by":"auto","created_at":"2021-12-06 15:18:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1815218,"visible":true,"origin":"","legend":"Decreased expression of ER binding chaperone protein BiP marker leads in the inactivation or down regulation of ATF6 ER stress sensors after Teriflunomide treatment (10mg/kg animal body weight)\nImmunostaining and microscopic evaluation of ER binding chaperone protein BiP and ER stress ATF6 sensors. Images a, vehicle group, n=6, mean-164.213, SEM-2.017; b, one-week BAD group, n=6, mean-176.691, SEM-3.319; c, self-recovery group, n=6, mean-178.975, SEM-3.574 ; and d, drug therapy group, n=6, mean-174.438, SEM-2.915 were immunostained with BiP marker and detected the expression in b and no self-recovery in c comparing with a and b. e, vehicle group, n=6, mean-54.492, SEM-1.922; f, one-week BAD group, n=6, mean-80.892. SEM- 2.111; g, self-recovery group, n=6, mean-82.107, SEM-2.500; and h, drug therapy group, n=6, mean-65.169, SEM-3.487 were immunostained with ATF6 marker and show the decreased expression in h, no self-recovery in g comparing with e and f. \n","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-1128634/v1/440aea78ff9db94f665e2a5a.png"},{"id":16211118,"identity":"f46535e6-ad60-45a3-ace9-f8a0b6388f33","added_by":"auto","created_at":"2021-12-06 15:18:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3770716,"visible":true,"origin":"","legend":"Regeneration or remyelination of demyelinated rats after drug Teriflunomide therapy for 2 week alternate days soon after 48 hour post BAD.\nImmunohistochemistry of MBP, CNPase, and S100 markers followed by microscopic evaluations. e, Vehicle group, n=6, mean-130.475, SEM-1.007; f, one-week BAD group, n=6, mean-112.168, SEM-3.451; g, self-recovery group, n=6, mean-99.294, SEM-1.485; and h, drug Teriflunomide treated group, n=6, mean-120.132, SEM-3.132 represents immunostain of MBP marker and reveals the increased expression in h, no self-recovery in g comparing with e and f; i, vehicle group, n=6, mean-77.651, SEM-2.143; j, one-week BAD group, n=6, mean-54.620, SEM-2.941; k, self-recovery group, n=6, mean-45.785, SEM-2.794; and l, drug Teriflunomide treated group, n=6, mean-56.596, SEM-3.611 were immunostained with CNPase marker and show the increased expression in l and no self-recovery k comparing with I and j; m, Vehicle group, n=6, mean-182.117, SEM-1.446; n, one-week BAD group, n=6, mean-138.398, SEM-4.450; o, self-recovery group, n=6, mean-134.982, SEM-3.103; and p, drug therapy group, n=6, mean-153.213, SEM-3.300 immunostained with S100 marker and observed the increased expression in p, no self-recovery o comparing with m and n. ","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-1128634/v1/f8aa1f6e7d8ecb58ca2f267b.png"},{"id":16211114,"identity":"8a8ece86-c8d7-452c-b0b4-f255460152fc","added_by":"auto","created_at":"2021-12-06 15:18:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2955108,"visible":true,"origin":"","legend":"2D – gel electrophoresis comparative studies of rat brain mitochondria \nrepresents sham, damage, and treatment groups. Red color indicates the detection of high-intensity protein spots and green color indicates the common protein spots in three groups. (All the gels are analyzed for the detection of up-regulated, down-regulated, and new proteins by IMP software version7 GE Healthcare and shortlisted proteins with the high intensity followed by MALDI TOF MS analysis).","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-1128634/v1/da2a2873da12de3f58096b6d.png"},{"id":16211119,"identity":"4523d6c6-6052-48cf-ab1f-4b0baf59d66f","added_by":"auto","created_at":"2021-12-06 15:18:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5675131,"visible":true,"origin":"","legend":"Identification of mitochondrial down-regulated proteins in inner membrane, cristae and matrix of rat trauma brain \nrepresents the down-regulated proteins in the rat trauma brain mitochondria and up-regulated in treatment groups analyzed by IMP software version7 GE Healthcare. COX7B- Cytochrome c oxidase subunit 7B, mass-9047, down-regulated after trauma and slightly noticeable up-regulation observed after treatment, localization-mitochondria, function- Component of the cytochrome c oxidase, the last enzyme in the mitochondrial electron transport chain which drives oxidative phosphorylation. Plays a role in proper central nervous system (CNS) development in vertebrates; PRDX3- Thioredoxin-dependent peroxide reductase, mass-28563, down-regulated after trauma and slightly noticeable up-regulation observed after treatment, localization-mitochondria, function- Thiol-specific peroxidase that catalyzes the reduction of hydrogen peroxide and organic hydroperoxides to water and alcohols, respectively. Plays a role in cell protection against oxidative stress by detoxifying peroxides; SPYA- Serine--pyruvate aminotransferase, mass-46204, down-regulated after trauma and up-regulation observed after treatment, localization-mitochondria, function- Dual metabolic roles of gluconeogenesis (in the mitochondria) and glyoxylate detoxification (in the peroxisomes); ACPM- Acyl carrier protein, mass-17644, down-regulated after trauma and slightly noticeable up-regulation observed after treatment, localization: Mitochondria, function: Carrier of the growing fatty acid chain in fatty acid biosynthesis which functions in the transfer of electrons from NADH to the respiratory chain; M2OM- mitochondrial 2-oxoglutarate/malate carrier protein, mass-34393, down-regulated after trauma and slightly noticeable up-regulation observed after treatment, localization-mitochondria, functions- maintains mitochondrial fusion and fission events, and the organization and morphology of cristae. Involved in the regulation of apoptosis; MTCH1- Mitochondrial carrier homolog 1, mass-41880, down-regulated in trauma brain group and total protein degraded and disappeared in the treatment group, localization-mitochondria, function-potential mitochondrial transporter. May play a role in the regulation of apoptosis; TTC19- Tetratricopeptide repeat protein 19, mass-41649, disappeared in the trauma brain group and up-regulated after treatment, localization-mitochondria, function-required for the preservation of the structural and functional integrity of mitochondrial respiratory complex III by allowing the physiological turnover of the Rieske protein UQCRFS1. Involved in the clearance of UQCRFS1 N-terminal fragments, which are produced upon incorporation into the complex III and whose presence is detrimental for its catalytic activity; AATM- Aspartate aminotransferase, mass-47683, degraded or absent in trauma group and up-regulated in the treatment group, localization-mitochondria, function-catalyses the irreversible transamination of the L-tryptophan metabolite L-kynurenine to form kynurenic acid (KA) and is important for metabolite exchange between mitochondria and cytosol, and amino acid metabolism. Facilitates cellular uptake of long-chain free fatty acids; IPYR2- Inorganic pyrophosphatase 2, mass-38546, down-regulated in traumatic brain and up-regulated in the treatment group, localization-mitochondria, function-hydrolyses inorganic pyrophosphate. This activity is essential for correct regulation of mitochondrial membrane potential, and mitochondrial organization and function; All the mention protein are with high peptide coverage sequences (see supporting file for the down-regulated proteins peptide sequences). Identifications are done by MALDI/TOF MS, flex analysis 3.2 followed by mascot search engine software tools.","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-1128634/v1/ec179b8847d7a627dd3b37b7.png"},{"id":16211115,"identity":"0e60cdaa-4e39-4e41-9b21-2f3d8910ecd8","added_by":"auto","created_at":"2021-12-06 15:18:16","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3346941,"visible":true,"origin":"","legend":"Identification of mitochondrial up-regulated proteins in inner membrane and matrix of rat trauma brain \nrepresents down-regulated protein in the trauma brain group compared with the sham group. ACADL- long-chain specific acyl-CoA dehydrogenase, mass-48277, down-regulated in the trauma group and noticeable up-regulation in the treatment group, localization-mitochondria, function-long-chain specific acyl-CoA dehydrogenase is one of the acyl-CoA dehydrogenases that catalyze the first step of mitochondrial fatty acid beta-oxidation, an aerobic process breaking down fatty acids into acetyl-CoA and allowing the production of energy from fats; GCSH-glycine cleavage system H protein, mass-18701, up-regulated in the trauma brain group and down-regulated in the treatment group which is equal to the sham group, localization-mitochondria, function-the glycine cleavage system catalyzes the degradation of glycine. The H protein (GCSH) shuttles the methylamine group of glycine from the P protein (GLDC) to the T protein (GCST); RM55- 39S ribosomal protein L55, mitochondrial, mass-15226, totally absent or degraded in the trauma brain group but high increased expression levels are observed in the treatment group, localization-mitochondria, function- Predicted to be involved in translation and localized to mitochondrial large ribosomal subunit which involves in the protein synthesis within the mitochondrion; CH10, Hspe1- 10 kDa Heat shock protein, mass-10956, up-regulated in the trauma brain group and increased level of expression observed in the treatment group, localization-mitochondria, function-co-chaperonin implicated in mitochondrial protein import and macromolecular assembly. Together with Hsp60, facilitates the correct folding of imported proteins. May prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix; All the mention protein are with high peptide coverage sequences (see supporting file for the down-regulated proteins peptide sequences), identifications are done by MALDI/TOF MS, flex analysis 3.2 followed by mascot search engine software tools.\n","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-1128634/v1/39bc6118263f701c68d51e0c.png"},{"id":17544629,"identity":"d0f0fe0b-64be-4af9-8c95-963d730b654d","added_by":"auto","created_at":"2022-01-21 19:44:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9369341,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1128634/v1/c2d9434e-315a-4548-8a8f-6e13b85c4685.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eTraumatic Brain Injury: Crosstalk Between ER and Mitochondria Contributes to Oligodendrocyte Cell Death and Demyelination\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTraumatic brain injury (TBI) usually results from a violent blow to the head caused by concussive head injury (external mechanical force) or diffuse injury (crash injuries, athletic head injuries)[\u003ca href=\"#_ENREF_1\" title=\"Shi, 2015 #25\"\u003e1-3\u003c/a\u003e]. TBI causes the pathological alterations in the neuronal cells within the grey matter. But, according to recent studies the pathological changes depend on the impact or intensity of damage occurring to the brain and have highlighted the equal importance of white matter integrity which has axons[\u003ca href=\"#_ENREF_4\" title=\"Williamson, 2018 #5\"\u003e4-6\u003c/a\u003e]. White matter atrophy has a low probability of repair, due to the loss of neuron cell bodies in the corresponding regions[\u003ca href=\"#_ENREF_7\" title=\"Marion, 2018 #8\"\u003e7-9\u003c/a\u003e]. Demyelination is the term used in white matter injuries and is characterized by loss of the myelin sheath and oligodendrocytic cell death.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the injured brain activated macrophages trigger cytokines causing inflammation at the injured site inducing ER stress sensors[\u003ca href=\"#_ENREF_10\" title=\"Sprenkle, 2017 #51\"\u003e10\u003c/a\u003e]. \u0026nbsp;ER mediates the unfolded protein response (UPR) signaling pathway and is classified into three domain cascade sensors, protein kinase R (PKR)-like ER R kinase (PERK), activating transcription factor6 (ATF6), and inositol requiring enzyme 1 (IRE1)[\u003ca href=\"#_ENREF_11\" title=\"Logsdon, 2014 #11\"\u003e11\u003c/a\u003e]. BiP which is a protein chaperone, maintains the physical interactions between the ER stress sensors and unfolded proteins in the ER lumen to prevent their activation[\u003ca href=\"#_ENREF_12\" title=\"Shen, 2002 #37\"\u003e12\u003c/a\u003e]. During ER stress the abnormal accumulation of unfolded proteins results in the release of BiP from the ER stress sensors and activates ATF6 which translocate into the cytosol[\u003ca href=\"#_ENREF_13\" title=\"Hood, 2018 #44\"\u003e13\u003c/a\u003e]. Proteases S1P and S2P at golgi apparatus cleaves the translocated ATF6 sensors into fragments which moves into the nucleus that causes more inflammation in several neurodegenerative diseases[\u003ca href=\"#_ENREF_14\" title=\"Reverendo, 2019 #14\"\u003e14\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_15\" title=\"Sprenkle, 2017 #15\"\u003e15\u003c/a\u003e].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMitochondria play an important role in cell survival and cell death mechanisms. The outer membrane is composed of integral proteins and voltage-dependent anion channels (VDAC) which is the most common pathway in exchanging the ions from the cytosol like Ca\u003csup\u003e2+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, and Na\u003csup\u003e+\u003c/sup\u003e through OM by ER-mitochondrial junction called mitochondrial associated membrane (MAM). The inner membrane is composed of many compartmentalized functional proteins and molecules that require special membrane transporters to enter and exit the mitochondrial matrix. IM proteins involved in oxidative phosphorylation, ATP synthase, transporter proteins which regulate enter and exit metabolites exchanges in the mitochondrial matrix, import machinery, mitochondrial fusion, and fission. \u0026nbsp;Whereas the respiratory chain protein complexes are accumulated in the cristae matrix[\u003ca href=\"#_ENREF_16\" title=\"Kühlbrandt, 2015 #18\"\u003e16\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_17\" title=\"Zorova, 2018 #19\"\u003e17\u003c/a\u003e].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTF is an immunomodulatory, anti-inflammatory and multi-functional drug for multiple sclerosis (MS) patients[\u003ca href=\"#_ENREF_18\" title=\"Bar-Or, 2014 #41\"\u003e18\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_19\" title=\"Rommer, 2019 #21\"\u003e19\u003c/a\u003e]. Treatment with TF inactivates the immune cells and reduces the cytokine secretion from macrophages. In addition, TF promotes oligodendroglia precursor cells (OPCs) maturation which results in recovery and remyelination[\u003ca href=\"#_ENREF_20\" title=\"Göttle, 2018 #22\"\u003e20-22\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eIn this study, we elucidated the role of cytokines secretion from the activated macrophages followed by ER stress activation. Further, we evaluated the impact of ER stress on mitochondrial damage and observed the ER-mitochondria cross-talk promotes oligodendroglia cell death and aggravates demyelination. In addition, we have evaluated the remyelination and recovery by treatment with TF.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal selection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSixty Sprague Dawley (SD) rats (3 months old, 220 \u0026ndash; 250gms male) were purchased from the National Institute of Nutrition (NIN), Hyderabad. As per the experimental requirement, animals were acclimatized in the Animal house facility, University of Hyderabad before 10 days of the experiments, (Reg number: 151/1999/CPCSEA). Experiments were approved by the institutional animal ethical committee (UH/IAEC/PBP/2019 \u0026ndash; I/03). The animals were housed in cages with an ambient temperature of 24\u0026deg;C, constant and standard air humidity and natural day/night cycles, quality food and water \u003cem\u003ead libitum.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTBI animal model of brain axonal damage (BAD)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSD rats were anesthetized and BAD were induced by a well-established weight-drop method. In Sham group (n=6) animals were placed on the weight drop platform without inducing BAD, post-experimental 48-hour point (n=6), post-experimental 1-week point (n=6), Vehicle treated (n=6) [carboxymethylcellulose made up to 0.06% (w/v) in water, to which Tween 80 was added to reach a final concentration of 0.5% (v/v)] administrated orally for two weeks after post BAD. One group of rats were allowed for two weeks for self-recovery (n=6) and Teriflunomide therapy (n=6). Before performing the weight drop experiment, all the experimental instruments were sterilized using 70% ethyl alcohol. Animals were weighed and anesthetized through intraperitoneal administration of anesthesia: Avertin (2,2,2 - Tribromoethanol 1.25gms, 2 - Methyl 2 - Butanol - 2.5ml per 100ml) Dose: 150mg/kg or 12.4ml/kg body weight of the animal model. Site: Intraperitoneal, Volumes: 3ml/250gms of selected SD rats. 200gms of stainless steel iron ball was dropped on the anesthetized rat head from a height of 60cms to induce BAD. BAD-induced rats were shifted to the recovery chamber which maintains the temperature 37\u0026deg;C followed by accommodating in cages with food and water in different day points 48 hours, 1 week, 2 weeks self-recovery, and Teriflunomide therapy for a 2-week point post BAD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTF administration to the BAD induced rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter inducing BAD, a batch of n=6 rats was maintained for the TF treatment. TF was dissolved in vehicle: carboxymethylcellulose made up to 0.06% (w/v) in water, to which Tween 80 was added to reach a final concentration of 0.5% (v/v). TF was administrated at a concentration of 10mg/ml orally using rat oral gavage at a volume of 1.0ml/kg body weight for two weeks every alternate day after post-48-hour injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEuthanasia followed by animal perfusion fixation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter BAD, rats were euthanized by an overdose of sodium pentobarbital (100mg/kg) injected intraperitoneally at different time points. Rats were then perfused and the brain samples were excised for further studies. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePerfusion fixation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChemical composition: i) Stock solution: 8% paraformaldehyde: Add 80gm paraformaldehyde to 1000ml distilled water. Stir the solution while heating (not exceed more than 60- 65\u0026deg;c). Reduce heat and add 2-3ml of 1.0M NaOH with a dropper. Filter and store at 4\u0026deg;c or up to 1 month.\u003c/p\u003e\n\u003cp\u003eii) Prepare 0.2 M Sodium Phosphate Buffer, pH 7.4: Add 27.8gm of NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e to 1litre distilled water for sodium phosphate monobasic stock; add 28.4gm of Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e to 1litre of distilled water for sodium phosphate dibasic stock. The final stock of sodium phosphate buffer was prepared by adding 810ml of the monobasic stock to 190ml of the dibasic stock and pH adjusted at 7.4\u003c/p\u003e\n\u003cp\u003eiii) 4% Paraformaldehyde Fixative preparation: Add equal parts 1:1 ratio of 8% paraformaldehyde stock to 0.2M Sodium Phosphate Buffer. 4% paraformaldehyde was prepared freshly before 72hour experimentation plans.\u003c/p\u003e\n\u003cp\u003eiv) Phosphate buffered saline preparation, pH 7.4: Add NaCl-9gms, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e-144mg, Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e-795mg to the 1litre of distilled water and pH adjusted to 7.4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProcedure\u0026nbsp;\u003c/strong\u003eEuthanized animals were placed on the shallow tray filled with crushed ice. Beneath the rib cage lateral incisions of 5-6 cm were made through the integument and abdominal wall carefully, to separate the liver from the diaphragm and followed by another small incision in the diaphragm using the curved, blunt scissors which continued till the entire rib cage along with the pleural cavity. Lung displacement was done using curved, blunt scissors and a cut was made through the rib cage up to the collarbone followed by a similar cut on the contralateral side. The tip of the sternum was clamped with the haemostat and placed over the head. A small incision to the posterior end of the left ventricle using iris scissors was made and a 15-gauge blunt- or olive-tipped perfusion needle had inserted through the cut ventricle into the ascending aorta. The heart was clamped using a haemostat to secure the needle and prevents leakage. Finally, an incision was made to the animal\u0026apos;s right atrium using iris scissors to create as large an outlet as possible without damaging the descending aorta. Outlet port perfusion equipment was attached to the needle base by avoiding air bubbles and 80mm Hg pressure of the manometer bulb was maintained throughout the buffer infusion period with a proper needle angle adjustment to achieve the maximum flow rate. Fixation was almost pumped to the animal and the clear running of the fluid was monitored. The clearance of the liver was observed which is indicative of proper perfusion. Fixation tremors had been observed within seconds; which can be considered as the true time of fixation indicator. Further, the pressure was gradually increased up to a maximum of 130mm Hg\u003csub\u003e2\u003c/sub\u003e to maintain a steady flow rate. The outlet valve was closed soon after the completion of the fixation process followed by the ending time recordings and the stiffness of the animal was observed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParaffin-embedded tissue sections\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFreshly collected brain samples were sliced into 3mm slices and fixed with 10% paraformaldehyde or formalin for 48 hr at room temperature. After fixation samples were washed under running tap water for 1 hrfollowed by dehydration steps using 70%, 80%, 95% alcohol changes 30min each and 3 changes of 100% alcohol for 1hour each. Brain samples were cleared in 1 change of xylene for 5min and another step of xylene + melted paraffin 1: 1 ratio for 5mins followed by immersing the samples in 3 changes of paraffin (Paraplast\u0026reg; - polyisobutylene mixture, catalogue no P3558 \u0026ndash; SIGMA \u0026ndash; ALDRICH\u0026reg;) 1hour each and the brain slices were embedded in a paraffin block. Brain slice blocks were fixed to the microtome \u003cem\u003e(LEICA RM2145)\u003c/em\u003e and 10\u0026micro;m sections were done and floated in a 40\u0026deg;c maintained water bath containing clear distilled water. Tissue sections were transferred carefully to the glass slides for further neuropathological studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematoxylin and eosin staining (H \u0026amp; E)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFormalin-fixed paraffin-embedded FFPE brain sections were firstly deparaffinized on the heating pad or heating plate and followed by tissue clearing for 2min each in two xylene changes. Slides containing FFPE sections were rehydrated accordingly in two changes of 100% alcohol for 3min each, thereafter 95%, 80%, and 70% alcohol for 3min and followed by a 10min water wash. Processing slides were then dipped in nuclear stain (Haematoxylin) containing coupling jars for 3 \u0026ndash; 5min and shifted to the stain (eosin) for extracellular matrix and cytoplasmic staining for 2min followed by a tap water wash for 6min. After the staining procedures, sections were transferred to 30%, 50% alcohol for 6min each, 70% alcohol for 10 min, 95% alcohol, and 2 changes of 100% alcohol 6min each for dehydration and followed by tissue clearing with 2 changes of xylene, 3min each and DPX mounting followed by microscopic evaluations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrain sections were deparaffinized using a heating pad and placed or dipped in 2 changes of xylene containing coupling jars for 2min and quickly transferred to a 1:1 ratio of xylene: 100% ethanol for 3min. Tissue sections were further rehydrated in 2 changes of 100%, 95%, 70%, 50%, and 30% ethanol for 3min and rinsed in tap water for 5min. The tissue sections were transferred to antigen retrieval buffer (trypsin 0.05% in 100 ml of PBS) for 15min at 37\u0026deg;c and permeabilized (triton x \u0026ndash; 100 0.2% in PBS) for 7 \u0026ndash; 10min followed by 1 change of wash in PBS for 5min. Tissue section slides were transferred into blocking solution (BSA 1%, NGS 5% in PBS) and left for 1hour at room temperature. Approximately 100\u0026micro;l of diluted primary antibodies myelin basic protein MBP (AB clonal Catalogue No: A1664, 86 Cummings Park Dr, Woburn, MA 01801, United States), CNPase -2, 3-cyclic nucleotide 3-phosphodiesterase, GenScript, Catalogue no: A01308-40, 860 Centennial Ave., Piscataway, NJ 08854, USA), S100 Beta EP 32 (PathnSitu Catalog No: CR070 \u0026ndash; 0.1 ML Concentrated, USA-Registered Office 538, Selby Ln, Livermore, CA-94551 USA) as myelin and glial markers. \u0026nbsp;GRP 78 (76-E6): Catalogue no: Sc-13539 and ATF-6\u0026alpha; Antibody (H-280): Catalogue no: sc-22799 from Santa Cruz Biotechnology, Inc. 10410 Finnell Street, Dalla, Texas 75220, U.S.A, as ER stress markers. CD68 KP 1 (PathnSitu Catalogue no: PM113 \u0026ndash; 0.1 ML concentrated, USA-Registered Office 538, Selby Ln, Livermore, CA-94551 USA, and TNF\u0026alpha; antibody-NBP1-19532, Novus Biologicals, LLC-10730 E. Briarwood Avenue, Building IV Centennial, CO 80112, USA. (dilution 1:200 PBS with 0.02% sodium azide, 50% glycerol, Ph 7.3) were applied to the tissue sections and incubated in a humidified chamber at room temperature for 30min. Sections were transferred for washing with PBS for 2 changes of 5min. Approximately 100\u0026micro;l of diluted biotinylated secondary antibody was applied (using the antibody dilution buffer) to the sections on the slides and incubated in a humidified chamber at room temperature for 30min (protected from sunlight) followed by washing with 2 changes of PBS for 5min. DAB substrate solution (freshly prepared before use: 0.05% DAB, 0.015% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in PBS) was applied to the sections on the slides to reveal the color of antibody staining. The color development was done for less than 5min until the desired color intensity was obtained followed by washing in 3 changes of PBS for 2min each. For counterstaining slides were immersed in haematoxylin for 2 \u0026ndash; 3min as per the thickness of tissue sections followed by water wash for 15 \u0026ndash; 20min. Tissue slides were dehydrated in graded alcohol solutions of 2 changes 95% and 2 changes 100% alcohol 5min. Tissue sections were mounted by using DPX mounting solution and covered with coverslips.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondria isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRat brains were collected and finely minced by adding around 5 ml of buffer: 1 \u0026ndash; 200 \u0026mu;l of 500 mM EGTA for a final concentration of 10 mM, 0.392 g of D-mannitol for a final concentration of 215 mM, 1.25 ml of 8X mitochondria buffer (10.28 g of sucrose for a final concentration of 0.6 M, 400 mg of free-fatty acid bovine serum albumin (BSA) for a final concentration of 0.8%, 2.08 g of HEPES for a final concentration of 160 mM, pH to 7.4 and made up to 50 ml with distilled water) pH to 7.4 and made-up to 10 ml with distilled water. Minced brain with the solution was then transferred to the polytron homogenizer. Homogenized tissue samples were collected into pre-chilled microcentrifuge tubes and centrifuged at 700 \u003cem\u003eg\u0026nbsp;\u003c/em\u003efor 10 minutes at 4\u0026deg;c. Supernatant was transferred to new pre-chilled microcentrifuge tubes and pellets were discarded. The supernatant was centrifuged at 10,500 \u003cem\u003eg\u0026nbsp;\u003c/em\u003efor 10 minutes at 4\u0026deg;c and the pellet was resuspended in 500 \u0026mu;l of (buffer: 2 - Add 60 \u0026mu;l of 500 mM EGTA for a final concentration of 3 mM, 0.392 g of D-mannitol for a final concentration of 215 mM, 1.25 ml of 8X mitochondria buffer, pH to 7.4 and makeup to 10 ml with distilled water) and centrifuged at 10,500 \u003cem\u003eg\u0026nbsp;\u003c/em\u003efor 10 minutes at 4\u0026deg;c. \u0026nbsp;Final mitochondrial pellet was suspended in 100 \u0026mu;l of Buffer: 2 followed by quick-spin for final mitochondrion for a few seconds. Supplement Lysis Buffer: 1 and Buffer: 2, 1/100 volume of Protease Inhibitor Solution (100x) (i.e., if using 2 ml Disruption Buffer, add 20 \u0026mu;l Protease Inhibitor Solution. Determine protein concentration using the Bradford assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2D \u0026ndash; gel electrophoresis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRehydration of IPG strips and First dimension separation IEF (Isoelectric focusing)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe first \u0026ndash; dimension separation of 2Dgel is IEF, in this proteins are separated based on differences in their isoelectric point (pI)[\u003ca href=\"#_ENREF_23\" title=\"Kwok, 2020 #76\"\u003e23\u003c/a\u003e].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSamples were added to the rehydration tray and cover removed from the selected IPG strip pH 4-10 and gel was allowed to slide down onto the rehydration buffer in the tray. The tray was overlaid with the mineral oil to prevent the evaporation and precipitation of urea during rehydration and left for 20 hours for complete rehydration. The rehydrated IPG strips were transferred to the IEF tray by gel side up using two notches and IPG strips were overlaid with mineral oil and run the IEF accordingly to the protocol \u003cem\u003e(GE Healthcare).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIPG strip equilibration and running second dimension by SDS - PAGE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter IEF the strips were transferred to the new tray gel side up and filled with the recommended volume of equilibration buffer 1 followed by incubation for 10 min. Buffer 1 was removed and equilibration buffer 2 was added for 10 min incubation. After equilibration, IPG strips were removed and followed by a rinse with SDS \u0026ndash; PAGE running buffer, and the strips were placed on the prepared 12% SDS \u0026ndash; PAGE gel and sealed with 0.5% of agarose solution, and the electrophoresis unit was run. Comparative studies of the gels for the up-regulation and down-regulation for the detection of the protein was analyzed by \u003cem\u003eIMR software version7 GE Healthcare\u003c/em\u003e (all the above experimental protocols and buffers are from \u003cem\u003eGE Healthcare\u003c/em\u003e)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn-gel digestion and mass spectrometric analysis MS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn automated spot cutter was used to collect the spots of interest. For in-gel digestion, the gel plugs were washed with water twice for 5 min and incubated with 100% acetonitrile for 10 min. Then, the gels were dried completely for at least 30 min. The protein in the gel was digested by treatment with TPCK \u0026ndash; treated trypsin in 50 mM ammonium bicarbonate at 37\u0026deg;c overnight with gentle agitation. After digestion, peptides were extracted with 50 \u0026micro;l of 50% acetonitrile, 0.1% Trifluoroacetic acid \u0026nbsp;twice, concentrated, and extensively treated with ZipTip. Then trypsin digests were mixed with an equal volume of matrix solution, comprising of saturated dihydroxy benzoic acid in 50% acetonitrile/0.1% TFA. The samples were spotted on a MALDI target plate and subjected to mass spectrometric analysis. Mass spectrometric analysis of trypsin digestion was performed using a Q-STAR Pulser-i equipped with a MALDI ion source. Raw data to mzml file conversion was done using \u003cem\u003eFlex analysis 3.2 software\u003c/em\u003e and followed by \u003cem\u003emascot online search engine tool\u003c/em\u003e for the protein identification and peptide sequencing (Fig B).\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCytokines secretion from activated macrophages in traumatic brain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCellular damage triggered the macrophage\u0026apos;s activation and secretion of cytokines at the site of injury when compared to the normal brain. Immunostaining with macrophage marker CD68 and cytokine marker TNF\u0026alpha; demonstrated early expressions, 48 hours post BAD (Fig1). CD68 and TNF\u0026alpha; were highly expressed in the late stages, around one-week, post-BAD. Treatment using TF for two weeks on alternate days resulted in the decreased expression levels of CD68 and TNF\u0026alpha; respectively as shown in Fig4.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eER stress sensors activation via BiP ER chaperone binding protein \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the traumatic brain, secreted cytokines from the activated macrophages translocate ATF6 sensor from ER lumen to the cytosol by releasing BiP, ER chaperone binding protein (Fig2). BiP expression was observed soon after 48 hours post BAD and further increased after one-week post BAD (Fig2). \u0026nbsp;ATF6 expression was observed at 48 hours and slightly increased after one-week post BAD (Fig2). However, BiP and ATF6 expressions decreased after TF treatment (Fig5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondrial dysfunction under stress condition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe confirmed mitochondrial damage induces apoptotic cell death upon ER stress. We observed differential expression of mitochondrial proteins in the traumatic brain which were restored upon treatment with teriflunomide. Proteins were analysed using 2D-gel electrophoresis and identified by MALDI MS. \u0026nbsp;Peptide sequences were identified as \u0026nbsp;\u003cstrong\u003eCOX7B\u003c/strong\u003e- Cytochrome c oxidase subunit 7B,\u0026nbsp;\u003cstrong\u003ePRDX3\u003c/strong\u003e-\u0026nbsp;Thioredoxin-dependent peroxide reductase, \u003cstrong\u003eSPYA\u003c/strong\u003e- Serine--pyruvate aminotransferase, \u003cstrong\u003eACPM\u003c/strong\u003e- Acyl carrier protein, \u003cstrong\u003eM2OM\u003c/strong\u003e- mitochondrial 2-oxoglutarate/malate carrier protein, \u003cstrong\u003eMTCH1\u003c/strong\u003e- Mitochondrial carrier homolog 1, \u003cstrong\u003eTTC19\u003c/strong\u003e- Tetratricopeptide repeat protein 19, \u003cstrong\u003eAATM\u003c/strong\u003e- Aspartate aminotransferase, \u003cstrong\u003eIPYR2\u003c/strong\u003e- Inorganic pyrophosphatase 2, \u003cstrong\u003eGCSH\u003c/strong\u003e-glycine cleavage system H protein, \u003cstrong\u003eCH10, Hspe1\u003c/strong\u003e- 10 kDa Heat shock protein, and Mitochondrial matrix proteins as \u003cstrong\u003eACADL\u003c/strong\u003e- long-chain specific acyl-CoA dehydrogenase, \u003cstrong\u003eRM55\u003c/strong\u003e- 39S ribosomal protein L55 (Fig7, 8\u0026amp;9). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDemyelination and remyelination after Teriflunomide treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunostaining with myelin markers, myelin basic protein (MBP), CNPase, and Neuroglia S100 detected high expression in white matter tracts, in the sham group (Fig3) and the vehicle group (Fig6). Early myelin loss was observed in the post 48-hour BAD group and moderate in the one-week post BAD group (Fig3). Remyelination or recovery was observed after TF treatment by the activation, recruitment, and differentiation of resident OPCs as shown in Fig6.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe brain consists of grey and white matter regions which majorly consist of glial cells. Grey matter contains neuronal cell bodies which serve as an information processor for the CNS[\u003ca href=\"#_ENREF_1\" title=\"Shi, 2015 #25\"\u003e1\u003c/a\u003e]. On the other hand, white matter is composed of myelin-producing oligodendrocytes, and other glial cells which maintains myelin, signal transmission, and communication[\u003ca href=\"#_ENREF_7\" title=\"Marion, 2018 #8\"\u003e7\u003c/a\u003e]. Oligodendrocytes are the primary cells that are richly present in white matter and are responsible for maintaining the myelin sheath under normal conditions and for remyelination after axonal damage[\u003ca href=\"#_ENREF_24\" title=\"Simons, 2013 #27\"\u003e24\u003c/a\u003e]. Oligodendrocyte cell death causes the myelin loss and is known to be a significant factor underlying demyelination after brain injury. Increased demyelination is a major pathological condition of white matter injury-causing BADs which contributes to significant long-term sensorimotor and cognitive deficits[\u003ca href=\"#_ENREF_24\" title=\"Simons, 2013 #27\"\u003e24\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_25\" title=\"Armstrong, 2016 #28\"\u003e25\u003c/a\u003e].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo date, many TBI animal models are in use to induce neurodegeneration and help in studying the mechanism of cell death[\u003ca href=\"#_ENREF_26\" title=\"Xiong, 2013 #29\"\u003e26\u003c/a\u003e]. However, to the best of our knowledge, there are no reports representing demyelination in TBI during ER stress induced mitochondrial protein alterations. Here in the present study, we used the weight-drop method as a tool to understand the mechanism of cell death leading to demyelination. Further, we attempted to reveal role of inflammation in aggravating the demyelination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe demonstrated the early pro-inflammation by CD68 and TNF\u0026alpha; at forty-eight hour and moderate at one-week injured rat brain white matter lesions. In similar studies increased expression levels of CD68 and TNF\u0026alpha; markers were found to detect the activated macrophages and secreted cytokines[\u003ca href=\"#_ENREF_27\" title=\"Turtzo, 2014 #32\"\u003e27-29\u003c/a\u003e]. ATF6 and BiP markers detected the initial expression at forty eight hour and increased expression at one week TBI. Pro-inflammatory cytokines mediates ER stress sensor ATF6 activation, initiates the release of BiP chaperone binding protein which commences the cell death[\u003ca href=\"#_ENREF_14\" title=\"Reverendo, 2019 #14\"\u003e14\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_30\" title=\"Zhang, 2008 #34\"\u003e30\u003c/a\u003e]\u003csup\u003e,\u003c/sup\u003e [\u003ca href=\"#_ENREF_12\" title=\"Shen, 2002 #37\"\u003e12\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_31\" title=\"Bertolotti, 2000 #36\"\u003e31\u003c/a\u003e].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eMBP, CNPase (2\u0026apos;,3\u0026apos;-Cyclic-nucleotide 3\u0026apos;-phosphodiesterase) and S100 markers showed significant early myelin loss at forty eight hours which increased at one week intervals after inducing TBI. Comparably, myelin loss or demyelination was assessed by using MBP, CNPase and S100 markers in many neurological diseases[\u003ca href=\"#_ENREF_32\" title=\"Frid, 2015 #70\"\u003e32-35\u003c/a\u003e]. To our knowledge oligodendrocyte cell death under ER-stress promoting demyelination is not reported so far.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurther we demonstrated the decreased expression of CD68 and TNF\u0026alpha; markers after TF treatment; this influenced the expression of ER stress sensors ATF6 and BiP markers. Additionally, our results showed the increased expression of myelin markers MBP, CNPase, and S100 after treatment with TF. TF is an anti-inflammatory drug which helps in the inactivation of macrophages and reduces the cytokines secretion at the injured site[\u003ca href=\"#_ENREF_18\" title=\"Bar-Or, 2014 #41\"\u003e18\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_19\" title=\"Rommer, 2019 #21\"\u003e19\u003c/a\u003e]. Similar reports indicates that TF promotes oligodendrocyte precursor cell differentiation which causes remyelination in white matter lesions[\u003ca href=\"#_ENREF_20\" title=\"Göttle, 2018 #22\"\u003e20\u003c/a\u003e]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThough the mitochondrial cell death has been reported in various pathological models but the involvement of specific proteins is spars. Mitochondrial dysfunction maybe considered to be one of the early events that can cause cell death in traumatic brain[\u003ca href=\"#_ENREF_36\" title=\"Kim, 2017 #57\"\u003e36\u003c/a\u003e]. Therefore, in the present study mitochondria has been isolated from the control, traumatic brain and treated brain samples. Proteins were subjected to 2D- gel electrophoresis and identified using MALDI-MS. In proteomic studies, we observed significant changes in the mitochondrial functional proteins. Mitochondrial carrier homolog 1 (MTCH1) acts as the receptor for truncated BID (tBID) which involves in apoptosis[\u003ca href=\"#_ENREF_37\" title=\"Cogliati, 2010 #64\"\u003e37\u003c/a\u003e]. Herein we found MTCH1 down-regulated in trauma brain group and total protein degraded and disappeared in the treatment. In TBI, MTCH1 down-regulation results in signalling the tBID (pro-apoptotic protein) on the surface of mitochondria. tBID triggers the release of cytochrome c by causing oligomerization of BAX/BAK (apoptotic regulators) and results in outer membrane permeabilization. On the other hand MTCH1 down-regulation increases the opening of voltage dependent anion channels (VDAC) leading to the membrane potential loss and release of cytochrome c. Mitochondrial 2-oxoglutarate/malate carrier protein (M2OM) involves in the regulation of apoptosis[\u003ca href=\"#_ENREF_38\" title=\"Xu, 2006 #65\"\u003e38\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_39\" title=\"Pizzorno, 2014 #66\"\u003e39\u003c/a\u003e]. This was down-regulated after TBI and slightly noticeable up-regulation was observed after treatment. Reduced expression of M2OM mitochondrial inner membrane protein in traumatic brain can transport the glutathione (GSH) from the cytosol into the mitochondrial matrix promotes apoptosis. Additionally, over accumulation of the GSH in the mitochondrial matrix promotes the BAX/BAK activation (apoptotic regulars) and cytochrome c release. Restoration of M20M after treatment can help in normalising the transportation of GSH from cytosol to mitochondrial matrix. Cytochrome c oxidase subunit 7B (COX7B) functionally drives oxidative phosphorylation and plays an important role in proper central nervous system development in vertebrates. We demonstrated COX7B down-regulation after TBI and slight noticeable up-regulation was observed after treatment, the reduced expression of COX7B in the mitochondria indicates that this must have released into the cytosol and forms apoptosis protease activating factor (APAF1) complex thereby activating caspase cascade promoting the apoptosis. Whereas restored COX7B after treatment in the mitochondria reduces the activation of apoptotic pathway. Thioredoxin-dependent peroxide reductase (PRDX3) functionally protects the cell against oxidative stress by detoxifying peroxides[\u003ca href=\"#_ENREF_40\" title=\"Szeliga, 2020 #59\"\u003e40\u003c/a\u003e]. In the present study PRDX3 down-regulated in traumatic brain samples and slightly up-regulated after treatment. Decreased expression of PRDX3 led to the increased oxidative stress in the mitochondria, this might have responsible for the cell death in our model. However, upon TF treatment PRDX3 expression \u0026nbsp;restored, this appeared to have a protective role against traumatic brain mitochondrial damage. \u0026nbsp;Serine pyruvate amino transferase (SPYA) involves in gluconeogenesis and glyoxylate detoxification[\u003ca href=\"#_ENREF_41\" title=\"Ichiyama, 2011 #74\"\u003e41\u003c/a\u003e]. We noticed SPYA down-regulated in TBI and up-regulated after treatment. Down-regulation of SPYA influences in the breakdown of metabolic processes and must have aggravated the cell death leading to demyelination in TBI whereas recovery was observed after the treatment. Long chain specific acyl-CoA dehydrogenase (ACADL) catalyses the first step of mitochondrial fatty acid beta-oxidation, an aerobic process[\u003ca href=\"#_ENREF_42\" title=\"Zhao, 2020 #61\"\u003e42\u003c/a\u003e]. In our study ACADL was down-regulated in the trauma brain and up-regulated after treatment. \u0026nbsp;After using TF, we showed the restoration of ACADL expression can promote cell cycle arrest and inhibit cell proliferation and growth. Acyl carrier protein (ACPM) involves in the transfers of electrons from NADH to the respiratory chain[\u003ca href=\"#_ENREF_43\" title=\"Feng, 2009 #62\"\u003e43\u003c/a\u003e]. ACPM down-regulation was noticed in TBI and slight up-regulation after treatment. Down-regulated ACPM imbalances the functions of electron transport chain. Whereas functioning of the electron transport chain and maintenance of normal mitochondrial membrane potential can be restored after TF treatment. Tetratricopeptide repeat protein (TTC19) plays a key role in the preservation of structural and functional integrity of mitochondrial respiratory complex III[\u003ca href=\"#_ENREF_44\" title=\"Bottani, 2017 #63\"\u003e44\u003c/a\u003e]. TTC19 disappeared in the trauma brain group and up-regulated after treatment. Disappearance of TTC19 can lead to mitochondrial respiratory complex chain III deficiency. However, after TF treatment, reappearance of the TTC19 can restore the complex III deficiencies, progressive neurological and metabolic decline. 39S ribosomal protein (RM55) involves in the protein synthesis within the mitochondria[\u003ca href=\"#_ENREF_45\" title=\"Shcherbik, 2019 #67\"\u003e45\u003c/a\u003e]. We found RM55 totally absent or degraded in the trauma brain group but increased expressions were found in the treated. In traumatic brain RM55 fails\u0026nbsp;in protein synthesis and causes adenosine tri-phosphate (ATP) deficiency within the mitochondria. TF treatment can help in mitochondrial protein synthesis recovery and ATP production. \u0026nbsp;10 kDa heat shock protein (CH10, Hspe1) prevents misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix[\u003ca href=\"#_ENREF_46\" title=\"Kim, 2020 #68\"\u003e46\u003c/a\u003e]. CH10 Hspe1 up-regulated in the traumatic brain group and increased expression was observed in the treated. Up-regulated CH10, Hspe1 represents the prevention of protein denaturation and aggregation by refolding. After treatment with TF, normalisation of CH10, Hspe1 was noted. Aspartate aminotransferase (AATM) exchanges metabolites between mitochondria and cytosol, including amino acid metabolism. We observed the degradation or absence of AATM in traumatic brain and up-regulation after treatment. \u0026nbsp;In similar studies AATM levels were decreased soon after post blast exposure TBI[\u003ca href=\"#_ENREF_47\" title=\"Arun, 2013 #69\"\u003e47\u003c/a\u003e]. Inorganic pyrophosphatase 2 (IPYR2) regulates mitochondrial membrane potential, and mitochondrial organization and function[\u003ca href=\"#_ENREF_48\" title=\"Kennedy, 2016 #75\"\u003e48\u003c/a\u003e]. We demonstrated down-regulation of IPYR2 in traumatic brain and up-regulation after treatment. Down-regulation of IPYR2 influences in the functional loss of mitochondria and restores after the treatment with TF. Proteomics of mitochondrial expression level in sham, traumatic brain and treated groups reveal the prominent changes of proteins which are mainly involved in activating the caspase cascade, cytochrome c release, induction of the apoptosis, detoxification, respiratory chain, exchange of metabolite and restoration or refolding of denatured proteins in the mitochondria. Earlier research predicts ER releases more metabolites under stress conditions and mitochondria uptakes the excess Ca\u003csup\u003e2+\u003c/sup\u003e through VDAC at the MAM junction[\u003ca href=\"#_ENREF_49\" title=\"Manalo, 2018 #53\"\u003e49\u003c/a\u003e]. Calcium overload in mitochondria induces permeability transition pore which promotes apoptotic cell death at the site of injury[\u003ca href=\"#_ENREF_50\" title=\"Marchi, 2014 #39\"\u003e50\u003c/a\u003e]. After treatment with TF significant changes were noticed, which suggests restoration of mitochondrial functional proteins.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOverall, our findings explain the involvement of activated macrophages in ER stress sensors activation. Proteomics studies demonstrated the cross-talk between ER and mitochondria through mitochondrial dysfunction followed by oligodendrocyte cell death in white matter lesions causing demyelination. Treatment with TF reduced the inflammation by immunomodulation and promoted recovery or remyelination and can be used as a remyelination therapy for TBI patients. \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eCNS:\u0026nbsp;\u003c/strong\u003eCentral nervous system\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTBI or TBIs:\u0026nbsp;\u003c/strong\u003eTraumatic brain injury or Injuries\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;BAD:\u0026nbsp;\u003c/strong\u003eBrain axonal damage\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eER:\u0026nbsp;\u003c/strong\u003eEndoplasmic reticulum\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiP:\u0026nbsp;\u003c/strong\u003eImmunoglobulin-heavy-chain-binding protein\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATF6:\u0026nbsp;\u003c/strong\u003eActivating transcription factor 6\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTNF\u0026alpha;:\u0026nbsp;\u003c/strong\u003eTumour necrosis factor alpha\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMAM:\u0026nbsp;\u003c/strong\u003eMitochondrial associated membrane\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVDAC:\u0026nbsp;\u003c/strong\u003eVoltage dependent anion channels\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePTP:\u0026nbsp;\u003c/strong\u003ePermeability transition pores\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD68:\u0026nbsp;\u003c/strong\u003eCluster of differentiation 68\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMBP:\u0026nbsp;\u003c/strong\u003eMyelin basic protein\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTF:\u0026nbsp;\u003c/strong\u003eTeriflunomide\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUPR:\u0026nbsp;\u003c/strong\u003eUnfolded protein response\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePKR:\u0026nbsp;\u003c/strong\u003eProtein kinase R\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePERK:\u0026nbsp;\u003c/strong\u003e(PKR) \u0026ndash; Like endoplasmic reticulum kinase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIRE1:\u0026nbsp;\u003c/strong\u003eInositol requiring enzyme1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSD:\u0026nbsp;\u003c/strong\u003eSprague Dawley\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFFPE:\u0026nbsp;\u003c/strong\u003eFormalin fixed paraffin embedded\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOPCs:\u0026nbsp;\u003c/strong\u003eOligodendroglia precursor cells\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePPB and RRSR thank NATCO Pharma for Teriflunomide. The authors acknowledge the help of Dr. Ranjani Chakravarthy, MD (Pathology), FRCPath (UK), Managing Director \u0026amp; Consultant Histopathologist, Cpathlabs (India). Tirumala chari, Histopathologists, Cpathlabs \u0026amp; PathnSitu for their help in Neuropathological studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinancial assistance from the Department of Science and Technology (DST- India) (Grant no: SR/CSRI/196/2016), Department of Biotechnology (DBT-India) (Grant No. BT/PR18168/MED/29/1064/2016). RRSR is thankful to DST \u0026ndash; Inspire Fellowship under the Ref.no: DST/INSPIRE/03/2015/003047\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData generated and analysed during this study are available from the authors on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAffiliation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Biotechnology and Bioinformatics, School of Life sciences, University of Hyderabad, Hyderabad-500046 (T.S), INDIA;\u003cstrong\u003e\u0026nbsp;Contact details: Ph. No:\u0026nbsp;\u003c/strong\u003e7993379999, 040-23134584/4684;\u003cstrong\u003e\u0026nbsp;Email id:\u0026nbsp;\u003c/strong\[email protected]; [email protected];\u003c/p\u003e\n\u003cp\u003eRahul Shankar Rao Rayilla and Prakash Babu Phanithi\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRRSR performed animal experiments; PPB and RRSR analyzed and interpreted the data. RRSR drafted the work, PPB revised it critically and both the authors have approved the submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrakash Babu Phanithi\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval for animal experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiments were approved by the Institutional animal ethical committee IAEC (UH/IAEC/PBP/2019 \u0026ndash; I/03). As per the experimental requirement, animals were acclimatized in the Animal house facility, University of Hyderabad before 10 days of the experiments, (Reg number: 151/1999/CPCSEA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors provide consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShi H, Hu X, Leak RK, Shi Y, An C, Suenaga J, Chen J, Gao Y: \u003cstrong\u003eDemyelination as a rational therapeutic target for ischemic or traumatic brain injury.\u003c/strong\u003e \u003cem\u003eExp Neurol \u003c/em\u003e2015, \u003cstrong\u003e272:\u003c/strong\u003e17-25.\u003c/li\u003e\n\u003cli\u003eCernak I: \u003cstrong\u003eUnderstanding blast-induced neurotrauma: how far have we 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pro-inflammation, Teriflunomide therapy, remyelination","lastPublishedDoi":"10.21203/rs.3.rs-1128634/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1128634/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eDemyelination is one of the manifestations of traumatic brain injury (TBI). Mechanisms underlying oligodendrocyte cell death in white matter lesions have not been fully studied. In the present study our focus is to investigate how the pro-inflammation contributes to the ER stress, mitochondrial protein alteration leading to oligodendrocyte cell death that aggravates demyelination in TBI.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eWeight drop method was used to induce the TBI. Immunohistochemical studies were done to understand the role of pro-inflammation in activating ER-stress leading to cell death and demyelination. Proteomic analysis of mitochondria was done using 2D-gel electrophoresis. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to identify the mitochondrial functional protein changes and their role in cell death causing demyelination. Remyelination was assessed by treating with teriflunomide (TF).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eHistopathology studies revealed the infiltration of activated macrophages leading to cytokines secretion which initiates ATF6 activation by releasing BiP, the endoplasmic reticulum (ER) chaperone binding protein and unfolded proteins at the site of ER lumen. Activated ER stress alters the mitochondrial functional proteins leading to oligodendrocyte cell death causing white matter lesions and demyelination. Treatment with TF inactivates the macrophages and reduces the cytokines secretion at the site of injury promoting recovery and remyelination.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eOur findings confirm pro-inflammation activates the ER stress sensors. The cross-talk between ER and mitochondria leads to cell death followed by demyelination. TF acts as an anti-inflammatory drug promoting oligodendroglial precursor cells maturation for recovery and secretion of myelin.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Traumatic Brain Injury: Crosstalk Between ER and Mitochondria Contributes to Oligodendrocyte Cell Death and Demyelination","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-12-06 15:18:14","doi":"10.21203/rs.3.rs-1128634/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43387c62-1c45-4946-b4a4-12951b30f4c0","owner":[],"postedDate":"December 6th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":8963773,"name":"Neurology"},{"id":8963774,"name":"Immunology"}],"tags":[],"updatedAt":"2022-01-21T19:44:07+00:00","versionOfRecord":[],"versionCreatedAt":"2021-12-06 15:18:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1128634","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1128634","identity":"rs-1128634","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}