Krajewska Maryla, Rosenthal Robert E, Mikolajczyk Jowita, Stennicke Henning R, Wiesenthal Thomas, Mai Juergen, Naito Mikihiko, Salvesen Guy S, Reed John C, Fiskum Gary, Krajewski Stan
The Burnham Institute, La Jolla, CA 92037, USA.
Exp Neurol. 2004 Oct;189(2):261-79. doi: 10.1016/j.expneurol.2004.05.020.
A clinically relevant model of transient global brain ischemia involving cardiac arrest followed by resuscitation in dogs was utilized to study the expression and proteolytic processing of apoptosis-regulatory proteins. In the hippocampus, an increase in pro-apoptotic Bcl-2 family proteins Bcl-XS and Bak was detected, concomitant with proteolysis of Bcl-XL and Bcl-2, following ischemia-reperfusion injury. Also, biphasic cleavage of Bid was found in this region of the brain, with early generation of tBid-p11 within 10 min of cardiac arrest, followed by generation of tBid-p15 within 30-min reperfusion, consistent with activation of this pro-apoptotic protein. In addition, cardiac arrest and resuscitation induced early, reperfusion-dependent proteolytic processing of pro-caspase-6, -8, -10, and -14, which preceded caspase-3 activation. Immunohistochemical analysis using antibodies, which preferentially recognize processed caspase-3, -6, -8, and -10, provided evidence of time-dependent activation of these proteases in both neurons and glia in ischemia-sensitive regions of the brain. In conclusion, extremely rapid, cell-selective processing of apoptosis-regulatory proteins occurs in a clinically relevant model of ischemic brain injury caused by cardiac arrest and resuscitation. The early cleavage of Bid and rapid depletion of 32-kDa pro-caspase-14 from the canine hippocampus after induction of ischemia suggests the involvement of calpains in the processing of these proteins. Demonstration of in vitro cleavage of recombinant mouse caspase-14 by calpain I in the present study lends support to this hypothesis, further implicating cross-talk between different protease families in the pathophysiology of ischemic neural cell death.
利用一种涉及犬心脏骤停后复苏的短暂性全脑缺血的临床相关模型，来研究凋亡调节蛋白的表达和蛋白水解过程。在海马体中，缺血再灌注损伤后，检测到促凋亡Bcl-2家族蛋白Bcl-XS和Bak增加，同时伴有Bcl-XL和Bcl-2的蛋白水解。此外，在该脑区发现Bid的双相切割，心脏骤停后10分钟内早期产生tBid-p11，随后在再灌注30分钟内产生tBid-p15，这与该促凋亡蛋白的激活一致。另外，心脏骤停和复苏诱导了前体半胱天冬酶-6、-8、-10和-14的早期、再灌注依赖性蛋白水解过程，这先于半胱天冬酶-3的激活。使用优先识别加工后的半胱天冬酶-3、-6、-8和-10的抗体进行免疫组织化学分析，提供了这些蛋白酶在脑缺血敏感区域的神经元和神经胶质细胞中随时间激活的证据。总之，在心脏骤停和复苏引起的缺血性脑损伤的临床相关模型中，凋亡调节蛋白发生了极其快速、细胞选择性的加工过程。缺血诱导后犬海马体中Bid的早期切割和32-kDa前体半胱天冬酶-14的快速消耗表明钙蛋白酶参与了这些蛋白的加工过程。本研究中钙蛋白酶I对重组小鼠半胱天冬酶-14的体外切割证明支持了这一假设，进一步表明不同蛋白酶家族之间的相互作用参与了缺血性神经细胞死亡的病理生理学过程。
