Proteomic Identification of Nitrated
Brain Proteins in Traumatic Brain-Injured
Rats Treated Postinjury With
Gamma-Glutamylc...
years have the highest incidence of TBI (Rutland-
Brown et al., 2006). The outcome can range from com-
plete patient recov...
centration in the supernatant was determined by the BCA
protein assay.
Measurement of Protein Carbonyls
Five microliters o...
ferred into clean microcentrifuge tubes. The protein spots
were then washed with 0.1 M ammonium bicarbonate
(NH4HCO3; Sigm...
Identification of Nitrated Proteins in
GCEE-Treated TBI Rats
Two-dimensional gels were performed for TBI,
sham, and TBI rat...
et al., 2006). Phosphoglycerate mutase 1 (PGM1) cata-
lyzes the interconversion of 3-phosphoglycerate to 2-
phosphoglycera...
tabolism, etc.). In TBI, glycogen levels are increased
compared with control, supporting the downstream
effects impaired b...
Alpha-spectrin plays a critical role in maintaining
cytoskeletal structure and membrane integrity. This pro-
tein interact...
Ekegren T, Hanrieder J, Aquilonius SM, Bergquist J. 2006. Focused pro-
teomics in post-mortem human spinal cord. J Proteom...
Sawyer E, Mauro LS, Ohlinger MJ. 2008. Amantadine enhancement of
arousal and cognition after traumatic brain injury. Ann P...
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  1. 1. Proteomic Identification of Nitrated Brain Proteins in Traumatic Brain-Injured Rats Treated Postinjury With Gamma-Glutamylcysteine Ethyl Ester: Insights Into the Role of Elevation of Glutathione as a Potential Therapeutic Strategy for Traumatic Brain Injury Tanea T. Reed,1 Joshua Owen,1 William M. Pierce,2 Andrea Sebastian,3 Patrick G. Sullivan,3 and D. Allan Butterfield1,4* 1 Department of Chemistry, University of Kentucky, Lexington, Kentucky 2 Department of Pharmacology, University of Louisville, Louisville, Kentucky 3 Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, Kentucky 4 Center of Membrane Sciences, University of Kentucky, Lexington, Kentucky Traumatic brain injury (TBI) occurs suddenly and has damaging effects to the brain that are dependent on the severity of insult. Symptoms can be mild, moder- ate, or severe. Oxidative damage is associated with traumatic brain injury through reactive oxygen/nitrogen species production. One such species, peroxynitrite, is elevated in TBI brain tissue (Orihara et al. [2001] Foren- sic Sci. Int. 123:142–149; Deng et al. [2007] Exp. Neu- rol. 205:154–165). Peroxynitrite can react with carbon dioxide and decompose to produce NO2 and carbonate radicals, which in turn can lead to 3-nitrotyrosine, an index of protein nitration. Gamma-glutamylcysteine ethyl ester (GCEE) is an ethyl ester moiety of gamma- glutamylcysteine, an agent that up-regulates glutathi- one (GSH) production in brain (Drake et al. [2002] J. Neurosci. Res. 68:776–784). Many preclinical studies of TBI have employed pretreatment of animals with proposed beneficial agents prior to the injury itself. However, in the real world of TBI, treatment begins postinjury. Hence, insights into agents that improve out- come following injury are desperately needed. This study is one of the first to investigate a potential GSH- based therapy for TBI postinjury. Protein carbonyls, an index of protein oxidation, were significantly elevated in brain of animals subjected to TBI. However, if, after TBI, GCEE was administered i.p., protein carbonyl lev- els were significantly reduced. Similarly, 3-nitrotyrosine levels were elevated in brain following TBI but signifi- cantly decreased following TBI if GCEE was adminis- tered i.p. Redox proteomics analysis showed that sev- eral brain proteins were nitrated after TBI. However, if GCEE was given i.p. following TBI, many of these pro- teins were protected from nitration. The results are encouraging and are discussed with reference to potential therapeutic strategies for TBI involving ele- vated GSH. VVC 2008 Wiley-Liss, Inc. Key words: oxidative stress; brain; antioxidant Traumatic brain injury (TBI) is defined as an event in which sudden trauma and secondary injury cause brain damage. Symptoms of TBI can be mild, moderate, or severe, depending on extent of brain damage. Mild symptoms can include but are not limited to confusion, lightheadedness, double vision, personality changes, and memory impairment. Moderate and severe TBI cases show the same symptoms but worsened, slurred speech, seizures, nausea, unresponsiveness, and possible death. TBI can be focused on one particular area (focal) or involve more than one area of the brain (diffuse). Worldwide, TBI occurs in 10 million people annually, with 1.4 million cases in the United States alone (Lan- glois et al., 2006). Males between the ages of 15 and 24 Tanea T. Reed’s current address is Department of Chemistry, Eastern Kentucky University, Richmond, Kentucky. Contract grant sponsor: NIH; Contract grant number: AG-10836 (to D.A.B.). *Correspondence to: Prof. D. Allan Butterfield, Department of Chemis- try, Center of Membrane Sciences, University of Kentucky, Lexington, KY 40506. E-mail: dabcns@uky.edu Received 9 May 2008; Revised 22 June 2008; Accepted 5 July 2008 Published online 19 September 2008 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21872 Journal of Neuroscience Research 87:408–417 (2009) ' 2008 Wiley-Liss, Inc.
  2. 2. years have the highest incidence of TBI (Rutland- Brown et al., 2006). The outcome can range from com- plete patient recovery to permanent neurological dys- function. There is no known cure for TBI, although fluoxetine, neurosteroids, bromocriptine, and dexanabi- nol show therapeutic promise (Gorska, 2000; Bedell and Prough, 2002; Djebaili et al., 2005; Mostert et al., 2008); however, immediate medical care after injury is most advantageous for patient recovery. Oxidative damage has been associated with TBI through formation of reactive oxygen species (ROS), including O2 Á2 (Kontos and Povlishock, 1986; Kontos and Wei, 1986; Opii et al., 2007), peroxynitrite (Hall et al., 2004), and OHÁ (Hall et al., 1994). Peroxynitrite is a potent nitrating agent involved in neurodegenerative diseases (Butterfield, 2006; Sultana et al., 2006b). Perox- ynitrite is formed by the combination of the two radi- cals, nitric oxide and superoxide. In the presence of CO2, peroxynitrite forms NO2 Á and CO3 Á radicals. NO2 Á radicals can react with DNA, lipids, and proteins, includ- ing MnSOD (Jackson et al., 1998; Tangpong et al., 2007), Cu/ZnSOD (Alvarez et al., 2004), creatine kinase (Wendt et al., 2003), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Szabo, 2003). Inactivation of these proteins can result in inefficient energy metabo- lism, reduced ATP production, and an excess hydrogen peroxide, leading to overall increase in ROS production and oxidative stress (Sultana et al., 2006a; Reed et al., 2008). Peroxynitrite is elevated in brain tissue following TBI (Orihara et al., 2001; Deng et al., 2007). This ele- vation likely is due to superoxide leakage from the mito- chondria reacting with NOÁ and is supported by research using nitric oxide synthase (NOS) inhibitors to decrease 3-NT levels in TBI-treated mice (Gahm et al., 2005). Peroxynitrite- derived radicals can lead to cellular dam- age in proteins, lipids, RNA, and DNA (Szabo et al., 1996; Aoyama et al., 2000; Masuda et al., 2002; Botti et al., 2005; Sultana et al., 2006b, 2007). Increased oxi- dative damage in the form of elevated levels of protein carbonyls, protein nitration, and lipid peroxidation was observed in a diffuse, closed-head-injury mouse model (Hall et al., 2004). Gamma-glutamylcysteine ethyl ester (GCEE) is a modified molecule of gamma-glutamylcysteine, the first two amino acids of the tripeptide glutathione (GSH). Gamma-glutamylcysteine synthetase catalyzes the forma- tion of gamma-glutamylcysteine. The ethyl ester moiety increases its efficacy to cross both the plasma membrane of cells and the blood–brain barrier. GCEE leads to up- regulated GSH in the brain and protects the brain against peroxynitrite-mediated oxidative stress (Drake et al., 2002). Glutathione (g-Glu-Cys-Gly) is a powerful anti- oxidant found in brain. Cysteine (Cys) is the limiting amino acid in GSH biosynthesis. Cys is in micromolar levels in the brain, whereas glutamate and glycine are in millimolar concentrations (Cooper and Kristal, 1997). It has well documented that several free radical scavengers are neuroprotective in various TBI animal models (Ozdemir et al., 2005; Dohi et al., 2006; Singh et al., 2007; Sonmez et al., 2007). Importantly, these and other agents were administered prior to TBI. In contrast, we investigated nitrated brain proteins following TBI and their protection against oxidative stress induced by post- treatment with GCEE. Only a few drugs have shown cognitive benefit when administered post-TBI, making this study imperative in the investigation of TBI thera- peutics postinjury. MATERIALS AND METHODS Chemicals All chemicals were of the highest quality and were pur- chased from Sigma-Aldrich (St. Louis, MO) unless otherwise mentioned. The OxyBlot kit used for protein carbonyl deter- mination was purchased from Intergen (Purchase, NY). GCEE was purchased from Bachem (Torrance, CA). Surgical Procedures and Brain Processing All the surgical, injury, and animal care protocols described below have been approved by the University of Kentucky Institutional Animal Care and Use Committee and are consistent with the animal care procedures set forth in the guidelines of the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals. Eighteen Wistar adult male rats (Harlan Laboratories, In- dianapolis, IN) wieghing 300–350 g were used in this study. The rats were anesthetized with isoflurane (3.0%), shaved, and then placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). Surgery was completed in the same fashion as previously described (Sullivan et al., 2002). A 1.5-mm drop of a metal ball was used to produce TBI in each rat as described elsewhere (Sullivan et al., 2002), except for sham animals. Each group (sham, TBI, and GCEE-treated) consisted of six animals. After surgery and injury, a 4-mm disk made from dental cement was placed over the craniotomy site and adhered to the skull with cyanoacrylate. To prevent immedi- ate hypothermia following the skin suturing, rats were placed on a warm mat until they regained consciousness (increased attention and mobility). Six rats were given GCEE (150 mg/ kg) approximately 10 min postinjury. This concentration was chosen based on prior studies (Joshi et al., 2007). Six injured rats were given saline 10 min after injury. The remaining six rats were treated as sham controls, in which they received a craniotomy but not cortical contusion. All rats were kept alive 24 hr postinjury and then sacrificed. This time point was cho- sen based on prior research in which improvement with post- neurosteroidal treatment was observed (Djebaili et al., 2005). Also, maximal glutathione deletion from TBI is observed at this time point (Ansari et al., 2008b). Upon sacrifice, rats were decapitated, and the whole brain was rapidly removed and placed in a –808C freezer until use. Brain samples were minced and suspended in 10 mM HEPES buffer (pH 7.4) containing 137 mM NaCl, 4.6 mM KCl, 1.1 mM KH2PO4, 0.1 mM EDTA, and 0.6 mM MgSO4 as well as proteinase inhibitors: leupeptin (0.5 mg/ml), pepstatin (0.7 lg/ml), type II S soybean trypsin inhibitor (0.5 lg/ml), and phenylmethyl- sulfonyl fluoride (PMSF; 40 lg/ml). Homogenates were cen- trifuged at 14,000g for 10 min to remove debris. Protein con- Elevated Glutathione as a Potential Therapy for TBI 409 Journal of Neuroscience Research
  3. 3. centration in the supernatant was determined by the BCA protein assay. Measurement of Protein Carbonyls Five microliters of sample were derivatized with 10 mM 2,4-dinitrophenylhydrazine (DNPH) in the presence of 5 ll 12% sodium dodecyl sulfate for 20 min at room temperature. The samples were then neutralized with 7.5 ll neutralization solution (2 M Tris in 30% glycerol). Derivatized samples (250 ng) were loaded onto a nitrocellulose membrane with a slot blot apparatus. The blot was then blocked with 3% bovine serum albumin (BSA) and incubated with rabbit polyclonal anti-DNPH antibody (1:100 dilution) for 2 hr. The blot was washed in wash blot buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) three times before incubation with a secondary antibody (goat anti-rabbit antibody conju- gated with alkaline phosphatase). The blot was washed again and then developed using SigmaFast tablets (Sigma Aldrich), and densiometric levels were quantified in Scion Image software. Measurement of HNE and 3-NT Levels Measurement of HNE-bound proteins is one way of indexing lipid peroxidation, whereas measuring 3-NT levels is an effective method for quantifying the level of nitrated pro- teins. For both measurements, 5 ll brain homogenate was incubated with 5 ll Laemmli buffer [0.125 M Tris base, pH 6.8, 4% (v/v) sodium dodecyl sulfate (SDS), and 20% (v/v) glycerol] for 20 min at room temperature. The resulting sam- ples (250 ng) were loaded per well in the slot blot apparatus containing a nitrocellulose membrane under pressure. Each sample was loaded in duplicate. After the sample was loaded, the membrane was blocked with 3% (w/v) BSA in phos- phate-buffered saline containing 0.01% (w/v) sodium azide and 0.2% (v/v) Tween 20 (PBST) for 1 hr and then incubated with a 1:5,000 dilution of anti-HNE (or a 1:2,000 dilution of anti-3NT) polyclonal antibody in PBST for 2 hr. After primary antibody incubation, the membrane was carefully washed three times for 5 min each. A secondary antibody of anti-rabbit IgG alkaline phosphatase in PBST (1:3,000 dilu- tion) was added to the membrane for 1 hr. The membrane was again washed three times in PBST and developed using SigmaFast BCIP/NBT (5-bromo-4-chloro-3-indolyl phos- phate/nitro blue tetrazolium) tablets (Sigma). Blots were dried, scanned in Adobe Photoshop (San Jose, CA), and quantitated in Scion Image software. Two-Dimensional Gel Electrophoresis Two-dimensional (2D) gel electrophoresis was per- formed to separate proteins into single detectable protein spots based on size and isoelectric point. Proteins (200 lg) were precipitated by addition of cold 100% trichloroacetic acid (TCA) to obtain a final concentration of 15% TCA and placed on ice for 10 min. Precipitates were centrifuged at 14,000g for 2 min at 48C. The pellets were washed three times with 1 ml of 1:1 (v/v) ethanol:ethyl acetate solution. The samples were then dissolved with 200 ll of rehydration buffer [8 M urea, 2 M thiourea, 20 mM dithiothreitol, 2.0% (w/v) CHAPS, 0.2% Biolytes, and bromophenol blue]. Iso- electric focusing separates proteins according to their isoelec- tric point (pI). For first-dimension electrophoresis, 200 ll of sample solution was applied to a 110-mm ReadyStrip IPG strip, pH 3–10 (Bio-Rad, Hercules, CA). The strips were allowed to soak in the sample solution for 1 hr to allow uptake of the proteins. Two milliliters of mineral oil were added on top of each strip to prevent evaporation throughout this entire process. The strip was then actively rehydrated in a Protean IEF cell (Bio-Rad) for 16 hr at 50 V. The isoelectric focusing was performed at 300 V for 2 hr linearly, 500 V for 2 hr linearly, 1,000 V for 2 hr linearly, 8,000 V for 8 hr line- arly, and 8,000 V for 10 hr rapidly. All the processes described above were carried out at 208C. The focused IEF strip was stored at –808C until 2D gel electrophoresis was performed. SDS-PAGE separates proteins based on size and shape. For 2D electrophoresis, thawed strips were equilibrated for 10 min in equilibration buffer A, which consisted of 50 mM Tris- HCl (pH 6.8) containing 6 M urea, 1% (w/v) SDS, 30% (v/ v) glycerol, and 0.5% dithiothreitol, then reequilibrated for 10 min in the same buffer containing 4.5% iodacetamide instead of dithiothreitol. Linear Gradient (8–16%) Precast Criterion Tris-HCl gels (Bio-Rad) were used to perform 2D electro- phoresis. Precision Protein Standards (Bio-Rad) were run along with the sample at 200 V for 65 min. SYPRO Ruby Staining After electrophoresis, the gels were incubated in fixing solution [7% (v/v) acetic acid, 10% (v/v) methanol] for 20 min. Approximately 40 ml of SYPRO Ruby Gel Stain (Bio- Rad) was used to stain the gels for 2 hr, on a gently continu- ous rocker. The gels were placed in deionized water overnight for destaining. SYPRO Ruby fluorescent stain achieves a lin- ear and sensitive staining of gel slabs. Western Blotting and Immunochemical Detection Western immunoblotting was performed to detect pro- teins immunochemically on a nitrocellulose membrane. Pro- tein samples (200 lg) were loaded onto an 8–16% Criterion gel (Bio-Rad), and electrophoresis was carried out as described above. The proteins from the 2D gel were transferred to a nitrocellulose membrane (Bio-Rad) using a TransBlot SD Semi-Dry Transfer Cell (Bio-Rad) at 15 V for 2 hr. Nitrated proteins were detected on nitrocellulose paper using anti-3-ty- rosine antibody (1:2,000) for 2 hr at room temperature while rocking, followed by a secondary goat anti-rabbit IgG (Sigma) antibody conjugated to alkaline phosphatase (1:3,000) diluted in wash blot buffer for 1 hr at RT. The resultant membrane was developed using 5-bromo-4-chloro-3-indolyl phosphate/ nitroblue tetrazolium (BCIP/NBT) solution (SigmaFast tablets; Sigma). In-Gel Digestion In-gel digestion of selected gel spots was performed according to techniques established by Thongboonkerd et al. (2002). The significant protein spots were excised from SYPRO Ruby-stained 2D gels with a clean blade and trans- 410 Reed et al. Journal of Neuroscience Research
  4. 4. ferred into clean microcentrifuge tubes. The protein spots were then washed with 0.1 M ammonium bicarbonate (NH4HCO3; Sigma) at room temperature for 15 min. Aceto- nitrile (Sigma) was added to the gel pieces and incubated at room temperature for 15 min. The solvent was removed, and the gel pieces were dried in a flow hood. The protein spots were incubated with 20 ll of 20 mM dithiothreitol (DTT; Bio-Rad) in 0.1 M NH4HCO3 at 568C for 45 min. The DTT solution was then removed and replaced with 20 ll of 55 mM iodoacetamide (Bio-Rad) in 0.1 M NH4HCO3. The solution was incubated at room temperature in the dark for 30 min. The excess iodoacetamide was removed and replaced with 0.2 ml of 50 mM NH4HCO3 and incubated at room temperature for 15 min. Two hundred microliters of acetoni- trile was added. After a 15-min incubation, the solvent was removed, and the gel spots were dried for 30 min in a flow hood. The gel pieces were rehydrated with 20 ng/ll modified trypsin (Promega, Madison, WI) in 50 mM NH4HCO3 with the minimal volume to cover the gel pieces. The gel pieces were chopped into smaller pieces and incubated with shaking overnight at 378C. Image Analysis The immunoreactivity of the Western blot was normal- ized to the actual protein content as measured by the intensity of a SYPRO Ruby protein stain (Bio-Rad). Images from SYPRO Ruby-stained gels, used to measure protein content, were obtained with a UV transilluminator (kex 5 470 nm, kem 5 618 nm; Molecular Dynamics, Sunnyvale, CA). Gels were then stored in deionized water until spot excision for in- gel trypsin digestion. Western blots were scanned in Adobe Photoshop on a Microtek Scanmaker 4900. PDQuest 2-D Analysis software (Bio-Rad) was used to match and analyze visualized protein spots among differential gels and membranes to compare protein and immunoreactivity content between injured animal samples and age-matched controls. Powerful automatching algorithms quickly and accurately match gels or blots, and sophisticated statistical analysis tools allow spots of interest to be identified. The principles of measuring intensity values by 2D analysis software were similar to those of densi- tometric measurement. The average mode of background sub- traction was used to normalize intensity values, which repre- sent the amount of protein (total protein on gel and protein bound on membrane) per spot. After completion of spot matching, the normalized intensity of each protein spot from individual gels (or membranes) was compared between groups using statistical analysis (Student’s t-test). Mass Spectrometry All mass spectrometry was performed at the University of Louisville’s Mass Spectrometry Facility (ULMSF) and Vet- eran’s Affairs Medical Center (VAMC). A Bruker Autoflex matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer (Bruker Daltonics, Biller- ica, MA) operated in the reflectron mode was used to gener- ate peptide mass fingerprints. Peptides resulting from in-gel digestion with trypsin were analyzed on a 384-position, 600- lm AnchorChip Target (Bruker Daltonics, Bremen, Germany) and prepared according to AnchorChip recommendations (AnchorChip Technology, Rev. 2; Bruker Daltonics). Briefly, 1 ll digestate was mixed with 1 ll a-cyano-4-hydroxycin- namic acid (0.3 mg/ml in ethanol:acetone, 2:1 ratio) directly on the target and allowed to dry at room temperature. The sample spot was washed with 1 ll of a 1% TFA solution for approximately 60 sec. The TFA droplet was gently blown off the sample spot with compressed air. The resulting dif- fuse sample spot was recrystallized (refocused) using 1 ll of a solution of ethanol:acetone:0.1% TFA (6:3:1 ratio). Reported spectra are a summation of 100 laser shots. External calibra- tion of the mass axis was used for acquisition and internal calibration using either trypsin autolysis ions or matrix clus- ters and was applied postacquisition for accurate mass determination. Analysis of Peptide Mass Fingerprints Results from MALDI spectra used for protein identifica- tion from tryptic fragments were searched against the NCBI database using the MASCOT search engine (http://www.ma- trixscience.com). The search parameters used were previously described by Castegna et al. (2002a). Peptide mass fingerprint- ing used the assumption that peptides are monoisotopic, oxi- dized at methionine residues, and carbamidomethylated at cysteine residues (Castegna et al., 2002a,b; Butterfield et al., 2003). Carbamylation occurs when urea is broken down in a sample solution. Urea can degrade to cyanate, which can then react with protein amino groups and remove the positive charge, making them more acidic. Database searches allowed up to one missed trypsin cleavage. Mass tolerance of 100 ppm was the window of error allowed for matching the peptide mass values. Statistical Analysis Probability-based MOWSE scores were estimated by comparison of search results against estimated random match population and were reported as –10 Á log10 (P), where P is the probability that the protein identification is not correct. Proteins with a P value less than 0.05 were considered signifi- cant. All protein identifications were in the expected size and pI ranges based on their gel position. RESULTS There was a significant increase (19%) in protein carbonyls observed in TBI rats compared with sham ani- mals (Fig. 1). GCEE treatment 10 min postinjury pro- vided protection against TBI (70% of control). There was a significant increase (19%) in protein nitration in brain from TBI rats. Protein nitration was reduced to below control levels by administration of GCEE as shown in Figure 2. Protein-bound HNE levels were found to be elevated in TBI rats compared with sham (27%), but there was no significant decrease in HNE levels post-GCEE treatment (data not shown). Elevated Glutathione as a Potential Therapy for TBI 411 Journal of Neuroscience Research
  5. 5. Identification of Nitrated Proteins in GCEE-Treated TBI Rats Two-dimensional gels were performed for TBI, sham, and TBI rats treated with GCEE 10 min follow- ing TBI (Fig. 3a–c, respectively). The 2D Western blots probed with anti-3NT antibody to detect 3-NT immu- noreactivity are seen in Figure 4a–c, respectively. Pro- teomics analysis for excessively nitrated proteins in TBI was performed as described in Materials and Methods. Seven proteins were found to be significantly nitrated in TBI rats compared with sham rats. These proteins include synapsin 1, gamma enolase, guanosine diphos- phate dissociation inhibitor 1 (GDP), phosphoglycerate mutase 1, heat shock protein 70, ATP synthase, and a- spectrin. A list of these proteins can be found in Table I. If GCEE is given to animals that suffered a TBI, these proteins are no longer excessively nitrated compared with sham controls. DISCUSSION Sudden brain trauma is described as TBI. There is no known cure, but immediate medical care after injury is most advantageous for patient recovery. ROS produc- tion occurs in TBI, as can be observed by increased pro- tein nitration and protein carbonyls (Hall et al., 2004; Opii et al., 2007). Ansari et al. (2008b) demonstrated that oxidative stress occurs within 3 hr postinjury. Cur- rently, several drugs show promise in treating TBI, including fluoxetine (Mostert et al., 2008), progesterone (Djebaili et al., 2005), bromocriptine (Gorska, 2000), and dexanabinol (Bedell and Prough, 2002). Nicotina- mide, atomoxetine, erythropoietin, and amandatine have shown cognitive improvement when administered post- injury (Grasso et al., 2007; Hoane et al., 2008; Reid and Hamm, 2008; Sawyer et al., 2008). The most investi- gated therapeutic strategy for TBI patients over the past 10 years has been the use of mild hypotheremia expo- sure. This lowers core body temperature in an effort to prevent the likelihood of further complications. How- ever, there is little efficacy in this method of treatment (Tisherman et al., 1999; Ginsberg, 2002; Polderman, 2004; Qiu et al., 2007). GCEE, an ester moiety of the dipeptide gamma- glutamylcysteine, is a vital antioxidant that can easily cross the plasma membrane and up-regulate GSH in the brain (Drake et al., 2002). GCEE prevents oxidative stress induced by amyloid-b peptide and other moieties by scavenging and free radicals (Boyd-Kimball et al., 2005b; Joshi et al., 2007). In the current study, GCEE reduced levels of 3NT and protein carbonyls to those of control. In this study, posttreatment of GCEE appears to lower protein oxidation significantly in TBI rats, leading to possible promise as a potential postinjury therapeutic. In this work, seven proteins were found by redox proteomics analysis to be nitrated in TBI. The functions of these proteins include energy metabolism, neurotrans- mission, and chaperone function. Synapsin 1 is a synaptic protein that binds to synaptic vesicles and regulates neu- rotransmission. Synapsin aids in maintaining an ample supply of ‘‘reserve’’ vesicles at the synapse for release. This phosphoprotein binds to the SH3 (high-affinity phosphotyrosine binding site) domain of the c-Src kinase in synaptic vesicles, causing stimulation of c-Src activity (Onofri et al., 2000). Src kinase phosphorylates Tyr residues on proteins rather than Ser or Thr residues. Synapsin-deficient vesicles show significantly reduced Src activity and Tyr phosphorylation (Onofri et al., 2007). This synaptic protein has also been found to be modified oxidatively in recent work involving traumati- cally brain-injured animals (Ansari et al., 2008a). Nitra- tion of synapsin 1 is consistent with reported reduced neurotransmission and synaptic plasticity exhibited by TBI patients (Schwarzbach et al., 2006). Several proteins (g-enolase, phosphoglycerate mu- tase, and ATP synthase) nitrated in TBI are associated with energy metabolism. Enolase catalyzes the conver- sion of 2-phosphoglycerate to phosphoenolpyruvate, finally leading to ATP production. Gamma-enolase is one of the three isoforms (a, b, and g) of enolase, a protein frequently modified in several in vivo and in vitro systems of neurodegeneration (Schonberger et al., 2001; Poon et al., 2004, 2005a,b; Boyd-Kimball et al., 2005a; Perluigi et al., 2005; Butterfield, 2006; Ekegren Fig. 2. 3-Nitrotyrosine levels in traumatically brain-injured rats and remedied by GCEE treatment. Percentage 6 SEM; N 5 6; *P < 0.01,**P < 0.05, ***P < 0.009. Fig. 1. Protein carbonyl content in traumatically brain-injured rats and remedied by GCEE treatment. Percentage 6 SEM; N 5 6; *P < 0.04, **P < 1.45 3 1025 , ***P < 7.87 3 10–6 . 412 Reed et al. Journal of Neuroscience Research
  6. 6. et al., 2006). Phosphoglycerate mutase 1 (PGM1) cata- lyzes the interconversion of 3-phosphoglycerate to 2- phosphoglycerate, leading to a second equivalent of ATP produced in glycolysis. Protein dysfunction and increased glycolytic intermediates lead to decreased pyruvate pro- duction and decreased ATP production. PGM1 is found to be modified oxidatively in several models of oxidative stress and genetic mutation in neurodegenerative diseases (Bowling and Beal, 1995; Castegna et al., 2004). ATP synthase a-chain is a mitochondrial regulating subunit of complex V that plays a key role in energy production. ATP synthase goes through a sequence of coordinated conformational changes of its major subunits (a, b) to produce ATP. ATP synthase a-subunit has been previ- ously shown to be modified oxidatively in various mod- els of aging and in neurodegeneration (Boyd-Kimball et al., 2005c; Butterfield, 2006). The oxidation of ATP synthase leads to the inactivation of this mitochondrial complex, resulting in decreased electron transport chain activity and impaired ATP production. ATP, the energy source of the cell, is extremely important at nerve termi- nals for normal neural communication. Decreased levels of cellular ATP at nerve terminals may lead to loss of synapses and synaptic function, decreased function of ion pumps, loss of ion gradients, and ATPase dysfunction and may ultimately contribute to cell death and memory loss observed in TBI patients. Energy metabolism is sig- nificantly reduced in TBI patients (Marklund et al., 2006), and nitration of this protein can provide a reason for the lethargy associated with TBI. GTP-coupled proteins are lipid anchored to the plasma membrane and bind guanine nucleotides (GDP or GTP). G-proteins require binding to GTP for activa- tion. Normally, a guanosine exchange factor binds to the inactive G protein–GDP complex, alters the conforma- tion, and exchanges GDP for GTP, thereby activating the G protein. Once active, G proteins can bind to a target protein (effector) and stimulate signal transduction. Guanosine diphosphate dissociation inhibitors (GDI) in- hibit the exchange of GDP for GTP; therefore, the G protein will remain inactive. This inactivity prevents effector binding downstream signaling (i.e., glycogen ca- Fig. 3. Two-dimensional gel of TBI-treated rats (a), sham rats (b), and TBI rats treated with GCEE 10 min postinjury (c). Elevated Glutathione as a Potential Therapy for TBI 413 Journal of Neuroscience Research
  7. 7. tabolism, etc.). In TBI, glycogen levels are increased compared with control, supporting the downstream effects impaired by GDI (Otori et al., 2004). Heat shock protein 70 (Hsp70) is a member of the heat shock protein family. Heat shock proteins are cyto- protective to injury and various metabolic disturbances while also assisting in the stress response (Kiang and Tso- kos, 1998; Poon et al., 2004). The main function of heat shock proteins/cognates is to act as chaperone proteins by refolding misfolded proteins or shepherding these proteins for recycling through proteasomal degradation. Hsp70 is altered in TBI, bolstering the importance of functioning heat shock proteins in the cell (Lai et al., 2004; Poon et al., 2004; da Rocha et al., 2005). Hsp70 dysfunction may exacerbate protein misfolding, protein aggregation, and reduced effective proteasomal activity. Fig. 4. a: Two-dimensional Western blot of TBI rat probed with anti-3-nitrotyrosine antibody. b: Nitrated proteins in sham rat. c: Nitrated proteins in TBI rat treated with GCEE 10 min postinjury. TABLE I. Nitrated Proteins in TBI-Treated Rats Protein MOWSE score pI Apparent MW based on migration rate (Mr) Peptide coverage (%) Protein nitration (% control) Probability Synapsin 1 169 9.81 74,114 39 122.5 6 54.2 <0.05 Gamma-enolase 185 5.91 61,098 34 54.03 6 8.9 <0.02 Guanosine diphosphate dissociation inhibitor 1 (GDP) 140 5.00 51,074 50 161.4 6 13.4 <0.05 Phosphoglycerate mutase (PGM) 84 6.75 28,797 31 728.6 6 27.4 <0.04 Heat shock protein 70 (Hsp70) 186 5.07 72,402 36 280.8 6 28.6 <0.05 ATP synthase 72 5.54 50,548 37 164.2 6 49.5 <0.03 Alpha-spectrin 217 5.22 285,689 18 365.2 6 23.1 <0.05 414 Reed et al. Journal of Neuroscience Research
  8. 8. Alpha-spectrin plays a critical role in maintaining cytoskeletal structure and membrane integrity. This pro- tein interacts with actin and tropomyosin, two proteins found in thin filaments and associated with muscle con- traction, in a hexagonal arrangement. Spectrin also binds to synapsin 1 (also known as band 4.1 in RBC mem- branes), which enhances spectrin-actin binding. In dif- fuse TBI, spectrin is cleaved by calpain, a Ca21 -depend- ent cysteine protease causing membrane degradation and cell death (Pike et al., 1998; Buki et al., 1999; Kupina et al., 2003; Yu and Geddes, 2007). Calpain activity plays a dominant role in posttraumatic brain injury and neurodegeneration (Saatman et al., 1996). Oxidative modification of spectrin has also been observed in the senescence-accelerated (SAMP8) mouse model (Poon et al., 2004), which has diminished learning and mem- ory. Spectrin oxidative modification in TBI is consistent with the memory and learning deficits associated with TBI (Schwarzbach et al., 2006). CONCLUSIONS This study suggests that elevation of GSH by GCEE 10 min post-TBI is neuroprotective against oxi- dative stress associated with TBI. This is one of the first studies to demonstrate a potential postinjury therapeutic strategy for the treatment of TBI, a condition that affects millions of persons annually. Specific proteins are pro- tected against nitrosative stress associated with TBI, which conceivably modulates loss of function of the pro- teins in TBI. GCEE offers promise as a potential postin- jury therapeutic for TBI. ACKNOWLEDGMENTS We thank TBI nurse Marcia Butterfield, RN, for useful discussions. T.T.R. was supported by a T32 train- ing grant administered by the University of Kentucky Spinal Cord and Brain Injury Research Center and funded by NIH grant AG-1012806160. 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