Abstracts
Many studies of protein expression after traumatic brain injury (TBI) have identified biomarkers for diagnosing or determining the prognosis of TBI. In this study, we searched for additional protein markers of TBI using a fluid perfusion impact device to model TBI in S-D rats. Two-dimensional gel electrophoresis and mass spectrometry were used to identify differentially expressed proteins. After proteomic analysis, we detected 405 and 371 protein spots within a pH range of 3-10 from sham-treated and contused brain cortex, respectively. Eighty protein spots were differentially expressed in the two groups and 20 of these proteins were identified. This study validated the established biomarkers of TBI and identified potential biomarkers that could be examined in future work.
traumatic brain injury; biomarkers; proteomics; two-dimensional gel electrophoresis; mass spectrometry
Muitos estudos de expressão proteica após lesão cerebral traumática (LCT) identificam biomarcadores para determinação diagnóstica ou prognóstica do LCT. No presente estudo, foram investigados marcadores proteicos adicionais de LCT, através de um aparelho de impacto no fluxo e perfusão em ratos S-D. Eletroforese bidimensional em gel e espectrometria de massa foram utilizadas para identificar diferentes proteínas expressas. Após a análise proteômica, detectamos marcas de proteínas 405 e 371, com pH variando entre 3-10 no córtex de ratos sham e naqueles com contusão cerebral, respectivamente. Oitenta marcas proteicas foram expressas nos dois grupos e 20 destas proteínas foram identificadas. Este estudo validou o estabelecimento de biomarcadores de LCT e identificou potencial biomarcadores que poderão ser estudados em estudos futuros.
lesão traumática cerebral; biomarcadores; proteômica; eletroforese bidimensional em gel; espectrometria de massa
Traumatic brain injury (TBI) is the leading cause of traumatic death and disability
worldwide11 Chiu WT, Huang SJ, Tsai SH, Lin JW, Tsai MD, Lin TJ et al. The impact of
time, legislation, and geography on the epidemiology of traumatic brain injury. J Clin
Neurosci. 2007;14(10):930-5. http://dx.doi.org/10.1016/j.jocn.2006.08.004
https://doi.org/10.1016/j.jocn.2006.08.0...
. The main causes of TBI are
motor vehicle accidents (50%), falls (21%), assaults and violence (12%), sports and recreation
(10%), and other (7%)22 Centers for Disease Control and Prevention. TBI outcomes and consequences.
[Cited 2005 July]. Available from:
http://www.cdc.gov/node.do/id/0900f3ec8000dbdc/aspectId/A0400027
http://www.cdc.gov/node.do/id/0900f3ec80...
. TBI patients impose a
tremendous burden on their families and society, and increase demands on the healthcare
system. In order to achieve favorable outcomes, rapid diagnosis and treatment are
important.
TBI triggers complex changes to the central nervous system (CNS). A better understanding of
its complex pathobiology is required to further our ability to evaluate and care for brain
injury patients. However, an understanding of the mechanisms and biomarkers of TBI remains
elusive. Biomarkers reflecting the biological response to injury or disease have proven useful
for diagnosing many disorders, including responses to injury, cancer, heart failure,
infection, and genetic disorders (Table 1)33 Hergenroeder GW, Redell JB, Moore AN, Dash PK. Biomarkers in the clinical
diagnosis and management of traumatic brain injury. Mol Diagn Ther 2008;12(6):345-58.
http://dx.doi.org/10.2165/1250444-200812060-00002
https://doi.org/10.2165/1250444-20081206...
. Many biomarkers of TBI have been identified,
although they are not widely used clinically. These markers include S-100ß, neuron specific
enolase (NSE), glial fibrillary acid protein (GFAP), and myelin basic protein (MBP). Although
these proteins are still being assessed, they appear to lack either the sensitivity or
specificity (except GFAP) to be used effectively alone44 Bazarian JJ, Zemlan FP, Mookerjee S, Stigbrand T. Serum S-100B and
cleaved-tau are poor predictors of long-term outcome after mild traumatic brain injury.
Brain Inj 2006;20(7):759-65.. However, combinations of these markers can provide valuable
information and effectively diagnose and predict the outcome of TBI55 Berger RP, Beers SR, Richichi R, Wiesman D, Adelson PD. Serum biomarker
concentrations and outcome after pediatric traumatic brain injury. J Neurotraum.
2007;24(12):1793-801. http://dx.doi.org/10.1089/neu.2007.0316
https://doi.org/10.1089/neu.2007.0316...
. Given their potential utility for determining the diagnosis
and prognosis of brain injuries, their clinical utility should be explored further.
Proteomic analysis is a powerful tool for the global evaluation of protein expression and has
been applied widely in the analysis of disease66 Sun W, Xing B, Sun Y, Du X, Lu M, Hao C et al. Proteome analysis of
hepatocellular carcinoma by two-dimensional difference gel electrophoresis: novel protein
markers in hepatocellular carcinoma tissues. Mol Cell Proteomics.
2007;6(10):1798-808.. Analyses of the TBI proteome can aid in our understanding of the
association between protein changes and brain injury, and several studies have already applied
proteomics to the identification of biomarkers for TBI77 Ottens AK, Kobeissy FH, Fuller BF, Liu MC, Oli MW, Hayes RL et al. Novel
neuroproteomic approaches to studying traumatic brain injury. Prog Brain Res.
2007;161:401-18. http://dx.doi.org/10.1016/S0079-6123(06)61029-7
https://doi.org/10.1016/S0079-6123(06)61...
.
Proteomic analysis is a discovery-based method that invariably identifies many proteins that
differ in abundance between control and experimental samples. TBI studies use samples such as
brain tissue (often from animals), cerebrospinal fluid (CSF), and blood for proteomic
analysis. These samples contain information related to the CNS. Of the proteins found in CSF
samples, 76% were unique to that biofluid. By contrast, plasma and serum proteins have wide,
dynamic concentration ranges and it is often difficult to discover a disease-specific
biomarker within the background of blood-borne housekeeping proteins77 Ottens AK, Kobeissy FH, Fuller BF, Liu MC, Oli MW, Hayes RL et al. Novel
neuroproteomic approaches to studying traumatic brain injury. Prog Brain Res.
2007;161:401-18. http://dx.doi.org/10.1016/S0079-6123(06)61029-7
https://doi.org/10.1016/S0079-6123(06)61...
. Consequently, the ideal samples for proteomic analysis are
brain tissue and CSF.
METHOD
Animal model
Forty male Sprague-Dawley rats weighing 250-300 g were randomized into sham-operated or
injury groups (n = 20/group). Rats were acclimated in a humidified room and maintained on
a standard pellet diet at the Animal Center of Shanghai Jiao Tong University School of
Medicine for 10 days before the experiment. The temperature in both the feeding and
operating rooms was maintained at approximately 25 to 28°C. A fluid perfusion impact
device was used to model TBI in the rats, as described previously88 Finnie JW. Animal models of traumatic brain injury: a review. Aust Vet J.
2001;79(9):628-33. http://dx.doi.org/10.1111/j.1751-0813.2001.tb10785.x
https://doi.org/10.1111/j.1751-0813.2001...
. All of the surgical, injury, and animal care protocols
described below were approved by the Scientific and Ethics Committee of Shanghai Jiaotong
University affiliated Sixth People’s Hospital (Permit no. SYXK20130608). The rats in the
TBI group were anesthetized with pentobarbital (3.5%, 40 mg/kg), shaved, and then placed
in a stereotaxic frame. Surgery was performed as described by Sullivan and colleagues99 Sullivan PG, Keller JN, Bussen WL, Scheff SW. Cytochrome c release and
caspase activation after traumatic brain injury. Brain Res. 2002;949(1-2):88-96.
http://dx.doi.org/10.1016/S0006-8993(02)02968-2
https://doi.org/10.1016/S0006-8993(02)02...
. Briefly, a 6-mm ipsilateral craniotomy
tangential to the bregma and the sagittal suture was made, leaving the underlying dura
mater intact. A 5-mm-wide impactor tip was adjusted so that it just touched the exposed
dura mater and was secured over the right parietal cortex. The next day, the animals were
anesthetized, intubated, and then placed under a fluid-percussion brain injury device. A
moderate fluid-percussion pulse (2.0 ± 0.2 atmospheres) was delivered to the right
parietal cortex. Sham-operated rats underwent all of the surgical manipulations, but
without the fluid-percussion pulse, and were monitored under anesthesia for 30 min after
the sham operation. All of the animals were kept in the same environment after surgery for
48 hours.
Specimens
The sham-operated and TBI animals were anesthetized 48 h postoperatively and sacrificed by decapitation. Immediately following decapitation, the brain was removed and the cortex samples were dissected rapidly over dry ice. All of the samples were washed with ice-cold saline before freezing to reduce brain contamination with blood proteins. Samples were then stored in liquid nitrogen for further processing. Injured cortex samples were dissected to about 2 mm from the injury spot; sham samples were removed from the same location. The total time from decapitation to snap freezing of the samples was about 5 min for all animals in both groups.
Two-dimensional gel electrophoresis
First, 100 mg of tissue sample was ground into a powder in liquid nitrogen, homogenized in 1 ml of lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris-HCl, protease inhibitor mixture) on ice, and sonicated (10×10-s pulses) on ice. The homogenate was centrifuged at 12,000 rpm for 30 min at 4°C. The protein was precipitated with cold acetone at -20°C for 2 h and dissolved with rehydration buffer (8 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 2% ampholyte). Protein concentrations were determined using the Bradford method (Bio-Rad). Immobilized pH gradient (IPG) strips (18 cm, pH 4-7, non-linear; Bio-Rad) were rehydrated passively using 400 μl of rehydration buffer for 12 h at 17°C. Isoelectric focusing electrophoresis (IEF) was performed on an IEF cell (Bio-Rad). The strips were equilibrated in equilibration buffer (25 mM Tris-HCl (pH 8.8), 6 M urea, 20% glycerol, 2% SDS, 130 mM DTT) for 15 min and then in the same buffer containing 200 mM iodoacetamide instead of DTT for another 15 min. The two-dimensional gel electrophoresis (2-DE) separation was performed on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The gels were stained using Coomassie Brilliant Blue R-350 (Merck), according to the supplier’s protocol.
In-gel tryptic digestion and protein identification by mass spectrometry
In-gel tryptic digestion and protein identification by mass spectrometry (MS) were performed as described elsewhere66 Sun W, Xing B, Sun Y, Du X, Lu M, Hao C et al. Proteome analysis of hepatocellular carcinoma by two-dimensional difference gel electrophoresis: novel protein markers in hepatocellular carcinoma tissues. Mol Cell Proteomics. 2007;6(10):1798-808.. Briefly, protein spots of interest were excised and destained. In-gel digestion was performed with 0.01 μg/μl trypsin (Promega) for 20 h at 37°C. The tryptic peptides were extracted from the gel and dried by centrifugal lyophilization. The peptide mixtures were redissolved in 0.5% trifluoroacetic acid (TFA) and analyzed on an AB4700 Proteomics Analyzer (Bruker Daltonics Inc.). Peptide mass maps were acquired in positive reflection mode, averaging 1500 laser shots per matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectrum and 3,000 shots per TOF/TOF spectrum.
Data analysis
After gel staining, the protein spots were detected, quantified, and matched using PD-Quest 2D analysis software (Bio-Rad). Each sample was run in triplicate. Protein identificated by mass spectrometry (MS), and the MS/MS data were assigned using a MASCOT search against the NCBInr database. Redundant proteins that appeared in the database under different names and accession numbers were eliminated. If more than one protein was identified in one spot, the single protein with the highest protein score was selected from the multi-protein family. Statistical calculations were performed by SPSS statistical software (version 16.0; SPSS, Inc.). Comparison between two groups was performed by Wilcoxon two-sample test. Statistical significance was defined as p < 0.05.
RESULTS
Differentially expressed proteins in the sham-operated and TBI groups
Contused brain cortex from TBI rats and brain cortex from sham-operated rats were analyzed in triplicate using 2-DE. Coomassie staining of the gels detected 405 and 371 protein spots within a pH range from 3-10 from the sham-operated and contused cortex, respectively (Figure 1). Statistically, 80 protein spots were differentially expressed in the sham-operated and TBI rats brain cortices (p < 0.05).
Proteomic analysis of sham-operated and traumatic brain injury (TBI) brain cortex using 2-DE.
Mass spectrum identification of differentially expressed proteins
The differentially expressed protein spots were subjected to MS/MS analysis. Twenty proteins were identified from the 80 spots (Table 2). Of these, some were expressed more strongly in the sham-operated group (spot numbers: 49, 70, 109, 158, 231, 248, 254, 289 and 369), and others were expressed more strongly in the TBI group (spot numbers: 11, 31, 55, 97, 171, 300, 307, 358, 365, 387 and 388). MS/MS analysis showed these spots had different MASCOT scores and sequence coverage. The predicted molecular masses/pI values for the 20 spots are listed below, and fit the position of the corresponding spot on the 2-DE gel well (Table 2, Figure 2).
Of the 20 identified proteins, NSE, GFAP, S100B, αII-spectrin, and MAP2 have been studied as biomarkers of TBI (proteomic data not shown in Table 2). Tumor biomarkers such as 14-3-3 protein have not been studied in TBI. Proteins associated with cell apoptosis were also identified, such as GAPDH and RhoB. The other identified proteins have not been examined in relation to TBI and little is known regarding their function, such as nebulin-related anchoring protein and zinc finger protein 180.
DISCUSSION
This study used proteomic analyses to identify 20 proteins that were differentially expressed in the sham-operated and TBI groups of rats. These included proteins previously identified as biomarkers of TBI, such as NSE, GFAP, S100B, αII-spectrin, and MAP2. Others had not been identified in TBI patients or animals, including the tumor indicators M2-PK and 14-3-3 protein. The functions of some of the proteins that were identified after TBI have not been established.
NSE (spot no. 11) is a biomarker of TBI and has been studied in infants, children, and
adults, and in mild and severe TBI. In clinical studies, serum NSE levels have been
frequently studied. Berger et al. found that NSE was markedly increased in the CSF after
severe TBI1010 Berger RP, Pierce MC, Wisniewski SR, Adelson PD, Clark RS, Ruppel RA et al.
Neuron-specific enolase, and S100B in cerebrospinal fluid after severe traumatic brain
injury in infants and children. Pediatrics. 2002;109(2):E31.. NSE levels were
significantly elevated in non-survivors as compared with survivors in adults1111 Böhmer AE, Oses JP, Schmidt AP, Perón CS, Krebs CL, Oppitz PP et al.
Fibrillary acidic protein levels as outcome predictors in patients with severe traumatic
brain injury. Neurosurg. 2011;68(6):1624-31.
http://dx.doi.org/10.1227/NEU.0b013e318214a81f
https://doi.org/10.1227/NEU.0b013e318214...
. Early elevation (≤ 3 days) of NSE
secondary to severe TBI predicts deterioration to brain death. Topolovec-Vranic found that
NSE was abnormally elevated in 65% of patients with TBI and predicted a poor outcome at 6
weeks post-injury1212 Topolovec-Vranic J, Pollmann-Mudryj MA, Ouchterlony D, Klein D, Spence J,
Romaschin A et al. The value of serum biomarkers in prediction models of outcome after
mild traumatic brain injury. J Trauma. 2011;71(5 Suppl 1):S478-86.
http://dx.doi.org/10.1097/TA.0b013e318232fa70
https://doi.org/10.1097/TA.0b013e318232f...
. NSE is also a
candidate serum marker of impending cerebral hypoxia, which is elevated before the onset of
clinical manifestations1313 Stein DM, Lindell AL, Murdock KR, Kufera JA, Menaker J, Bochicchio GV et al.
Use of serum biomarkers to predict cerebral hypoxia after severe traumatic brain injury. J
Neurotrauma. 2012;29(6):1140-9. http://dx.doi.org/10.1089/neu.2011.2149
https://doi.org/10.1089/neu.2011.2149...
. After multiple
trauma, elevated NSE levels have been observed, but systemic NSE increased by similar
degrees with and without TBI, limiting its ability to discriminate brain injury magnitude.
Reports on correlations of serum NSE levels alone with clinical and neurological measures of
brain injury magnitude and outcome have been controversial. NSE is not used widely
clinically, perhaps because of the above and the lack of a large-scale, multi-center,
randomized controlled study.
GFAP (spot no. 307) is a filament protein found in the astroglial cytoskeleton, is not
found outside the CNS. GFAP could predictably discriminate between severe disability and
vegetative state versus good and moderate outcomes as evaluated by the Glasgow Outcome Scale
(GOS). A study of severe TBI patients confirmed its ability to predict mortality, and it was
found to discriminate outcome categories of the GOS and Marshall CT classification. It also
could discriminate between patients that had intracranial pressure (ICP) greater or less
than 25 mm Hg, patients that had cerebral perfusion pressure greater or less than 60 mm Hg,
and patients with mean arterial pressure greater or less than 60 mm Hg. GFAP shows good
diagnostic potential to predict outcome after injury, and may also be valuable for
diagnosing injury magnitude1414 Pelinka LE, Kroepfl A, Leixnering M, Buchinger W, Raabe, A, Redl H. GFAP
versus S100B in serum after traumatic brain injury: relationship to brain damage and
outcome. J Neurotrauma. 2004;21(11):1553-61.
http://dx.doi.org/10.1159/000103304
https://doi.org/10.1159/000103304...
.
S100B (spot no. 387) is the most well studied proteins for TBI and is considered a
promising, non-proprietary brain injury biomarker. S100B is most abundant in glial cells of
the CNS and peripheral nervous system (Schwann cells). But this marker is that it is not
exclusive to the brain, it can be found in other cells such as adipocytes and chondrocytes.
A number of studies have demonstrated S100B’s relationship to injury magnitude and outcome
in TBI55 Berger RP, Beers SR, Richichi R, Wiesman D, Adelson PD. Serum biomarker
concentrations and outcome after pediatric traumatic brain injury. J Neurotraum.
2007;24(12):1793-801. http://dx.doi.org/10.1089/neu.2007.0316
https://doi.org/10.1089/neu.2007.0316...
,1414 Pelinka LE, Kroepfl A, Leixnering M, Buchinger W, Raabe, A, Redl H. GFAP
versus S100B in serum after traumatic brain injury: relationship to brain damage and
outcome. J Neurotrauma. 2004;21(11):1553-61.
http://dx.doi.org/10.1159/000103304
https://doi.org/10.1159/000103304...
. However, some studies reported a poor value of S100B as a
predictor of outcome after brain injury, particularly mild and pediatric TBI. A poor
correlation was found between serum and brain S100B values, suggesting that the serum levels
may depend primarily on the integrity of the blood-brain-barrier and do not reflect the
S100B levels in the brain66 Sun W, Xing B, Sun Y, Du X, Lu M, Hao C et al. Proteome analysis of
hepatocellular carcinoma by two-dimensional difference gel electrophoresis: novel protein
markers in hepatocellular carcinoma tissues. Mol Cell Proteomics.
2007;6(10):1798-808.,1515 Piazza O, Storti MP, Cotena S, Stoppa F, Perrotta D, Esposito G et al. S100B
is not a reliable prognostic index in paediatric TBI. Pediatr. Neurosurg.
2007;43(4):258-64.. Despite apparent controversy, S100B still
has potential as a brain injury biomarker, and its preclinical and clinical utility should
be further explored.
αII-spectrin (spot no. 97) is a major structural component of the neuron axonal
cytoskeleton and a major proteolytic substrate for cysteine proteases involved in necrotic
and apoptotic cell death. Many authors have examined αII-spectrin after TBI in rats or
humans. αII-spectrin levels in the CSF were shown to increase after TBI in a rat model1616 Aikmana J, O’Steen B, Silver X, Torres R, Boslaugh S, Blackband S et al.
Alpha-II-spectrin after controlled cortical impact in the immature rat brain. Dev
Neurosci. 2006;28(4-5):457-65. http://dx.doi.org/10.1159/000094171
https://doi.org/10.1159/000094171...
. In patients, Brophy found that
αII-spectrin breakdown products (SBDPs) were significantly elevated in patients with worse
Glasgow Coma Scale (GCS) scores 24 h after injury compared to those whose GCS scores
improved1717 Brophy GM, Pineda JA, Papa L, Lewis SB, Valadka AB, Hannay HJ et al.
alphaII-Spectrin breakdown product cerebrospinal fluid exposure metrics suggest
differences in cellular injury mechanisms after severe traumatic brain injury. J
Neurotraum. 2009;26(4):471-9. http://dx.doi.org/10.1089/neu.2008.0657
https://doi.org/10.1089/neu.2008.0657...
. The mean CSF levels of SBDPs
were significantly higher in TBI patients than in controls and in patients who died than in
those who survived, and the SBDP concentration was significantly greater in TBI patients
than in controls and was correlated with the GCS score1818 Berger RP, Hayes R, Richichi R, Beers SR, Wang KK. Serum concentrations of
ubiquitin C-terminal hydrolase-L1 and alII-spectrin breakdown product 145 kDa correlate
with outcome after pediatric TBI. J Neurotrauma. 2012;29(1):162-7.
http://dx.doi.org/10.1089/neu.2011.1989
https://doi.org/10.1089/neu.2011.1989...
. Therefore, αII-spectrin is an established biomarker of TBI.
MAP2 (spot no. 388) is important for microtubule stability and neural plasticity and
appears to be among the most vulnerable of the cytoskeletal proteins following neuronal
injury. Huh et al. found that MAP2 is an early, sensitive marker of neuronal damage
following TBI. Early after TBI (2 hours) MAP2 expression decreased than that of control
group. But in this study, we found that MAP2 was increased in TBI group. We speculate that
the time of specimen harvesting led to this difference. In addition, MAP2 is an early blood
marker in ischemic brain injury1919 Park D, Joo SS, Lee HJ, Choi KC, Kim SU, Kim YB. Microtubule-associated
protein 2, an early blood marker of ischemic brain injury. J Neurosci Res.
2012;90(2):461-7. http://dx.doi.org/10.1002/jnr.22769
https://doi.org/10.1002/jnr.22769...
.
Therefore, MAP2 can be used as a marker for detecting neurotoxic insults, including ischemia
and TBI.
There is a growing awareness that RhoB (spot no. 31) is also important signaling molecules
in the CNS. RhoB is a member of the Rho GTPase family that is dramatically induced by brain
ischemia or trauma. It seems likely that the increase in RhoB level plays a major role in
determining the fate of the neurons, but there is little evidence as to whether the RhoB
induction is beneficial or detrimental to neuronal survival. The increased RhoB expression
promotes neurite outgrowth; but this increase is likely to make a substantial contribution
to the neurodegenerative process, and also promotes caspase 3 activation and DNA
fragmentation, key contributors to cell apoptosis2020 Barberan S, McNair K, Iqbal K, Smith NC, Prendergast GC, Stone TW et al.
Altered apoptotic responses in neurons lacking RhoB GTPase. Eur J Neurosci.
2011;34(11):1737-46. http://dx.doi.org/10.1111/j.1460-9568.2011.07891.x
https://doi.org/10.1111/j.1460-9568.2011...
. RhoB mediates apoptosis in neoplastically transformed cells after
DNA damage. Of the 20 proteins, GAPDH (spot no. 171) also participates in nuclear events,
including transcription, RNA transport, DNA replication, and cell apoptosis2121 Tisdale EJ. Glyceraldehyde-3-phosphate dehydrogenase is phosphorylated by
protein kinase Ciota /lambda and plays a role in microtubule dynamics in the early
secretory pathway. J Biol Chem. 2002;277(5):3334-41.
http://dx.doi.org/10.1074/jbc.M109744200
https://doi.org/10.1074/jbc.M109744200...
. Therefore, RhoB and GAPDH may play roles
in neuro-cell apoptosis after TBI.
M2-PK (spot no. 49) is a phosphotyrosine-binding protein that has been studied in tumors,
but not identified in TBI. M2-PK expression is critical for rapid growth in cancer
cells2222 Christofk HR, Heiden MG, Wu N, Asara JM, Cantley LC. Pyruvate kinase M2 is a
phosphotyrosine-binding protein. Nature. 2008;452(7184):181-6.
http://dx.doi.org/10.1038/nature06667
https://doi.org/10.1038/nature06667...
. It has been used in the
diagnosis and surveillance of a variety of malignant diseases. With respect to injury,
Oehler found that M2-PK expression and activity were increased in polytrauma patients
compared to controls2323 Luo W, Hu H, Chang R, Zhong J, Knabel M, O’Meally R et al. Pyruvate kinase
M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1. Cell.
2011;145(5):732-44. http://dx.doi.org/10.1016/j.cell.2011.03.054
https://doi.org/10.1016/j.cell.2011.03.0...
. M2-PK is the
product of the PKM2 gene. PKM2 gene transcription is
activated by hypoxia-inducible factor 1 (HIF-1), i.e., when hypoxia occurs, such as after
TBI, the PKM2 gene produces M2-PK. Pyruvate kinase (PK) orthologs in many
organisms are inhibited by oxidants. Anastasiou found that the inhibition of M2-PK by
reactive oxygen species contributes to cellular antioxidant responses2424 Anastasiou D, Poulogiannis G, Asara JM, Boxer MB, Jiang JK, Shen M et al.
Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular
antioxidant responses. Science. 2011;334(6060):1278-83.
http://dx.doi.org/10.1126/science.1211485
https://doi.org/10.1126/science.1211485...
. Given the presence of hypoxia after TBI, M2-PK is a
potential biomarker of TBI.
Another protein that has not previously been found after TBI is ATP-binding cassette
protein C12 (spot no. 254). The gene for ATP-binding cassette protein C12 is
Abcc12 in rats and ABCC12 in humans. The product of
ABCC12 is MRP9, which is expressed in breast tissue, brain, bone, and
ovary2525 Bera TK, Iavarone C, Kumar V, Lee S, Lee B, Pastan I. MRP9, an unusual
truncated member of the ABC transporter superfamily, is highly expressed in breast cancer.
Proc Natl Acad Sci USA. 2002;99(10):6997-7002.
http://dx.doi.org/10.1073/pnas.102187299
https://doi.org/10.1073/pnas.102187299...
. SUR1, the product of
Abcc8, has been found in the CNS after TBI and has an effect on
progressive hemorrhagic necrosis (PHN)2626 Simard JM, Tsymbalyuk O, Ivanov A, Ivanova S, Bhatta S, Geng Z et al.
Endothelial sulfonylurea receptor 1– regulated NC Ca-ATP channels mediate progressive
hemorrhagic necrosis following spinal cord injury. J Clin Invest. 2007;117(8):2105-13.
http://dx.doi.org/10.1172/JCI32041
https://doi.org/10.1172/JCI32041...
.
Abcc8 and Abcc12 are members of the Abcc superfamily and
their products, SUR1 and MRP9, respectively, have been found in brain. Therefore, similar to
SUR1, MRP9 might be an indicator of TBI.
The 14-3-3 protein (spot no. 387) family plays an important role in tumorigenesis and
development. In addition, 14-3-3 protein is a biomarker of neurodegenerative diseases2727 Bersano A, Fiorini M, Allaria S, Zanusso G, Fasoli E, Gelati M et al.
Detection of CSF 14-3-3 protein in Guillain-Barré syndrome. Neurology. 2006;67(12):2211-6.
http://dx.doi.org/10.1212/01.wnl.0000249150.98891.d1
https://doi.org/10.1212/01.wnl.000024915...
. In the motoneurons of rats, Namikawa
reported that enhanced expression of the molecules involved in Ras-Erk signaling, such as
14-3-3 protein, is required for peripheral nerve regeneration2828 Namikawa K, Su Q, Kiryu-Seo S, Kiyama H. Enhanced expression of 14-3-3
family members in injured motoneurons. Brain Res Mol Brain Res. 1998;55(2):315-20.
http://dx.doi.org/10.1016/S0169-328X(98)00012-6
https://doi.org/10.1016/S0169-328X(98)00...
. Apoptosis is important in cell death after TBI and is associated
with B-cell lymphoma 2 (Bcl-2). 14-3-3 protein can bind Bcl-2-antagonist cell death (BAD)
and then trigger cell apoptosis2929 Datta SR, Katsov A, Hu L, Petros A, Fesik SW, Yaffe MB et al. 14-3-3
proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation.
Mol Cell. 2000;6(1):41-51.
http://dx.doi.org/10.1016/S1097-2765(00)00006-X
https://doi.org/10.1016/S1097-2765(00)00...
.
Consequently, 14-3-3 protein is a potential marker of apoptosis after TBI.
The remaining proteins, including leucine-, glutamate-, and lysine-rich protein 1, H(+)-transporting ATP synthase, nebulin-related anchoring protein, and zinc finger protein 180, have not been studied sufficiently and their functions after TBI have not been established. We can find no conclusive published relationships between these proteins and TBI. Therefore, more studies should target these proteins.
This study has several limitations. First, the study examined one time point after TBI (48 h), and the animal model was for moderate TBI. We did not determine protein expression at other times after TBI, nor did we determine protein expression in mild or severe TBI models. Second, the 20 proteins identified in the proteomic analysis have not been confirmed using Western blotting, immunohistochemistry, or reverse transcription polymerase chain reaction (RT-PCR) in animals or patients. Future work should focus on these limitations and identify protein expression at different time points in mild, moderate, and severe TBI, in animal models or in patients at one or more centers.
In general, this study validated several established biomarkers of TBI and identified other potential biomarkers that can be evaluated in the future.
References
-
1Chiu WT, Huang SJ, Tsai SH, Lin JW, Tsai MD, Lin TJ et al. The impact of time, legislation, and geography on the epidemiology of traumatic brain injury. J Clin Neurosci. 2007;14(10):930-5. http://dx.doi.org/10.1016/j.jocn.2006.08.004
» https://doi.org/10.1016/j.jocn.2006.08.004 -
2Centers for Disease Control and Prevention. TBI outcomes and consequences. [Cited 2005 July]. Available from: http://www.cdc.gov/node.do/id/0900f3ec8000dbdc/aspectId/A0400027
» http://www.cdc.gov/node.do/id/0900f3ec8000dbdc/aspectId/A0400027 -
3Hergenroeder GW, Redell JB, Moore AN, Dash PK. Biomarkers in the clinical diagnosis and management of traumatic brain injury. Mol Diagn Ther 2008;12(6):345-58. http://dx.doi.org/10.2165/1250444-200812060-00002
» https://doi.org/10.2165/1250444-200812060-00002 -
4Bazarian JJ, Zemlan FP, Mookerjee S, Stigbrand T. Serum S-100B and cleaved-tau are poor predictors of long-term outcome after mild traumatic brain injury. Brain Inj 2006;20(7):759-65.
-
5Berger RP, Beers SR, Richichi R, Wiesman D, Adelson PD. Serum biomarker concentrations and outcome after pediatric traumatic brain injury. J Neurotraum. 2007;24(12):1793-801. http://dx.doi.org/10.1089/neu.2007.0316
» https://doi.org/10.1089/neu.2007.0316 -
6Sun W, Xing B, Sun Y, Du X, Lu M, Hao C et al. Proteome analysis of hepatocellular carcinoma by two-dimensional difference gel electrophoresis: novel protein markers in hepatocellular carcinoma tissues. Mol Cell Proteomics. 2007;6(10):1798-808.
-
7Ottens AK, Kobeissy FH, Fuller BF, Liu MC, Oli MW, Hayes RL et al. Novel neuroproteomic approaches to studying traumatic brain injury. Prog Brain Res. 2007;161:401-18. http://dx.doi.org/10.1016/S0079-6123(06)61029-7
» https://doi.org/10.1016/S0079-6123(06)61029-7 -
8Finnie JW. Animal models of traumatic brain injury: a review. Aust Vet J. 2001;79(9):628-33. http://dx.doi.org/10.1111/j.1751-0813.2001.tb10785.x
» https://doi.org/10.1111/j.1751-0813.2001.tb10785.x -
9Sullivan PG, Keller JN, Bussen WL, Scheff SW. Cytochrome c release and caspase activation after traumatic brain injury. Brain Res. 2002;949(1-2):88-96. http://dx.doi.org/10.1016/S0006-8993(02)02968-2
» https://doi.org/10.1016/S0006-8993(02)02968-2 -
10Berger RP, Pierce MC, Wisniewski SR, Adelson PD, Clark RS, Ruppel RA et al. Neuron-specific enolase, and S100B in cerebrospinal fluid after severe traumatic brain injury in infants and children. Pediatrics. 2002;109(2):E31.
-
11Böhmer AE, Oses JP, Schmidt AP, Perón CS, Krebs CL, Oppitz PP et al. Fibrillary acidic protein levels as outcome predictors in patients with severe traumatic brain injury. Neurosurg. 2011;68(6):1624-31. http://dx.doi.org/10.1227/NEU.0b013e318214a81f
» https://doi.org/10.1227/NEU.0b013e318214a81f -
12Topolovec-Vranic J, Pollmann-Mudryj MA, Ouchterlony D, Klein D, Spence J, Romaschin A et al. The value of serum biomarkers in prediction models of outcome after mild traumatic brain injury. J Trauma. 2011;71(5 Suppl 1):S478-86. http://dx.doi.org/10.1097/TA.0b013e318232fa70
» https://doi.org/10.1097/TA.0b013e318232fa70 -
13Stein DM, Lindell AL, Murdock KR, Kufera JA, Menaker J, Bochicchio GV et al. Use of serum biomarkers to predict cerebral hypoxia after severe traumatic brain injury. J Neurotrauma. 2012;29(6):1140-9. http://dx.doi.org/10.1089/neu.2011.2149
» https://doi.org/10.1089/neu.2011.2149 -
14Pelinka LE, Kroepfl A, Leixnering M, Buchinger W, Raabe, A, Redl H. GFAP versus S100B in serum after traumatic brain injury: relationship to brain damage and outcome. J Neurotrauma. 2004;21(11):1553-61. http://dx.doi.org/10.1159/000103304
» https://doi.org/10.1159/000103304 -
15Piazza O, Storti MP, Cotena S, Stoppa F, Perrotta D, Esposito G et al. S100B is not a reliable prognostic index in paediatric TBI. Pediatr. Neurosurg. 2007;43(4):258-64.
-
16Aikmana J, O’Steen B, Silver X, Torres R, Boslaugh S, Blackband S et al. Alpha-II-spectrin after controlled cortical impact in the immature rat brain. Dev Neurosci. 2006;28(4-5):457-65. http://dx.doi.org/10.1159/000094171
» https://doi.org/10.1159/000094171 -
17Brophy GM, Pineda JA, Papa L, Lewis SB, Valadka AB, Hannay HJ et al. alphaII-Spectrin breakdown product cerebrospinal fluid exposure metrics suggest differences in cellular injury mechanisms after severe traumatic brain injury. J Neurotraum. 2009;26(4):471-9. http://dx.doi.org/10.1089/neu.2008.0657
» https://doi.org/10.1089/neu.2008.0657 -
18Berger RP, Hayes R, Richichi R, Beers SR, Wang KK. Serum concentrations of ubiquitin C-terminal hydrolase-L1 and alII-spectrin breakdown product 145 kDa correlate with outcome after pediatric TBI. J Neurotrauma. 2012;29(1):162-7. http://dx.doi.org/10.1089/neu.2011.1989
» https://doi.org/10.1089/neu.2011.1989 -
19Park D, Joo SS, Lee HJ, Choi KC, Kim SU, Kim YB. Microtubule-associated protein 2, an early blood marker of ischemic brain injury. J Neurosci Res. 2012;90(2):461-7. http://dx.doi.org/10.1002/jnr.22769
» https://doi.org/10.1002/jnr.22769 -
20Barberan S, McNair K, Iqbal K, Smith NC, Prendergast GC, Stone TW et al. Altered apoptotic responses in neurons lacking RhoB GTPase. Eur J Neurosci. 2011;34(11):1737-46. http://dx.doi.org/10.1111/j.1460-9568.2011.07891.x
» https://doi.org/10.1111/j.1460-9568.2011.07891.x -
21Tisdale EJ. Glyceraldehyde-3-phosphate dehydrogenase is phosphorylated by protein kinase Ciota /lambda and plays a role in microtubule dynamics in the early secretory pathway. J Biol Chem. 2002;277(5):3334-41. http://dx.doi.org/10.1074/jbc.M109744200
» https://doi.org/10.1074/jbc.M109744200 -
22Christofk HR, Heiden MG, Wu N, Asara JM, Cantley LC. Pyruvate kinase M2 is a phosphotyrosine-binding protein. Nature. 2008;452(7184):181-6. http://dx.doi.org/10.1038/nature06667
» https://doi.org/10.1038/nature06667 -
23Luo W, Hu H, Chang R, Zhong J, Knabel M, O’Meally R et al. Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1. Cell. 2011;145(5):732-44. http://dx.doi.org/10.1016/j.cell.2011.03.054
» https://doi.org/10.1016/j.cell.2011.03.054 -
24Anastasiou D, Poulogiannis G, Asara JM, Boxer MB, Jiang JK, Shen M et al. Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Science. 2011;334(6060):1278-83. http://dx.doi.org/10.1126/science.1211485
» https://doi.org/10.1126/science.1211485 -
25Bera TK, Iavarone C, Kumar V, Lee S, Lee B, Pastan I. MRP9, an unusual truncated member of the ABC transporter superfamily, is highly expressed in breast cancer. Proc Natl Acad Sci USA. 2002;99(10):6997-7002. http://dx.doi.org/10.1073/pnas.102187299
» https://doi.org/10.1073/pnas.102187299 -
26Simard JM, Tsymbalyuk O, Ivanov A, Ivanova S, Bhatta S, Geng Z et al. Endothelial sulfonylurea receptor 1– regulated NC Ca-ATP channels mediate progressive hemorrhagic necrosis following spinal cord injury. J Clin Invest. 2007;117(8):2105-13. http://dx.doi.org/10.1172/JCI32041
» https://doi.org/10.1172/JCI32041 -
27Bersano A, Fiorini M, Allaria S, Zanusso G, Fasoli E, Gelati M et al. Detection of CSF 14-3-3 protein in Guillain-Barré syndrome. Neurology. 2006;67(12):2211-6. http://dx.doi.org/10.1212/01.wnl.0000249150.98891.d1
» https://doi.org/10.1212/01.wnl.0000249150.98891.d1 -
28Namikawa K, Su Q, Kiryu-Seo S, Kiyama H. Enhanced expression of 14-3-3 family members in injured motoneurons. Brain Res Mol Brain Res. 1998;55(2):315-20. http://dx.doi.org/10.1016/S0169-328X(98)00012-6
» https://doi.org/10.1016/S0169-328X(98)00012-6 -
29Datta SR, Katsov A, Hu L, Petros A, Fesik SW, Yaffe MB et al. 14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol Cell. 2000;6(1):41-51. http://dx.doi.org/10.1016/S1097-2765(00)00006-X
» https://doi.org/10.1016/S1097-2765(00)00006-X
-
*
These authors contributed equally to this work.
-
Support: National Nature and Science Foundation of China (grant no. 81271383, 81471245), the Shanghai Science and Technique Committee (grant no. 13411951401) and the Shanghai Municipal Health Bureau project (grant no. 20114242).
Publication Dates
-
Publication in this collection
Apr 2015
History
-
Received
15 July 2014 -
Reviewed
07 Nov 2014 -
Accepted
27 Nov 2014