J Clin Neurol.  2014 Apr;10(2):84-93. 10.3988/jcn.2014.10.2.84.

Analysis of Spatial and Temporal Protein Expression in the Cerebral Cortex after Ischemia-Reperfusion Injury

Affiliations
  • 1Department of Neurological Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China. novamhi@gmail.com
  • 2Section of Neurosurgery, Department of Surgery, Taipei Medical University Hospital, Taipei Medical University, Taipei, Taiwan, Republic of China.

Abstract

BACKGROUND AND PURPOSE
Hypoxia, or ischemia, is a common cause of neurological deficits in the elderly. This study elucidated the mechanisms underlying ischemia-induced brain injury that results in neurological sequelae.
METHODS
Cerebral ischemia was induced in male Sprague-Dawley rats by transient ligation of the left carotid artery followed by 60 min of hypoxia. A two-dimensional differential proteome analysis was performed using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to compare changes in protein expression on the lesioned side of the cortex relative to that on the contralateral side at 0, 6, and 24 h after ischemia.
RESULTS
The expressions of the following five proteins were up-regulated in the ipsilateral cortex at 24 h after ischemia-reperfusion injury compared to the contralateral (i.e., control) side: aconitase 2, neurotensin-related peptide, hypothetical protein XP-212759, 60-kDa heat-shock protein, and aldolase A. The expression of one protein, dynamin-1, was up-regulated only at the 6-h time point. The level of 78-kDa glucose-regulated protein precursor on the lesioned side of the cerebral cortex was found to be high initially, but then down-regulated by 24 h after the induction of ischemia-reperfusion injury. The expressions of several metabolic enzymes and translational factors were also perturbed soon after brain ischemia.
CONCLUSIONS
These findings provide insights into the mechanisms underlying the neurodegenerative events that occur following cerebral ischemia.

Keyword

reperfusion injury; proteomics; protein expression; cerebral ischemia; neurodegenerative mechanisms; gerontology

MeSH Terms

Aconitate Hydratase
Aged
Anoxia
Brain Injuries
Brain Ischemia
Carotid Arteries
Cerebral Cortex*
Dynamin I
Fructose-Bisphosphate Aldolase
Geriatrics
Heat-Shock Proteins
Humans
Ischemia
Ligation
Male
Mass Spectrometry
Proteome
Proteomics
Rats, Sprague-Dawley
Reperfusion Injury*
Aconitate Hydratase
Dynamin I
Fructose-Bisphosphate Aldolase
Heat-Shock Proteins
Proteome

Figure

  • Fig. 1 Typical appearance of infarct areas in the rat brain at 24 h after transient occlusion of the middle cerebral artery. Normal tissues are stained purple with 2,3,5-triphenyltetrazolium chloride; infarcted tissue remains unstained. The appearance of this tissue relative to the sham-operated controls (data not shown) was similar to that of the nonlesioned contralateral hemispheres. The percentage volume of the hemispheric true infarct was typically 18-22%.

  • Fig. 2 Silver staining of 2-D gels of cerebrocortical tissue in different regions, revealing some different protein expression patterns.

  • Fig. 3 Temporal proteomes of ischemia-reperfusion injury in the cerebrocortex: two types of stain (silver stain and SYPRO Ruby) were used on 2-D gels at various time points after ischemia-reperfusion injury.

  • Fig. 4 Histogram comparing more than 400 spots to reveal pattern changes. SYPRO Ruby staining revealed 8 spots of down-regulated proteins and 39 spots of up-regulated proteins at 24 h after reperfusion. Conversely, 11 protein spots were up-regulated at 6 h and then returned to noninjury levels by 24 h.

  • Fig. 5 After in-gel digestion, the protein was eluted from spot and fingerprint patterns of peptides and described using matrix-assisted laser desorption ionization-time-of-flight analysis. The proteins were identified by Mascot search comparison.


Cited by  1 articles

The Role of the PI3K Pathway in the Regeneration of the Damaged Brain by Neural Stem Cells after Cerebral Infarction
Seong-Ho Koh, Eng H. Lo
J Clin Neurol. 2015;11(4):297-304.    doi: 10.3988/jcn.2015.11.4.297.


Reference

1. Swinbanks D. Government backs proteome proposal. Nature. 1995; 378:653.
Article
2. Hirano H, Kawasaki H, Sassa H. Two-dimensional gel electrophoresis using immobilized pH gradient tube gels. Electrophoresis. 2000; 21:440–445.
Article
3. Righetti PG, Castagna A. Recent trends in proteome analysis. Adv Chromatogr. 2003; 42:269–321.
4. Johnston-Wilson NL, Sims CD, Hofmann JP, Anderson L, Shore AD, Torrey EF, et al. The Stanley Neuropathology Consortium. Disease-specific alterations in frontal cortex brain proteins in schizophrenia, bipolar disorder, and major depressive disorder. Mol Psychiatry. 2000; 5:142–149.
Article
5. Krapfenbauer K, Engidawork E, Cairns N, Fountoulakis M, Lubec G. Aberrant expression of peroxiredoxin subtypes in neurodegenerative disorders. Brain Res. 2003; 967:152–160.
Article
6. Zabel C, Klose J. Influence of Huntington's disease on the human and mouse proteome. Int Rev Neurobiol. 2004; 61:241–283.
Article
7. Schonberger SJ, Edgar PF, Kydd R, Faull RL, Cooper GJ. Proteomic analysis of the brain in Alzheimer's disease: molecular phenotype of a complex disease process. Proteomics. 2001; 1:1519–1528.
Article
8. Maurer MH, Berger C, Wolf M, Fütterer CD, Feldmann RE Jr, Schwab S, et al. The proteome of human brain microdialysate. Proteome Sci. 2003; 1:7.
Article
9. Küry P, Schroeter M, Jander S. Transcriptional response to circumscribed cortical brain ischemia: spatiotemporal patterns in ischemic vs. remote non-ischemic cortex. Eur J Neurosci. 2004; 19:1708–1720.
Article
10. Chen A, Liao WP, Lu Q, Wong WS, Wong PT. Upregulation of dihydropyrimidinase-related protein 2, spectrin alpha II chain, heat shock cognate protein 70 pseudogene 1 and tropomodulin 2 after focal cerebral ischemia in rats--a proteomics approach. Neurochem Int. 2007; 50:1078–1086.
Article
11. Chen ST, Hsu CY, Hogan EL, Halushka PV, Linet OI, Yatsu FM. Thromboxane, prostacyclin, and leukotrienes in cerebral ischemia. Neurology. 1986; 36:466–470.
Article
12. Wang Y, Lin SZ, Chiou AL, Williams LR, Hoffer BJ. Glial cell line-derived neurotrophic factor protects against ischemia-induced injury in the cerebral cortex. J Neurosci. 1997; 17:4341–4348.
Article
13. Klein JB, Gozal D, Pierce WM, Thongboonkerd V, Scherzer JA, Sachleben LR, et al. Proteomic identification of a novel protein regulated in CA1 and CA3 hippocampal regions during intermittent hypoxia. Respir Physiol Neurobiol. 2003; 136:91–103.
Article
14. Kettritz R, Xu YX, Faass B, Klein JB, Müller EC, Otto A, et al. TNF-alpha-mediated neutrophil apoptosis involves Ly-GDI, a Rho GTPase regulator. J Leukoc Biol. 2000; 68:277–283.
15. Pappin DJ. Peptide mass fingerprinting using MALDI-TOF mass spectrometry. Methods Mol Biol. 1997; 64:165–173.
Article
16. Berg F, Gustafson U, Andersson L. The uncoupling protein 1 gene (UCP1) is disrupted in the pig lineage: a genetic explanation for poor thermoregulation in piglets. PLoS Genet. 2006; 2:e129.
Article
17. Turrens JF, Boveris A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem J. 1980; 191:421–427.
Article
18. Du Y, Miller CM, Kern TS. Hyperglycemia increases mitochondrial superoxide in retina and retinal cells. Free Radic Biol Med. 2003; 35:1491–1499.
Article
19. Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol. 2003; 552(Pt 2):335–344.
Article
20. Robbins AH, Stout CD. The structure of aconitase. Proteins. 1989; 5:289–312.
Article
21. Robbins AH, Stout CD. Structure of activated aconitase: formation of the [4Fe-4S] cluster in the crystal. Proc Natl Acad Sci U S A. 1989; 86:3639–3643.
Article
22. Hausladen A, Fridovich I. Superoxide and peroxynitrite inactivate aconitases, but nitric oxide does not. J Biol Chem. 1994; 269:29405–29408.
Article
23. Armstrong JS, Whiteman M, Yang H, Jones DP. The redox regulation of intermediary metabolism by a superoxide-aconitase rheostat. Bioessays. 2004; 26:894–900.
Article
24. Gardner PR, Fridovich I. Effect of glutathione on aconitase in Escherichia coli. Arch Biochem Biophys. 1993; 301:98–102.
Article
25. Uhl GR, Kuhar MJ, Snyder SH. Neurotensin: immunohistochemical localization in rat central nervous system. Proc Natl Acad Sci U S A. 1977; 74:4059–4063.
Article
26. Carraway RE, Mitra SP, Cochrane DE. Structure of a biologically active neurotensin-related peptide obtained from pepsin-treated albumin(s). J Biol Chem. 1987; 262:5968–5973.
Article
27. Park SJ, Son WS, Lee BJ. Structural analysis of hypothetical proteins from helicobacter pylori: an approach to estimate functions of unknown or hypothetical proteins. Int J Mol Sci. 2012; 13:7109–7137.
Article
28. Mazandu GK, Mulder NJ. Function prediction and analysis of mycobacterium tuberculosis hypothetical proteins. Int J Mol Sci. 2012; 13:7283–7302.
Article
29. Itoh H, Komatsuda A, Ohtani H, Wakui H, Imai H, Sawada K, et al. Mammalian HSP60 is quickly sorted into the mitochondria under conditions of dehydration. Eur J Biochem. 2002; 269:5931–5938.
Article
30. Izaki K, Kinouchi H, Watanabe K, Owada Y, Okubo A, Itoh H, et al. Induction of mitochondrial heat shock protein 60 and 10 mRNAs following transient focal cerebral ischemia in the rat. Brain Res Mol Brain Res. 2001; 88:14–25.
Article
31. Okubo A, Kinouchi H, Owada Y, Kunizuka H, Itoh H, Izaki K, et al. Simultaneous induction of mitochondrial heat shock protein mRNAs in rat forebrain ischemia. Brain Res Mol Brain Res. 2000; 84:127–134.
Article
32. Flórez G, Cabeza A, Gonzalez JM, Garcia J, Ucar S. Changes in serum and cerebrospinal fluid enzyme activity after head injury. Acta Neurochir (Wien). 1976; 35:3–13.
Article
33. Linke S, Goertz P, Baader SL, Gieselmann V, Siebler M, Junghans U, et al. Aldolase C/zebrin II is released to the extracellular space after stroke and inhibits the network activity of cortical neurons. Neurochem Res. 2006; 31:1297–1303.
Article
34. Cook T, Mesa K, Urrutia R. Three dynamin-encoding genes are differentially expressed in developing rat brain. J Neurochem. 1996; 67:927–931.
Article
35. Cao H, Garcia F, McNiven MA. Differential distribution of dynamin isoforms in mammalian cells. Mol Biol Cell. 1998; 9:2595–2609.
Article
36. Ferguson SM, Brasnjo G, Hayashi M, Wölfel M, Collesi C, Giovedi S, et al. A selective activity-dependent requirement for dynamin 1 in synaptic vesicle endocytosis. Science. 2007; 316:570–574.
Article
37. Dikow AL, Lolova I, Ivanova A, Bojinov S. [Biochemical and histochemical studies of fructosephosphate aldolase in tumors of the nervous system. Isoenzymes of fructosephosphate aldolase. 8]. Z Klin Chem Klin Biochem. 1969; 7:606–613.
38. Praefcke GJ, McMahon HT. The dynamin superfamily: universal membrane tubulation and fission molecules? Nat Rev Mol Cell Biol. 2004; 5:133–147.
Article
39. Gao TM, Xu ZC. In vivo intracellular demonstration of an ischemiainduced postsynaptic potential from CA1 pyramidal neurons in rat hippocampus. Neuroscience. 1996; 75:665–669.
Article
40. Gao TM, Pulsinelli WA, Xu ZC. Prolonged enhancement and depression of synaptic transmission in CA1 pyramidal neurons induced by transient forebrain ischemia in vivo. Neuroscience. 1998; 87:371–383.
Article
41. Lee AS. Coordinated regulation of a set of genes by glucose and calcium ionophores in mammalian cells. Trends Biochem Sci. 1987; 12:20–23.
Article
42. Kaufman RJ. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev. 1999; 13:1211–1233.
Article
43. Lee AS. Mammalian stress response: induction of the glucose-regulated protein family. Curr Opin Cell Biol. 1992; 4:267–273.
Article
44. Little E, Ramakrishnan M, Roy B, Gazit G, Lee AS. The glucose-regulated proteins (GRP78 and GRP94): functions, gene regulation, and applications. Crit Rev Eukaryot Gene Expr. 1994; 4:1–18.
Article
45. Li LJ, Li X, Ferrario A, Rucker N, Liu ES, Wong S, et al. Establishment of a Chinese hamster ovary cell line that expresses grp78 antisense transcripts and suppresses A23187 induction of both GRP78 and GRP94. J Cell Physiol. 1992; 153:575–582.
Article
46. Yu Z, Luo H, Fu W, Mattson MP. The endoplasmic reticulum stress-responsive protein GRP78 protects neurons against excitotoxicity and apoptosis: suppression of oxidative stress and stabilization of calcium homeostasis. Exp Neurol. 1999; 155:302–314.
Article
47. Miyake H, Hara I, Arakawa S, Kamidono S. Stress protein GRP78 prevents apoptosis induced by calcium ionophore, ionomycin, but not by glycosylation inhibitor, tunicamycin, in human prostate cancer cells. J Cell Biochem. 2000; 77:396–408.
Article
48. Reddy RK, Mao C, Baumeister P, Austin RC, Kaufman RJ, Lee AS. Endoplasmic reticulum chaperone protein GRP78 protects cells from apoptosis induced by topoisomerase inhibitors: role of ATP binding site in suppression of caspase-7 activation. J Biol Chem. 2003; 278:20915–20924.
Article
49. Gazit G, Hung G, Chen X, Anderson WF, Lee AS. Use of the glucose starvation-inducible glucose-regulated protein 78 promoter in suicide gene therapy of murine fibrosarcoma. Cancer Res. 1999; 59:3100–3106.
50. Kokame K, Agarwala KL, Kato H, Miyata T. Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress. J Biol Chem. 2000; 275:32846–32853.
Article
51. Fornage M, Swank MW, Boerwinkle E, Doris PA. Gene expression profiling and functional proteomic analysis reveal perturbed kinase-mediated signaling in genetic stroke susceptibility. Physiol Genomics. 2003; 15:75–83.
Article
52. Bergerat A, Decano J, Wu CJ, Choi H, Nesvizhskii AI, Moran AM, et al. Prestroke proteomic changes in cerebral microvessels in stroke-prone, transgenic[hCETP]-Hyperlipidemic, Dahl salt-sensitive hypertensive ratss. Mol Med. 2011; 17:588–598.
Article
53. Chen R, Vendrell I, Chen CP, Cash D, O'Toole KG, Williams SA, et al. Proteomic analysis of rat plasma following transient focal cerebral ischemia. Biomark Med. 2011; 5:837–846.
Article
54. Liu KX, Li C, Li YS, Yuan BL, Xu M, Xia Z, et al. Proteomic analysis of intestinal ischemia/reperfusion injury and ischemic preconditioning in rats reveals the protective role of aldose reductase. Proteomics. 2010; 10:4463–4475.
Article
55. Millares P, Lacourse EJ, Perally S, Ward DA, Prescott MC, Hodgkinson JE, et al. Proteomic profiling and protein identification by MALDI-TOF mass spectrometry in unsequenced parasitic nematodes. PLoS One. 2012; 7:e33590.
Article
56. Kannan S, Hauth AM, Burger G. Function prediction of hypothetical proteins without sequence similarity to proteins of known function. Protein Pept Lett. 2008; 15:1107–1116.
Article
57. Kawabata T, Nishikawa K. Protein structure comparison using the markov transition model of evolution. Proteins. 2000; 41:108–122.
Article
58. Dundas J, Ouyang Z, Tseng J, Binkowski A, Turpaz Y, Liang J. CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucleic Acids Res. 2006; 34(Web Server issue):W116–W118.
Article
59. Holm L, Kääriäinen S, Rosenström P, Schenkel A. Searching protein structure databases with DaliLite v.3. Bioinformatics. 2008; 24:2780–2781.
Article
60. Holm L, Rosenström P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 2010; 38(Web Server issue):W545–W549.
Article
61. Aloy P, Querol E, Aviles FX, Sternberg MJ. Automated structure-based prediction of functional sites in proteins: applications to assessing the validity of inheriting protein function from homology in genome annotation and to protein docking. J Mol Biol. 2001; 311:395–408.
Article
62. Ondrechen MJ, Clifton JG, Ringe D. THEMATICS: a simple computational predictor of enzyme function from structure. Proc Natl Acad Sci U S A. 2001; 98:12473–12478.
Article
63. Pazos F, Sternberg MJ. Automated prediction of protein function and detection of functional sites from structure. Proc Natl Acad Sci U S A. 2004; 101:14754–14759.
Article
64. Nimrod G, Schushan M, Steinberg DM, Ben-Tal N. Detection of functionally important regions in "hypothetical proteins" of known structure. Structure. 2008; 16:1755–1763.
Article
Full Text Links
  • JCN
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr