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

doi:10.21203/rs.3.rs-1128634/v1

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Research Article

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

https://doi.org/10.21203/rs.3.rs-1128634/v1

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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

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 Ca²⁺, 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.

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 NaH₂PO₄ to 1litre distilled water for sodium phosphate monobasic stock; add 28.4gm of Na₂HPO₄ 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, KH₂PO₄-144mg, Na₂HPO₄-795mg 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 Hg₂ 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% H₂O₂ 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).

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.

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 Ca²⁺ 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.

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.

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

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

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No competing interests reported.

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

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