Monnerie Hubert, Le Roux Peter D
Department of Neurosurgery, University of Pennsylvania, 330 S 9th Street, 4th Floor, Philadelphia, PA, 19107, USA.
Exp Neurol. 2008 Sep;213(1):145-53. doi: 10.1016/j.expneurol.2008.05.013. Epub 2008 May 27.
Brain cell vulnerability to neurologic insults varies greatly, depending on their neuronal subpopulation. Among cells that survive a pathological insult such as ischemia or brain trauma, some may undergo morphological and/or biochemical changes that could compromise brain function. We previously reported that surviving cortical GABAergic neurons exposed to glutamate in vitro displayed an NMDA receptor (NMDAR)-mediated alteration in the levels of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67) [Monnerie, H., Le Roux, P., 2007. Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65- and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro. Exp. Neurol. 205, 367-382]. In this study, we examined the mechanisms by which glutamate excitotoxicity caused a change in cortical GABAergic neurons' GAD protein levels. Removing extracellular calcium prevented the NMDAR-mediated decrease in GAD protein levels, measured using Western blot techniques, whereas inhibiting calcium entry through voltage-gated calcium channels had no effect. Glutamate's effect on GAD protein isoforms was significantly attenuated by preincubation with the cysteine protease inhibitor N-Acetyl-L-Leucyl-L-Leucyl-L-norleucinal (ALLN). Using class-specific protease inhibitors, we observed that ALLN's effect resulted from the blockade of calpain and cathepsin protease activities. Cell-free proteolysis assay confirmed that both proteases were involved in glutamate-induced alteration in GAD protein levels. Together these results suggest that glutamate-induced excitotoxic stimulation of NMDAR in cultured cortical neurons leads to altered GAD protein levels from GABAergic neurons through intracellular calcium increase and protease activation including calpain and cathepsin. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered balance between excitation and inhibition that is often observed after injury.
脑细胞对神经损伤的易感性差异很大，这取决于它们的神经元亚群。在诸如缺血或脑外伤等病理损伤中存活下来的细胞中，一些细胞可能会发生形态和/或生化变化，从而损害脑功能。我们之前报道过，体外暴露于谷氨酸的存活皮质GABA能神经元显示出NMDA受体（NMDAR）介导的GABA合成酶谷氨酸脱羧酶（GAD65/67）水平改变[莫内里，H.，勒鲁，P.，2007年。体外兴奋性毒性损伤后小鼠皮质GABA能神经元树突生长减少以及谷氨酸脱羧酶（GAD）65和67 kDa亚型蛋白表达改变。《实验神经病学》205卷，367 - 382页]。在本研究中，我们研究了谷氨酸兴奋性毒性导致皮质GABA能神经元GAD蛋白水平变化的机制。去除细胞外钙可防止使用蛋白质印迹技术测量的NMDAR介导的GAD蛋白水平降低，而抑制通过电压门控钙通道的钙内流则没有效果。用半胱氨酸蛋白酶抑制剂N - 乙酰 - L - 亮氨酰 - L - 亮氨酰 - L - 正亮氨酸（ALLN）预孵育可显著减弱谷氨酸对GAD蛋白亚型的影响。使用类别特异性蛋白酶抑制剂，我们观察到ALLN的作用是由于钙蛋白酶和组织蛋白酶的蛋白酶活性被阻断。无细胞蛋白水解试验证实这两种蛋白酶都参与了谷氨酸诱导的GAD蛋白水平改变。这些结果共同表明，培养的皮质神经元中谷氨酸诱导的NMDAR兴奋性毒性刺激通过细胞内钙增加和包括钙蛋白酶和组织蛋白酶在内的蛋白酶激活，导致GABA能神经元的GAD蛋白水平改变。各种疾病状态下存活的皮质GABA能神经元中的生化改变可能导致损伤后经常观察到的兴奋与抑制之间平衡的改变。
