Mild in vitro trauma induces rapid Glur2 endocytosis, robustly augments calcium permeability and enhances susceptibility to secondary excitotoxic insult in cultured Purkinje cells
Mild in vitro trauma induces rapid Glur2 endocytosis, robustly augments calcium permeability and enhances susceptibility to secondary excitotoxic insult in cultured Purkinje cells
复制标题
DOI：
10.1093/brain/awm164
复制
发表时间：
2007-10-01
期刊：
影响因子：
14.5
通讯作者：
Baker, Andrew J.
中科院分区：
文献类型：
--
作者：
Bell, Joshua D.;Ai, Jinglu;Baker, Andrew J.
轻度脑外伤会导致广泛的神经系统症状，这些症状不容易通过原发性病理学解释。小脑的浦肯野神经元选择性地易受脑创伤，包括前脑的间接远程创伤。这种脆弱性表现为选择性和延迟性细胞损失，其潜在机制知之甚少。钙不可渗透的AMPA受体（含GluR 2）的表面表达的改变可能介导创伤后钙超载，并启动生化级联反应，最终导致进行性细胞死亡。我们目前的研究使用轻度浦肯野创伤的体外模型来检验这一假设，该模型通过2.5-2.9磅/平方英寸（psi）的弹性拉伸来传递。与未受伤的对照组相比，通过碘化丙啶（PI）摄取（在20小时）测量，这种轻度创伤单独没有增加细胞损失。然而，有一个显着增加细胞损失，当细胞轻度创伤后，暴露于10 μ M的AMPA 1小时相比，无论是轻度创伤或单独的AMPA暴露。轻度损伤使浦肯野神经元在损伤后15分钟对AMPA刺激的（4 μ M）钙内流显著更具渗透性，包括持续的钙平台。通过用2 μ M Go 6976抑制蛋白激酶C依赖性GluR 2内吞作用或用500 nM 1-naphthylacetyl spermine（Naspm）阻断含有AMPAR的GluR 1/3的钙孔来消除这种效应。硝苯地平（2 mM）消除了轻度损伤后的钙平台期，但没有消除Ca 2+增加的初始峰值。这些结果表明，轻度损伤导致快速AMPA受体亚型转换（GluR 1/3取代GluR 2），这反过来又导致Ca 2+渗透性增强。我们通过免疫细胞化学进一步证实了这一点。轻度牵张后15分钟，树突状GluR 2与突触前标记物突触素的共定位显著下调（P < 0.01），表明含有该亚基的受体的突触表达迅速减少。羧基荧光（CBF）分析表明，温和的拉伸并没有改变膜的完整性。最后，我们证明了500 nM Naspm和5 nM Go 6976的组合通过在95%的细胞中有效消除轻度拉伸与AMPA组合的影响，对浦肯野细胞具有强大的神经保护作用。这些结果代表了一种新的机制，使神经元易受创伤后继发性损伤。预防GluR 2内吞作用可能是开发针对轻度、看似无关紧要的创伤的药物治疗的关键，以避免随后的继发性损伤。
Mild brain trauma results in a wide range of neurological symptoms that are not easily explained by the primary pathology. Purkinje neurons of the cerebellum are selectively vulnerable to brain trauma, including indirect remote trauma to the forebrain. This vulnerability manifests itself as a selective and delayed cell loss, for which the underlying mechanisms are poorly understood. Alterations to the surface expression of calcium impermeable AMPA receptors (GluR2-containing) may mediate post-traumatic calcium overload, and initiate biochemical cascades that ultimately cause progressive cell death. Our current study examined this hypothesis using an in vitro model of mild Purkinje trauma, delivered by an elastic stretch at 2.5-2.9 pounds per square inch (psi). This mild trauma alone did not increase cell loss as measured by propidium iodide (PI) uptake (at 20 h) compared to uninjured controls. However, there was a marked increase in cell loss, when cells following mild trauma, were exposed to 10 mu M AMPA for 1 h compared to either mild trauma or AMPA exposure alone. Mild injury rendered Purkinje neurons significantly more permeable to AMPA-stimulated (4 mu M) calcium influx at 15 min post-injury, including a sustained calcium plateau. This effect was eliminated by inhibiting protein kinase C-dependent GluR2 endocytosis with 2 mu M Go6976 or blocking the calcium pore of GluR1/3 containing AMPARs with 500 nM 1-naphthylacetyl spermine (Naspm). Nifedipine (2 mM) eliminated the calcium plateau following mild injury but not the initial spike of Ca2+ increase. These results suggest that mild injuries resulted in a rapid AMPA receptor subtype switch (GluR2 was replaced by GluR1/3), which in turn resulted in an enhanced Ca2+ permeability. We further confirmed this by immunocytochemistry. Dendritic GluR2 co-localization with the pre-synaptic marker synaptophysin was markedly down-regulated at 15 min following mild stretch (P < 0.01), indicative of a rapid decrease in the synaptic expression of receptors containing this subunit. Carboxyfluorescence (CBF) assays revealed that mild stretch did not alter membrane integrity. Finally, we demonstrated that the combination of 500 nM Naspm and 5 nM Go6976 conferred a powerful neuroprotective effect on Purkinje cells by effectively eliminating the effects of mild stretch combined with AMPA in 95% of cells. These results represent a newly described mechanism rendering neurons susceptible to secondary injuries following trauma. Prevention of GluR2 endocytosis may be critical in the development of pharmacotherapies aimed at mild, seemingly inconsequential trauma, to avoid ensuing secondary damage.