subcellular stress response after traumatic brain injury
TRANSCRIPT
JOURNAL OF NEUROTRAUMAVolume 24, Number 4, 2007© Mary Ann Liebert, Inc.Pp. 599–612DOI: 10.1089/neu.2006.0186
Subcellular Stress Response after Traumatic Brain Injury
JESSIE S. TRUETTNER,1 BINGREN HU,2 OFELIA F. ALONSO,1 HELEN M. BRAMLETT,1KOICHI KOKAME,3 and W. DALTON DIETRICH1
ABSTRACT
Traumatic brain injury (TBI) initiates a complex genetic response that may include the expressionof organelle specific stress genes. We investigated the effects of brain trauma on the expression ofa number of stress genes by in situ hybridization and Western blot analysis including the endo-plasmic reticulum (ER) stress gene grp78, ER protein processing enzymes calnexin and protein disul-phide isomerase (PDI), the mitochondrial stress gene hsp60, and the cytoplasmic stress gene hsp70.Male Sprague-Dawley rats were subjected either to sham-surgery or moderate (1.8–2.2 atm)parasagittal fluid-percussion (F-P) brain injury followed by 30 min of either normoxic or hypoxic(30–40 mm Hg) gas levels. Expression of grp78 was increased in the ipsilateral cerebral cortex anddentate gyrus beginning 4 h after trauma plus hypoxia. Similarly, mRNA encoding the mitochon-drial hsp60 was induced in the ipsilateral outer cortical layers at 4–24 h after TBI plus hypoxia.Calnexin and PDI mRNAs were not significantly altered following TBI with or without secondaryhypoxia. In contrast, mRNA of the cytoplasmic hsp70 was strongly induced at 4 h after brain in-jury in multiple brain regions within the injured hemisphere, and this expression was greatly en-hanced by secondary hypoxia. Because subcellular stress gene expression may reflect where un-folded or damaged proteins are abundant, these findings suggest that abnormal proteins are localizedmainly in the cytoplasm, and to a lesser degree in the ER lumen and mitochondria after braintrauma. Thus, distinct parts of the cellular machinery respond to traumatic and metabolic stressesin specific ways.
Key words: hypoxia; stress genes; subcellular; traumatic brain injury; unfolded protein response
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Departments of 1Neurological Surgery and 2Neurology, Neurotrauma Research Center. The Miami Project to Cure Paralysis,University of Miami Miller School of Medicine Miami, Florida.
3National Cardiovascular Center Research Institute, Osaka, Japan.
INTRODUCTION
TRAUMATIC BRAIN INJURY (TBI) is characterized by pri-mary and secondary injury mechanisms (Bramlett
and Dietrich, 2004; Laurer and McIntosh, 1999). The pri-mary, immediate effect is the result of the mechanicaltrauma itself, which causes disruption of the brainparenchyma and blood vessels, leading to ionic pertur-
bations and a breakdown of the blood–brain barrier(Cortez et al., 1998; Dietrich et al., 1994; Katayama etal., 1990). This initial trauma triggers secondary post-traumatic cascades that can include excitotoxicity, freeradical production, ischemia, inflammation, and apopto-sis, all of which lead to expansion of the lesion and sub-sequent secondary cellular injury (Carlos et al., 1997;Chan 2001; Keane et al., 2001; Lee et al., 1999; McIn-
tosh et al., 1997). Evidence for early ischemic/hypoxicepisodes exacerbating injury has also been reported fol-lowing severe TBI, along with an increase in intracranialpressure and repeated episodes of cortical spreading de-pression (Clark et al., 1997; Ishige et al., 1987a,b; Mar-ion et al., 1991; Rogatsky 2003; Strong et al., 2002).Some of these secondary injuries are mediated bychanges in gene expression patterns and activation of sig-naling pathways (Dash et al., 2002; Hu et al., 2004; Truet-tner et al., 1999, 2005).
TBI may damage cellular proteins and their foldingprocesses, leading to an increase in the levels of abnor-mally folded proteins in the endoplasmic reticulum (ER)lumen, mitochondrial matrix, and cytoplasm. Cerebral is-chemia and reperfusion have been shown to lead to an ac-cumulation of unfolded or misfolded proteins and proteinaggregation in neurons and activation of the unfolded pro-tein response (UPR) (DeGracia and Montie, 2004; Hu etal., 2000, 2001). Unfolded proteins are highly toxic tocells. Accumulating evidence has convincingly demon-strated that the ER, mitochondria, and cytoplasm respondto the increase in toxic unfolded proteins through theirown compartment-specific signaling pathways that initi-ate transcription of particular stress genes (Paschen, 2003;Yoneda et al., 2004). Most stress genes encode molecu-lar chaperones, protein folding enzymes and componentsof the ubiquitin-proteasomal system, thus stabilizing pro-teins and increasing degradation of toxic proteins(Bertolotti et al., 2000; Kaufman, 1999; Siman et al.,2001; Yoshida et al., 2000). Therefore, stress gene ex-pression in particular cellular compartments after brain in-jury may be indicative of which cellular compartmentscontain high levels of toxic unfolded or damaged proteins.
Glucose-regulated protein 78 (GRP78) is a member ofthe HSP70 superfamily localized to the ER that is a keysensor of levels of unfolded proteins and is highly in-duced after ER stress (Lee, 2001). Accumulation of un-folded proteins in the ER lumen leads, through GRP78,to translocation of transcription factors to induce expres-sion of chaperones such as grp78 itself as well as cal-nexin and PDI (Bertolotti et al., 2000).
HSP70 is a major inducible cytoplasmic chaperone thatprotects unfolded proteins from aggregation after stress.Induction of hsp70 expression reflects the presence of un-folded or misfolded proteins in the cytoplasm (Kim andSchoffl, 2002; Knowlton and Sun, 2001). In addition toits role in the immune response, HSP60 acts as a mole-cular chaperone predominantly residing in the mito-chondria matrix, which assists in mitochondrial proteinfolding and assembly via interaction with the mitochon-drial form of HSP70 (mt HSP70). The hsp60 gene is se-lectively induced by perturbations of mitochondrial pro-tein folding and assembly (Lee et al., 2002; Martin, 1997;
Yoneda et al., 2004). Neither heat-shock (mainly elicit-ing cytoplasmic stress) nor ER stress induces hsp60 geneexpression (Corydon et al., 2005; Yoneda et al., 2004).Previous work has focused on the role HSP60 plays inimmune responses, auto immune diseases, ischemia, andatherosclerosis. Little is known about the role of HSP60after brain trauma.
This study utilized the fluid-percussion (F-P) brain in-jury model (Dixon et al., 1987) to study expression ofmRNAs encoding the ER stress genes grp78, calnexin,and pdi, mitochondrial stress gene hsp60, and cytoplas-mic stress gene hsp70. We hypothesized that TBI wouldalter the expression of organelle-specific stress genes invulnerable brain regions. Because secondary hypoxia isa clinically relevant secondary insult in head-injured pa-tients (Chestnut, 1995) and has been reported to worsenoutcome after moderate F-P injury (Bramlett et al.,1999a,b), we also tested whether the addition of a mildhypoxic insult would augment the stress response to TBI.The results of this study indicate that expression of hsp70is the most robust after trauma and is further augmentedby secondary hypoxia, whereas expression of hsp60,grp78, calnexin, and pdi genes are modest after TBI.
METHODS
Animals
Male Sprague-Dawley rats (n � 5–6 per group) weigh-ing 280–340 g obtained from Charles River Breederswere used for all experiments. Animal care was in ac-cordance with the guidelines set by the University of Mi-ami Animal Care and Use Committee, and all animal pro-tocols were approved by the above committee prior tostudy initiation. All animals were kept at a constant tem-perature (23–25°C) in an air conditioned room for at least7 days before the study and exposed to a 12-h light-darkcycle. Rats were allowed free access to water, but foodwas withheld overnight before surgery.
Surgical Preparation and Fluid Percussion Injury
Anesthesia was induced using 3.0% halothane in a gasmixture of 70% nitrous oxide and a balance of oxygen, toachieve deep sedation. The animals were then endotra-cheally intubated and mechanically ventilated with a Har-vard rodent ventilator, on a mixture of 70% nitrous oxide,0.5–1.5% halothane, with a balance of oxygen adjusted asdescribed below. Pancuronium bromide 0.5 mg/kg i.v. wasadministered every hour during the surgical procedure tofacilitate mechanical ventilation. Femoral artery and veinwere cannulated with PE-50 tubing for purposes of drugadministration, blood sampling for serum glucose and
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hematocrit measurements, arterial blood gas determina-tion, and continuous arterial blood pressure monitoring.Anesthetic concentrations and ventilation settings were ad-justed to maintain normal values of mean arterial pressure,pH, pO2, and pCO2. Rectal and temporalis muscle ther-mometers were placed, and self adjusting feedback exter-nal warming lamps used to maintain temperature. Arter-ial blood gases, blood glucose, and hematocrit weremeasured 15 min before and after F-P injury, and 1, 2, and3 h after TBI. Animals were placed in a stereotactic framefor introduction of a right parietal craniotomy. In TBI an-imals, a F-P injury device was used to induce moderateparasagittal F-P injury (1.8–2.2 atm), as previously de-scribed (Chatzipanteli et al., 2000; Dietrich et al., 1994).
Secondary Hypoxia
After moderate F-P injury, animals were given 30 minof either a normoxic or hypoxic gas levels to simulate sec-ondary hypoxia (Bramlett et al., 1999a,b). Hypoxia wasinduced by reducing O2 (11%) and increasing N2O (56%)and adding N2 (33%) to the gas mixture. A decrease inthe oxygen and increase in nitrogen content of the gasmixture resulted in a pO2 of 30–40 mm Hg. This level ofhypoxia is similar to that observed clinically (Chestnut,1995). Physiological measurements were taken through-out all experiments (Table 1). Sham animals underwentall surgical procedures including normoxic or hypoxic gaslevels, but were not subjected to injury.
At 30 min, 4 h, or 24 h after TBI, animals were sacri-ficed, and whole brains were removed and fresh frozen.Coronal sections were cryosectioned at 12 �m at fourbregma levels: 1.2, �2.3, �3.3, and �5.3 (Zilles, 1985).
In Situ Hybridization
Fresh frozen sections were thawed to room tempera-ture and fixed for 5 min in 4% formaldehyde in phos-phate-buffered saline (PBS). Sections were acetylated for10 min at room temperature in 0.25% acetic anhydrideand 0.1 M triethanolamine HCl (pH 8), then dehydratedthrough a series of graded ethanol solutions, delipidizedin 100% chloroform for 5 min, rinsed in 100% ethanol,and allowed to air dry.
Sections were hybridized to 35S-labeled riboprobesgenerated by in vitro transcription of the anti-sense (forpositive probe) and sense (for negative probe) strands ofcDNA clones subcloned into transcription vectors usingthe Promega Riboprobe™ System. cDNA clones for cal-nexin, hsp60, and pdi were cloned in the lab of KoichiKokame. The grp78 cDNA clone was supplied by AmyS. Lee, and the hsp70 cDNA clone was subcloned in thislab from the rat inducible hsp70 cDNA clone suppliedby Frank Sharp (Longo et al., 1993). Hybridization was
conducted as follows: the denatured probe (2 � 107
dpm/mL) was added to a solution containing 100 �g/mLsalmon sperm DNA (ssDNA), 250 �g/mL each of yeasttotal RNA and tRNA, 50% formamide, 20 mM Tris HCl(7.4), 1 mM EDTA, 300 mM NaCl, 10% dextran sulfate,and 1� Denhardts. The hybridization solution was addedto the sections, covered with coverslips, and hybridizedunder humid conditions at 55°C for 20 h. After removalof the coverslips, the sections were washed at room tem-perature in a series of decreasing amounts of standardsaline citrate (SSC) with 1 mM DTT to a final concen-tration of 0.1� SSC. The slides were treated with 20�g/mL RNase A for 30 min at 37°C. The final high-strin-gency wash was carried out in 0.1� SSC, 1 mM DTTfor 1 h at 65°C. Sections were dehydrated through a se-ries of ethanols containing 300 mM ammonium acetate,ending with 100% ethanol.
Data Analysis
In situ hybridization. Sections were exposed to KodakBIOMAX MR film at 4°C for various lengths of time.Negative control (sense strand) probes showed no hy-bridization signals (data not shown). Autoradiographswere digitized by a charge-coupled device camera (Xil-lix Technologies Corp., Canada) with a Micro Nikon 55mm lens, which was interfaced to an advanced image-analysis system (MCID Model M2, Interfocus, Ltd.,United Kingdom) and captured at 50-�m resolution.[14C]-Methylmethacrylate standards placed on the filmswere digitized as well. Image files were transferred to aDEC Alpha Station for analysis (Dietrich et al., 2000).Optical density values of delineated regions of interest(Fig. 1) were converted to activity values (�Ci/g 14C-equivalents) by means of the standards. Measurementswere taken in regions that showed a positive signal foreach probe and similar regions in the control animals.Means and standard deviations were calculated.
Western blots. Tissue homogenates from sham-oper-ated, 4-h TBI, and 24-h TBI ipsilateral injured parietalcortex were electrophoresed on 10% or 12.5% sodiumdodecyl sulfate (SDS)–polyacrylamide gel electrophore-sis (PGE) and then transferred to Immobilon-P mem-branes (Millipore, Billerica, MA; n � 4 per group). Themembranes were blocked with 3% bovine serum albu-min (BSA) in TBS for 30 min and then incubatedovernight at 4°C with the following primary antibodies(1:1000) HSP70 and HSP60 (Stressgen, Victoria,Canada), and GRP78 (Affinity BioReagents, Golden,CO). Total protein loading was assessed by �-actin West-ern blot analysis (1:2000; Sigma, St. Louis, MO). Themembranes were then incubated with horseradish-perox-
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idase conjugated anti-rabbit or anti-mouse secondary an-tibodies for 60–120 min at RT (1:1000; Cell SignalingTechnology, Beverly, MA). The blots were developed us-ing enhanced chemiluminescence (Amersham Bio-sciences, Piscataway, NJ) and developed on Kodak Xo-mat LS film (Eastman Kodak Co., New Haven, CT).Densitometry was performed with Labworks 4.0 imageanalysis software (UVP, Upland, CA). Means and stan-dard deviations were calculated.
Statistical Analysis
Statistical assessments were performed using one-wayanalysis of variance (ANOVA) followed by post hocanalysis or Student’s t-test where appropriate.
RESULTS
Physiological Parameters
Physiological parameters were assessed for each ex-perimental group after sham normoxic, sham hypoxic,TBI normoxic, and TBI hypoxic procedures (values areshown in Table 1). No significant differences were ob-served between normoxic groups with respect to tempo-ralis muscle temperature, arterial pH, pO2, pCO2, or ar-terial blood pressure. Physiological variables were within
normal ranges throughout the study periods. Arterial pO2
differed in hypoxic groups versus normoxic groups (p �0.001), as expected.
Overall mRNA Expression Patterns
Expression of molecular chaperones after hypoxiaalone, TBI normoxia, or TBI hypoxia showed somewhatsimilar patterns, with some significant differences. Insham-operated control studies, hsp70, hsp60, and grp78did not show any increase in mRNA following hypoxictreatment alone (Fig. 1). Moderate TBI normoxia inducedwidespread expression of hsp70 in the injured cortex, andthe addition of secondary hypoxia increased this expres-sion to include the hippocampus and portions of the thal-amus (Fig. 1A, 4 h). hsp60 mRNA was induced in theouter cortical layers ipsilateral to the injury in both TBInormoxic and TBI hypoxic animals (Fig. 1B, 24 h). grp78was upregulated in the outer ipsilateral cortical layers,and in the hippocampus in both the TBI normoxic andTBI hypoxic animals (Fig. 1C, 4 h).
HSP70
Region of interest (ROI) measurements were made ofinjured cerebral cortex, hippocampus, and thalamus fromin situ autoradiographs at 4 h after injury in sham, hy-poxia alone, TBI normoxia, and TBI hypoxia treated an-
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FIG. 1. Stress-induced gene responses after traumatic brain injury (TBI) and hypoxia. Representative autoradiographs of in situhybridization of rat brains with sham, hypoxia, TBI, and TBI plus secondary hypoxia treatments. (A) hsp70. Coronal sectionsshow no expression in sham or hypoxia only brains (left two panels). Moderate TBI induces expression in the ipsilateral cortex.TBI plus hypoxia (right panel) shows increased levels of mRNA in the ipsilateral cortex, hippocampus, and thalamus. (B) hsp60.is induced in the cortex of the TBI and TBI plus hypoxia brains, but not in the hypoxia alone brains. (C) grp78. Expression isconstitutive in the sham and hypoxia only brains (two left panels). TBI and TBI plus hypoxia (two right panels) show an increasein mRNA levels in the outer cortical layers ipsilateral to the injury and in the hippocampus. All sections shown are bregma level�3.3. Outlined areas denote the regions measured for quantification, including injured hemisphere cortex, hippocampus, and thal-amus. All images are shown from the timepoint that showed the highest levels of expression: hsp70 4 h, hsp60 24 h, grp78 4 h.
imals (n � 5–6 per group). Using a radioactive standard,values were converted to nCi/g, and means and standarddeviations were calculated. Figure 2 shows that there wasno increase in expression of hsp70 in those sham animalssubjected to 30 min of hypoxia versus sham-operated nor-moxic animals. hsp70 mRNA was induced over shamlevels by TBI in the cortex only (p � 0.05). TBI followedby 30 min of hypoxia (Fig. 2) induced expression ofhsp70 in the cortex (p � 0.015), and also in the hip-pocampus and thalamus (p � 0.05).
The extent of hsp70 induction by TBI plus hypoxiawas widespread. Expression was seen in the ipsilateral
cortex, hippocampus, and thalamus throughout the fore-brain (Fig. 3) from the striatal level (bregma 1.2) throughthe posterior regions (bregma level �5.3 and beyond).Some expression was also observed in the contralateralcaudate putamen at 4 h (Fig. 3). The temporal profile ofhsp70 expression showed a rapid and strong response tocellular stress. Widespread expression was observed asearly as 30 min after injury (Fig. 3). The level of this ex-pression increased dramatically at 4 h and still persistedat 24 h after injury, showing that hsp70 was strongly andpersistently induced throughout the brain in regions re-mote from the epicenter of mechanical injury.
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FIG. 2. hsp70 mRNA levels in the ipsilateral cortex, hippocampus, and thalamus. Measurements of regions of interest (ROI)were made at 4 h in sham (white), hypoxia (diagonal stripe), TBI (black), and TBI plus secondary hypoxia (gray). *p � 0.05,**p � 0.015. Values are mean � SD; n � 5–6 per group.
FIG. 3. hsp70 in situ hybridization timecourse. Representative autoradiographs at four bregma levels (1.2, �2.3, �3.3, �5.3)show the temporal and spatial distribution of the induced hsp70 mRNA in sham animals and in TBI plus secondary hypoxia an-imals 3 min, 4 h, and 24 h after injury.
Quantification of ROI measurements of the cortex(Fig. 4A), hippocampus (Fig. 4B), and thalamus (Fig. 4C)showed that maximum expression in these regions oc-curred at the 4 h timepoint (p � 0.01 cortex and hip-pocampus, p � 0.02 thalamus). In the cortex (Fig. 4A),mRNA levels were still elevated (p � 0.01) at 24 h afterinjury.
HSP60
Moderate TBI plus secondary hypoxia induced ex-pression of this mitochondrial matrix chaperone in theouter ipsilateral cortical layers beginning at 4 h after in-jury and increasing to significant levels (p � 0.05) at 24h post-injury (Fig. 5A). mRNA levels were decreased inthe lesion regions of the cortex at all time points (30 min,4 h, 24 h). In contrast, regions of the hippocampus andthalamus did not exhibit any changes in expression ofhsp60 after TBI plus hypoxia. ROI measurements (Fig.5B), showed a significant increase in expression in theouter cortical layers at 24 h (p � 0.05) with a decreasein the lesioned regions (p � 0.05).
GRP 78
grp78 was constitutively expressed throughout the brainas seen in sham-operated animals (Fig. 6A). At 30 min af-ter TBI hypoxia, there was a transient decrease in mRNAlevels in the cortex (Fig. 6A,B; *p � 0.05) and in the cor-tex lateral to the injury site (lesion **p � 0.01). By 4 hpost-injury, mRNA levels in the outer cortical layers in-creased (*p � 0.05) and remained elevated to 24 h insome, but not all, animals. The dentate gyrus also showedan increase in mRNA at 4 h after TBI plus hypoxia (Fig.6B; DG). The thalamus showed no difference in grp78levels compared to sham-operated animals (Fig. 6B; Thal).
Calnexin and Protein Disulphide Isomerase
Calnexin and PDI are both constitutive endoplasmicreticulum proteins that are involved with normal pro-cessing of proteins through the ER and may be increasedafter injury. In this study, F-P injury resulted in a tran-siently decreased expression of these genes in the lateralcortical region after TBI normoxia and TBI hypoxia (Fig. 7) in most animals. This cortical region correspondsto the area of contusion and selective neuronal damagepreviously reported in this F-P model (Bramlett et al.,1999a). Quantification of optical density showed thatthere was no upregulation of either of these genes in anybrain regions (data not shown).
Protein Expression
Western blot analysis of ipsilateral injured parietal cor-tex homogenate for HSP70 (Fig. 8A) showed a large in-crease in HSP70 protein at 24 h after TBI (*p � 0.05).Western analysis for HSP60 (Fig. 8B) demonstrated asignificant decrease of HSP60 protein at 4 h and 24 h(p � 0.05) in the injured cortical tissue homogenateswhich mirrored those mRNA changes observed with insitu hybridization for this region. Western blot analysisof total cortical homogenate for GRP78 (Fig. 8C) showedno significant difference in protein levels.
DISCUSSION
In the present study, we assessed the subcellular re-sponse to moderate F-P brain injury alone or coupled withsecondary hypoxia on the expression of several stress re-sponse genes. We found that members of the heat shocksuper family of genes that are involved in responding to unfolded protein in the cytoplasm (hsp70), the ER
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FIG. 4. Quantification of hsp70 in situ hybridization signals shown in Figure 3. Sham (white bars), 30 min after TBI plus hy-poxia (diagonal stripe), 4 h after TBI plus hypoxia (black), and 24 h after TBI plus hypoxia (gray) measurements of n � 5–6 an-imals per group. *p � 0.01, **p � 0.02. Means � SD.
(grp78), and the mitochondria (hsp60) all showed in-creases in expression following TBI. The intensity andlocalization of this response varied dramatically depend-ing on the specific genetic target. HSP70 was widely dis-tributed throughout the injured hemisphere includingbrain regions remote from the injury epicenter. hsp60 andgrp78 were upregulated mainly in the ipsilateral cortexadjacent to the area of trauma. These distinct subcellularresponses suggest that the localization of unfolded andmisfolded proteins in the cell is not homogeneous. In con-
trast, enzymes involved in normal processing of proteinsin the ER (calnexin and PDI) were not increased in thisstudy by TBI, even with the addition of a secondary hy-poxic insult.
The ER is involved mainly in the proper folding andprocessing of secreted and membrane bound proteins.About one-third of all proteins go to the ER (Ghaem-maghami et al., 2003), and as a consequence, the ER lu-men has a very high concentration of proteins, on the or-der of �100 mg/mL (Kleizen and Braakman, 2004).
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FIG. 5. hsp60 expression timecourse after TBI plus hypoxia. (A) Representative autoradiographs of in situ hybridization sig-nals at four bregma levels (1.2, �2.3, �3.3, �5.3) in sham animals and TBI plus hypoxia animals 30 min, 4 h, and 24 h afterinjury. (B) ROI measurements of the outer cortical region and lesion in sham (white bars) and TBI plus hypoxia (black bars),*p � 0.05. ROI measurements of dentate gyrus (DG) and thalamus (Thal) 24 h after injury in sham (white bars) and TBI plushypoxia (black bars). Means � SD.
Chaperone and folding enzymes in the ER far outnumbernewly synthesized proteins and work to prevent proteinaggregation and act as quality control to target misfoldedproteins to the ER associated degradation (ERAD) path-way. The ER also serves as the major Ca2� storage or-ganelle and is crucial for maintaining Ca2� homeostasis.Perturbations in the cell involving calcium storage, redoxstatus, or protein glycosolation can lead to accumulationof unfolded protein and ER stress (Shen et al., 2004).
Calnexin is an ER membrane-bound chaperone that as-sociates with newly translated glycoproteins and is in-
volved in the proper folding and assembly of these pro-teins. It serves to prevent aggregation of glycoproteins asthey are being folded. This study determined whether TBIcaused an upregulation of this chaperone in response toan increased accumulation of unfolded proteins in the ER.No increase in mRNA or protein (data not shown) wasobserved. Protein disulphide isomerase, PDI, anotherconstitutive ER protein, is involved in the formation ofdisulfide bonds in newly folding proteins. PDI also actsas a chaperone by preventing protein aggregation. PDIwas likewise unaffected by TBI.
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FIG. 6. grp78 expression timecourse after TBI plus hypoxia. (A) Representative autoradiographs of in situ hybridization sig-nals at four bregma levels (1.2, �2.3, �3.3, �5.3) in sham animals and TBI plus hypoxia animals 30 min, 4 h, and 24 h afterinjury. (B) ROI measurements of the outer cortical region and lesion in sham (white bars) and TBI plus hypoxia (black bars),*p � 0.05, **p � 0.01. ROI measurements of dentate gyrus (DG) and thalamus (Thal) 4 h after injury in sham (white bars) andTBI plus hypoxia (black bars), **p � 0.01. Means � SD.
In 1992, Mori et al. first described the unfolded pro-tein response (UPR) signaling pathway in the yeast S.cerevisiae. The transcriptional induction of the ER stressgenes (the unfolded protein response) appears to behighly conserved between yeast and mammals. The UPRin mammals involves the upregulation of at least 18 genes(Okada et al.,2002). Unlike yeast, which have only tran-scriptional regulation of the UPR mediated by HAC1 andIRE1 (Mori et al., 1993, 1996), mammalian cells haveadditional pathways that involve both translational andpro-apoptotic responses. Three ER transmembrane pro-teins mediate the UPR in mammals upon ER stress: IRE1,PERK, and ATF6. IRE1 and ATF6 are involved in tran-scriptional activation, while PERK acts an inhibitor ofprotein translation by phosphorylating eIF2, which in-hibits assembly of the 80S ribosome and blocks proteintranslation (Harding et al., 1999; Kaufman, 1999).
In a normal state, GRP78 binds ATF6, IRE1, andPERK on the ER membrane and keeps them inactive(Lee, 2005). When the ER is stressed and unfolded pro-teins begin to accumulate in the ER lumen, GRP78 is re-quired to assist in folding and disassociates from PERKand IRE1, which then autophosphorylate and become ac-tive (Kada et al., 2002). Phosphorylation of IRE1 leadsto activation of Xbp 1 (X box protein 1), which functionsas a transcription factor for ER stress genes such asGRP78 (Yoshida et al., 2001). Once enough GRP78 pro-tein has been synthesized to bind and refold the unfoldedproteins in the ER and to bind and inactivate IRE1 andPERK, the UPR is turned off. GRP78, therefore, is in-strumental in both the transcriptional (through IRE1 andATF6) and translational (through PERK) aspects of theUPR. Additionally, GRP78 has been shown to be anti-apoptotic by blocking caspases (Rao et al., 2002; Reddy
et al., 2002). Alterations in GRP78 expression may there-fore influence anti-apoptotic cell death mechanisms thathave been described to be activated after TBI (Keane etal., 2001).
GRP78 has been shown to be induced in several is-chemia models, including myocardial ischemia (Szegezdiet al., 2006), permanent middle cerebral artery occlusion(MCAO) (Xu et al., 2006), and transient MCAO (Rissa-nen et al., 2006). Although some previous studies had re-ported that severe, but not moderate, F-P injury producedan increase in GRP78 expression (Hayes et al., 1995), inthe present study, we also observed a clear response byGRP78 to moderate injury. mRNA levels increased in thepericontusional regions of the cortex and in the dentategyrus. Further studies to localize the GRP78 protein inthese regions are necessary.
However, if the ER stress response is insufficient topromote cell survival, and long-term or excessive accu-mulation of unfolded proteins exists, cell death can betriggered. Recent studies have shown that caspase-12may be the initiator of the ER stress-induced apoptoticpathway (Nakagawa et al., 2000; Rao et al., 2001; Yonedaet al., 2001) that is independent of either the intrinsic (in-duced by mitochondrial damage) or extrinsic ( mediatedby ligand binding to membrane receptors) pathways. Ina recent study, Larner et al. (2004) reported in a con-trolled cortical impact model increased expression of cas-pase-12 mRNA and protein levels in neurons. The pro-posed pathway of ER stress-induced apoptosis hascaspase-12 activating caspase-9 which then activates cas-pase-3 (Morishima et al., 2002; Rao et al., 2001). An-other ER stress pro-apoptotic pathway involves the acti-vation of CHOP which is a stress inducible nucleartranscription factor (DeGracia and Montie, 2004; Fornace
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FIG. 7. Representative in situ hybridization autoradiographs of calnexin (left) and pdi (right) at three bregma levels (1.2, �3.3,�5.3) in sham animals and TBI plus hypoxia after 4 and 24 h.
perimental TBI (Sullivan et al., 1998). These disruptionsinclude structural changes such as swelling and outermembrane disruption (Lifshitz et al., 2004), metabolicdysfunction, and the prolonged accumulation of calcium(Fineman et al., 1993; Hovda et al., 1990). When the ERis damaged and the ability to buffer calcium effectivelyis reduced, the demand on mitochondria to perform thiscalcium buffering function is increased (Paschen andDoutheil, 1999). Mitochondrial stress can be very dele-terious to the cell, even leading to cell death. HSP60 hasbeen implicated as being anti-apoptotic. Romano et al.(2004) stimulated HSP60 production in cells in cultureand then subjected them to an apoptosis-inducing agent;the cells with elevated HSP60 showed significantly lessapoptosis. In addition, induction of HSP60 protected mi-tochondrial membrane potential in rat liver cells from hy-poxia (Motoyama et al., 2004). Thus, the upregulation ofchaperone proteins such as HSP60 observed in the pre-sent study may be crucial for protecting the mitochon-dria from stresses produced by TBI in those brain regionsthat show no cell death (pericontusional). Within the le-sion areas, we observed a decrease in both mRNA andprotein indicating either cell loss in this region, or a shut-down of transcription and translation. Further studies us-ing immunohistochemistry to localize subcellular com-partmentalization of these proteins directly may shedmore light on the subcellular responses to TBI.
The unfolded protein response has been shown to beinduced by a number of pathological processes, includ-ing spreading depression (Schneeloch et al., 2004), hy-poxia (Pan et al., 2004), ischemia (DeGracia and Mon-tie, 2004; Paschen et al., 2003), kainate-induced seizure(Lowenstein et al., 1994), and TBI (Larner et al., 2004;Lowenstein et al., 1994; Paschen et al., 2004). Cerebralischemia and trauma share many of the same underlyingpathophysiological mechanisms (Bramlett and Dietrich,2004; Leker and Shohami, 2002). These processes, wheninitiated by trauma, may lead to secondary injury simi-lar to the penumbral neuronal cell death seen in ischemia(Back, 1998; Hossman, 1994). Although the UPR hasbeen demonstrated after cerebral ischemia, the exact rolethe UPR plays in the pathogenesis of secondary injuryfollowing brain trauma has yet to be determined.
In the present study, we assessed the effect of sec-ondary hypoxia following TBI as well as TBI alone. Wefound that secondary hypoxia significantly augmentedthe HSP70 response to TBI. This indicates that the addi-tion of this clinically relevant secondary insult may ini-tiate cytoplasmic events including protein aggregation.For example, it is possible that this stress response maybe an attempt to protect unfolded proteins from aggre-gating following the combined insult. Continued researchinto understanding the nature of the subcellular stress re-
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FIG. 8. Western blot analysis of total protein from injuredparietal cortex in sham (white), 4 h (gray), and 24 h (black) an-imals. (A) HSP70 protein levels increased at 24 h after TBI(*p � 0.05). (B) HSP60 protein levels were reduced comparedto sham-operated animals at both 4 and 24 h after TBI (*p �0.05). (C) GRP78 protein levels were not measurably changedin these samples of total ipsilateral cortex. Means � SD. (D)Representative Westerns are shown.
et al., 1989) that targets TRB 3, a novel ER stress in-ducible gene. Thus, depending on the degree of the ERstress response, both anti- and pro-apoptotic pathwayscan be activated and effect traumatic outcome.
Another organelle involved with calcium homeostasisand cell death is the mitochondria. It has been known thatmitochondrial disruption is an early consequence of ex-
sponse to TBI is vital in understanding the molecularmechanisms underlying cell survival and death and thecontinued development of novel therapeutic strategies.
ACKNOWLEDGMENTS
We would like to thank Amy S. Lee, Ph.D., USC/Nor-ris Cancer Center, for providing us with the GRP78 gene,and Charlaine Rowlette for expert editorial assistance.This investigation was supported in part by grants fromNIH/NINDS 30291 and 42133.
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