Zhang Zhiqun, Ottens Andrew K, Sadasivan Shankar, Kobeissy Firas H, Fang Tie, Hayes Ronald L, Wang Kevin K W
Department of Psychiatry, Center for Neuroproteomics and Biomarkers Research, McKnight Brain Institute, University of Florida, Gainesville, Florida 32610, USA.
J Neurotrauma. 2007 Mar;24(3):460-72. doi: 10.1089/neu.2006.0078.
Collapsin response mediator proteins (CRMPs) are important molecules in neurite outgrowth and axonal guidance. Within the CRMP family, CRMP-2 has been implicated in several neurological diseases (Alzheimer's, epilepsy, and ischemia). Here, we investigated the integrity of CRMPs (CRMP-1, -2, -4, -5) after in vitro neurotoxin treatment and in vivo traumatic brain injury (TBI). After maitotoxin (MTX) and NMDA treatment of primary cortical neurons, a dramatic decrease of intact CRMP-1, -2 and -4 proteins were observed, accompanied by the appearance of distinct 55-kDa and 58-kDa breakdown products (BDP) for CRMP-2 and -4, respectively. Inhibition of calpain activation prevented NMDA-induced CRMP-2 proteolysis and redistribution of CRMP-2 from the neurites to the cell body, while attenuating neurite damage and neuronal cell injury. Similarly, CRMP-1, -2, and -4 were also found degraded in rat cortex and hippocampus following controlled cortical impact (CCI), an in vivo model of TBI. The appearance of the 55-kDa CRMP-2 BDP was observed to increase, in a time-dependent manner, between 24 and 48 h in the ipsilateral cortex, and by 48 hours in the hippocampus. The observed 55-kDa CRMP-2 BDP following TBI was reproduced by in vitro incubation of naive brain lysate with activated calpain-2, but not activated caspase-3. Sequence analysis revealed several possible cleavage sites near the C-terminus of CRMP-2. Collectively, this study demonstrated that CRMP-1, -2, and -4 are degraded following both acute traumatic and neurotoxic injury. Furthermore, calpain-2 was identified as the possible proteolytic mediator of CRMP-2 following excitotoxic injury and TBI, which appears to correlate well with neuronal cell injury and neurite damage. It is possible that the calpain-mediated truncation of CRMPs following TBI may be an inhibiting factor for post-injury neurite regeneration.
塌陷反应介导蛋白（CRMPs）是神经突生长和轴突导向中的重要分子。在CRMP家族中，CRMP-2与多种神经系统疾病（阿尔茨海默病、癫痫和缺血）有关。在此，我们研究了体外神经毒素处理和体内创伤性脑损伤（TBI）后CRMPs（CRMP-1、-2、-4、-5）的完整性。在用 maitotoxin（MTX）和NMDA处理原代皮层神经元后，观察到完整的CRMP-1、-2和-4蛋白显著减少，同时分别出现了CRMP-2和-4特有的55 kDa和58 kDa降解产物（BDP）。抑制钙蛋白酶激活可防止NMDA诱导的CRMP-2蛋白水解以及CRMP-2从神经突向细胞体的重新分布，同时减轻神经突损伤和神经元细胞损伤。同样，在TBI的体内模型——控制性皮质撞击（CCI）后，大鼠皮质和海马中的CRMP-1、-2和-4也被发现降解。在同侧皮质中，55 kDa的CRMP-2 BDP在24至48小时之间呈时间依赖性增加，在海马中则在48小时时增加。TBI后观察到的55 kDa CRMP-2 BDP可通过用活化的钙蛋白酶-2而非活化的半胱天冬酶-3体外孵育未损伤的脑裂解物来重现。序列分析揭示了CRMP-2 C末端附近的几个可能的切割位点。总体而言，这项研究表明，CRMP-1、-2和-4在急性创伤性和神经毒性损伤后都会降解。此外，钙蛋白酶-2被确定为兴奋性毒性损伤和TBI后CRMP-2可能的蛋白水解介质，这似乎与神经元细胞损伤和神经突损伤密切相关。TBI后钙蛋白酶介导的CRMPs截短可能是损伤后神经突再生的抑制因素。
