Meller Robert, Thompson Simon John, Lusardi Theresa Ann, Ordonez Andrea Nicole, Ashley Michelle Dawn, Jessick Veronica, Wang Weihzen, Torrey Daniel John, Henshall David Clifford, Gafken Philip R, Saugstad Julie Anne, Xiong Zhi-Gang, Simon Roger Pancoast
Robert S. Dow Neurobiology Laboratories, Legacy Research, Portland, Oregon 97232, USA.
J Neurosci. 2008 Jan 2;28(1):50-9. doi: 10.1523/JNEUROSCI.3474-07.2008.
Ischemic tolerance is an endogenous neuroprotective mechanism in brain and other organs, whereby prior exposure to brief ischemia produces resilience to subsequent normally injurious ischemia. Although many molecular mechanisms mediate delayed (gene-mediated) ischemic tolerance, the mechanisms underlying rapid (protein synthesis-independent) ischemic tolerance are relatively unknown. Here we describe a novel mechanism for the induction of rapid ischemic tolerance mediated by the ubiquitin-proteasome system. Rapid ischemic tolerance is blocked by multiple proteasome inhibitors [carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), MG115 (carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal), and clasto-lactacystin-beta-lactone]. A proteomics strategy was used to identify ubiquitinated proteins after preconditioning ischemia. We focused our studies on two actin-binding proteins of the postsynaptic density that were ubiquitinated after rapid preconditioning: myristoylated, alanine-rich C-kinase substrate (MARCKS) and fascin. Immunoblots confirm the degradation of MARCKS and fascin after preconditioning ischemia. The loss of actin-binding proteins promoted actin reorganization in the postsynaptic density and transient retraction of dendritic spines. This rapid and reversible synaptic remodeling reduced NMDA-mediated electrophysiological responses and renders the cells refractory to NMDA receptor-mediated toxicity. The dendritic spine retraction and NMDA neuroprotection after preconditioning ischemia are blocked by actin stabilization with jasplakinolide, as well as proteasome inhibition with MG132. Together these data suggest that rapid tolerance results from changes to the postsynaptic density mediated by the ubiquitin-proteasome system, rendering neurons resistant to excitotoxicity.
缺血耐受是大脑和其他器官中的一种内源性神经保护机制，即预先短暂暴露于缺血状态可使机体对随后通常具有损伤性的缺血产生耐受性。尽管许多分子机制介导延迟性（基因介导）缺血耐受，但快速（不依赖蛋白质合成）缺血耐受的潜在机制仍相对不明。在此，我们描述了一种由泛素 - 蛋白酶体系统介导的诱导快速缺血耐受的新机制。多种蛋白酶体抑制剂[苄氧羰基 - L - 亮氨酰 - L - 亮氨酰 - L - 亮氨酸甲酯（MG132）、MG115（苄氧羰基 - L - 亮氨酰 - L - 亮氨酰 - L - 正缬氨酸甲酯）和弹状乳胞素 - β - 内酯]可阻断快速缺血耐受。我们采用蛋白质组学策略来鉴定预处理缺血后的泛素化蛋白。我们将研究重点放在快速预处理后被泛素化的突触后致密区的两种肌动蛋白结合蛋白上：肉豆蔻酰化富含丙氨酸的蛋白激酶C底物（MARCKS）和丝束蛋白。免疫印迹证实预处理缺血后MARCKS和丝束蛋白的降解。肌动蛋白结合蛋白的缺失促进了突触后致密区的肌动蛋白重组以及树突棘的短暂回缩。这种快速且可逆的突触重塑减少了NMDA介导的电生理反应，并使细胞对NMDA受体介导的毒性产生耐受。预处理缺血后的树突棘回缩和NMDA神经保护作用可被茉莉酮酸甲酯的肌动蛋白稳定作用以及MG132的蛋白酶体抑制作用所阻断。这些数据共同表明，快速耐受是由泛素 - 蛋白酶体系统介导的突触后致密区变化所致，使神经元对兴奋性毒性产生抗性。
