Knockdown of m-calpain increases survival of primary hippocampal neurons following NMDA excitotoxicity
Knockdown of m-calpain increases survival of primary hippocampal neurons following NMDA excitotoxicity
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DOI：
10.1111/j.1471-4159.2008.05860.x
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发表时间：
2009-03-01
影响因子：
4.7
通讯作者：
Neumar, Robert W.
中科院分区：
文献类型：
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作者：
Bevers, Matthew B.;Lawrence, Eric;Neumar, Robert W.
半胱氨酸蛋白酶的钙蛋白酶家族在急性脑损伤后的神经元细胞死亡中具有明确的因果作用。然而，由于可用的钙蛋白酶抑制剂不是异构体特异性的，因此尚未确定钙蛋白酶异构体对各种形式损伤的相对贡献。在这项研究中，我们评估了 m-钙蛋白酶和 mu-钙蛋白酶在 NMDA 介导的兴奋性毒性的原代海马神经元模型中的相对作用。基于定量实时 PCR，发现 m-钙蛋白酶催化亚基 (capn2) 的基线 mRNA 表达比 mu-钙蛋白酶催化亚基 (capn1) 高 50 倍。设计用于递送靶向 capn1 或 capn2 的短发夹 RNA 的腺相关病毒载体分别导致了 60% 和 90% 的信息敲低。体外暴露 NMDA 21 天后，敲除 capn2 但不敲除 capn1 会增加神经元存活率。在此模型中暴露于 NMDA 后，未检测到钙蛋白酶底物凋亡诱导因子、p35/p25 和塌陷素反应介导蛋白 (CRMP) 2-4 的核转位。然而，观察到了 CRMP-1 的核易位，并且通过敲低 capn2 来阻止该现象。这些发现提供了对钙蛋白酶介导的神经变性的潜在机制的深入了解，并对异构体特异性钙蛋白酶抑制剂疗法的开发具有重要意义。
The calpain family of cysteine proteases has a well-established causal role in neuronal cell death following acute brain injury. However, the relative contribution of calpain isoforms to the various forms of injury has not been determined as available calpain inhibitors are not isoform-specific. In this study, we evaluated the relative role of m-calpain and mu-calpain in a primary hippocampal neuron model of NMDA-mediated excitotoxicity. Baseline mRNA expression for the catalytic subunit of m-calpain (capn2 ) was found to be 50-fold higher than for the mu-calpain catalytic subunit (capn1) based on quantitative real-time PCR. Adeno-associated viral vectors designed to deliver short hairpin RNAs targeting capn1 or capn2 resulted in 60% and 90% knockdown of message respectively. Knockdown of capn2 but not capn1 increased neuronal survival after NMDA exposure at 21 days in vitro. Nuclear translocation of calpain substrates apoptosis inducing factor, p35/p25 and collapsin response mediator protein (CRMP) 2-4 was not detected after NMDA exposure in this model. However, nuclear translocation of CRMP-1 was observed and was prevented by capn2 knockdown. These findings provide insight into potential mechanisms of calpain-mediated neurodegeneration and have important implications for the development of isoform-specific calpain inhibitor therapy.