PDF(Pubmed)
Abstract：
The small GTPase RAS acts as a plasma membrane-anchored intracellular neurotrophin counteracting neuronal degeneration in the brain, but the underlying molecular mechanisms are largely unknown. In transgenic mice expressing constitutively activated V12-Ha-RAS selectively in neurons, proteome analysis uncovered a 70% decrease in voltage-dependent anion channel-1 (VDAC-1) in the cortex and hippocampus. We observed a corresponding reduction in the levels of mRNA splicing variant coding for plasma membrane-targeted VDAC-1 (pl-VDAC-1) while mRNA levels for mitochondrial membrane VDAC-1 (mt-VDAC-1) remained constant. In primary cortical neurons derived from V12-Ha-RAS animals, a decrease in pl-VDAC-1 mRNA levels was observed, accompanied by a concomitant reduction in the ferricyanide reductase activity associated with VDAC-1 protein. Application of MEK inhibitor U0126 to transgenic cortical neurons reconstituted pl-VDAC-1 mRNA to reach wild-type levels. Excitotoxic glutamate-induced cell death was strongly attenuated in transgenic V12-Ha-RAS overexpressing cortical cultures. Consistently, a neuroprotective effect could also be achieved in wild-type cortical cultures by the extracellular application of channel-blocking antibody targeting the N-terminus of VDAC-1. These results may encourage novel therapeutic approaches toward blocking pl-VDAC-1 by monoclonal antibody targeting for complementary treatments in transplantation and neurodegenerative disease.
摘要：
小GTP酶RAS充当质膜锚定的细胞内神经营养蛋白，可抵抗大脑中的神经元变性，但是潜在的分子机制在很大程度上是未知的。在神经元中选择性表达组成型激活的V12-Ha-RAS的转基因小鼠中，蛋白质组分析发现皮质和海马中电压依赖性阴离子通道1（VDAC-1）降低了70％。我们观察到编码质膜靶向VDAC-1(pl-VDAC-1)的mRNA剪接变体的水平相应降低,而线粒体膜VDAC-1(mt-VDAC-1)的mRNA水平保持恒定。在来自V12-Ha-RAS动物的原代皮层神经元中，观察到pl-VDAC-1mRNA水平降低，伴随着与VDAC-1蛋白相关的铁氰化物还原酶活性的降低。将MEK抑制剂U0126应用于转基因皮质神经元,重构pl-VDAC-1mRNA以达到野生型水平。在过表达V12-Ha-RAS的转基因皮质培养物中，兴奋性谷氨酸诱导的细胞死亡被强烈减弱。始终如一，通过细胞外应用靶向VDAC-1N端的通道阻断抗体,也可以在野生型皮质培养物中实现神经保护作用.这些结果可能会鼓励通过单克隆抗体靶向阻断pl-VDAC-1的新治疗方法，用于移植和神经退行性疾病的补充治疗。
