Role of cytotoxic protease granzyme-b in neuronal degeneration during human stroke.
Infiltration of leukocytes into post-ischemic cerebrum is a well-described phenomenon in stroke injury. Because CD-8(+) T-lymphocytes secrete cytotoxic proteases, including granzyme-b (Gra-b) that exacerbates post-ischemic brain damage, we investigated roles of Gra-b in human stroke. To study the role of Gra-b in stroke, ischemic and non-ischemic tissues (from post-mortem stroke patients) were analyzed using immunoblotting, co-immunoprecipitation, terminal deoxy uridine nick end labeling (TUNEL) and Annexin-V immunostaining, and in vitro neuron survival assays. Activated CG-SH cells and supernatants were used to model leukocyte-dependent injury. Non-ischemic brain tissues were used as non-pathological controls. Non-activated CG-SH cells and supernatants were used as controls for in vitro experiments. Human stroke (ischemic) samples contained significantly higher levels of Gra-b and interferon-gamma inducible protein-10 (IP-10/CXCL10) than non-ischemic controls. In stroke, poly (ADP-ribose) polymerase-1 and heat shock protein-70 were cleaved to canonical proteolytic "signature" fragments by Gra-b. Gra-b was also found to bind to Bid and caspase-3. Gra-b also co-localized with Annexin-V(+) /TUNEL(+) in degenerating neurons. Importantly, Gra-b inhibition protected both normal and ischemia-reperfused neurons against in vitro neurotoxicity mediated by activated CG-SH cells and supernatants. These results suggest that increased leukocyte infiltration and elevated Gra-b levels in the post-stroke brain can induce contact-dependent and independent post-ischemic neuronal death to aggravate stroke injury.
Chaitanya
Ganta Vijay
GV
Department of Molecular and Cellular Physiology, School of Medicine, Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, La, USA.
Eeka
Prabhakar
P
Munker
Reinhold
R
Alexander
Jonathan Steven
JS
Babu
Phanithi Prakash
PP
D000328:Adult; D000368:Aged; D000704:Analysis of Variance; D017209:Apoptosis; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D002545:Brain Ischemia / Q000378:metabolism* / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D054357:Chemokine CXCL10 / Q000378:metabolism; D005260:Female; D053804:Granzymes / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D020287:In Situ Nick-End Labeling; D008297:Male; D008875:Middle Aged; D009336:Necrosis; D009410:Nerve Degeneration / Q000378:metabolism* / Q000473:pathology; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D020521:Stroke / Q000378:metabolism* / Q000473:pathology
C114753:CXCL10 protein, human; D054357:Chemokine CXCL10; D053804:Granzymes; D053148:Caspase 3
J Leukoc Biol. 2010 Feb;87(2):237-43
19915166
Cell. 1994 Mar 25;76(6):977-87
8137431
Biochem Soc Trans. 2006 Dec;34(Pt 6):1267-70
17073799
Brain Behav Immun. 2005 Jan;19(1):12-9
15581733
Neurochem Res. 2008 Nov;33(11):2178-86
18338260
J Cell Biol. 2003 Mar 17;160(6):875-85
12629051
J Neurosci. 2004 Mar 10;24(10):2458-64
15014121
Brain Res. 2008 Dec 30;1246:178-90
18938146
Biochem Biophys Res Commun. 1996 Oct 23;227(3):658-65
8885990
Neuroscience. 2010 Feb 17;165(4):1203-16
19895873
Autophagy. 2008 Apr;4(3):364-7
18216498
ScientificWorldJournal. 2001 Oct 24;1:590-2
12805854
Int J Biochem Cell Biol. 2005 Feb;37(2):267-71
15474972
Neurology. 2001 Oct 23;57(8):1428-34
11673584
J Biol Chem. 1996 Nov 15;271(46):29073-9
8910561
Cell Death Differ. 2000 Jun;7(6):556-65
10822279
Biochem Biophys Res Commun. 1996 Dec 24;229(3):838-44
8954981
Synapse. 1996 Oct;24(2):104-14
8890452
Am J Pathol. 2001 Sep;159(3):809-15
11549572
J Biol Chem. 2006 Sep 22;281(38):28326-35
16798735
Nat Cell Biol. 2001 Sep;3(9):839-43
11533664
Stroke. 2001 Nov;32(11):2665-74
11692032
J Immunol. 2003 Jul 1;171(1):368-79
12817020
J Neurosci. 2003 Aug 27;23(21):7922-30
12944523
J Invest Surg. 2008 May-Jun;21(3):141-7
18569435
Circulation. 2006 May 2;113(17):2105-12
16636173
Lab Invest. 2007 Mar;87(3):241-50
17260002
Cell Death Differ. 2009 Apr;16(4):638-47
19148187
Cell Cycle. 2006 Nov;5(22):2592-601
17106261
J Exp Med. 2000 Nov 20;192(10):1403-14
11085743
Cancer Biol Ther. 2009 May;8(9):792-8
19270509
Eur J Neurosci. 1998 Feb;10(2):680-8
9749729
FEBS J. 2009 Jan;276(1):13-26
19087196
Cell Mol Neurobiol. 2009 Jun;29(4):563-73
19225880
J Cereb Blood Flow Metab. 2007 Nov;27(11):1798-805
17392692
FASEB J. 2006 Jun;20(8):1209-11
16636104
Neuropathol Appl Neurobiol. 2003 Oct;29(5):472-81
14507339
Neurosci Lett. 2007 Feb 27;414(1):45-50
17287085
Eur J Immunol. 2000 Dec;30(12):3623-33
11169405
Neurol Res. 2008 Oct;30(8):783-93
18826804
Proteomics. 2003 Apr;3(4):513-26
12687618
Arch Neurol. 1993 Jan;50(1):30-6
8093428
J Cereb Blood Flow Metab. 2001 Apr;21(4):321-33
11323518
Mol Cell Neurosci. 2004 Jan;25(1):172-80
14962750
J Neuroimmunol. 2001 Mar 1;114(1-2):232-41
11240037
Prog Neurobiol. 1998 Oct;56(2):149-71
9760699
Leukemia. 2005 Mar;19(3):484-5
15618955
Int J Dev Neurosci. 2008 Feb;26(1):37-45
17942266
J Biol Chem. 1999 Jan 8;274(2):1156-63
9873064
Curr Opin Immunol. 2010 Jun;22(3):308-13
20226643
J Biol Chem. 2005 Feb 11;280(6):4476-82
15574417
Blood. 1995 Nov 1;86(9):3500-6
7579456
J Neuroimmune Pharmacol. 2006 Mar;1(1):11-9
18040787
Oncogene. 2003 Oct 2;22(43):6669-78
14555980
Mol Biol Cell. 2006 Feb;17(2):623-33
16280358
J Cell Biol. 2006 Apr 10;173(1):133-44
16606695
Neuron. 2009 Oct 15;64(1):55-60
19840549
Inflammation. 1998 Dec;22(6):573-82
9824772
Curr Pharm Des. 2008;14(33):3565-73
19075733
Autophagy. 2009 Feb;5(2):273-6
19139632
J Biol Chem. 2001 Mar 9;276(10):6974-82
11114298
Mult Scler. 2004 Feb;10(1):26-34
14760949
J Neurosci. 2007 Aug 29;27(35):9301-9
17728444
Transplant Rev (Orlando). 2009 Jan;23(1):1-10
19027612
J Neuropathol Exp Neurol. 2003 Apr;62(4):329-39
12722825
Mol Cell Biol. 2000 Jun;20(11):3781-94
10805722
Leuk Res. 2009 Oct;33(10):1405-8
19414191
Cell Mol Immunol. 2009 Feb;6(1):15-25
19254476
Cell Death Differ. 2006 Sep;13(9):1423-33
16676004
Prog Med Chem. 2004;42:125-69
15003720
J Biol Chem. 2004 May 21;279(21):22020-9
15014070
Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5679-83
7777569
Neuroscience. 2007 Apr 14;145(4):1267-72
17084037
J Biol Chem. 2005 Apr 22;280(16):16383-92
15713684
Blood. 2004 May 15;103(10):3845-53
14739229
Autoimmunity. 2006 Feb;39(1):9-19
16455578
J Neurosci Res. 1993 Dec 15;36(6):681-93
8145296
FEBS Lett. 2007 Jan 9;581(1):109-17
17182042
Eur J Immunol. 1996 Sep;26(9):2271-4
8814277
Carcinogenesis. 2007 Mar;28(3):537-44
16950797
Curr Opin Immunol. 2003 Oct;15(5):533-43
14499262
Neuropathology. 2009 Jun;29(3):270-9
19170890