Mechanism of accelerating death of newborn neurons by ss-amyloid

在阿尔茨海默病（AD）中，脑内未成熟神经元标记蛋白增加。然而，增加的神经发生不足以修复神经系统，因为在AD脑中发生进行性神经元损失。AD脑内微环境的改变可能影响未成熟神经元的命运。SS-淀粉样蛋白（Ass）在AD的早期发病机制中起重要作用。推测Ass改变大脑微环境使其对新生神经元有毒是合理的。为了解决这些问题，我们试图在诱导基因中鉴定抑制新生神经元存活的基因。在7个As诱导的基因中，只有Chat（Cas/HEF 1相关信号转导子）加速新生神经元的死亡。其他6个基因促进神经元存活或不影响神经元存活。接下来，我们重点研究了Chat基因，试图阐明其对新生神经元的毒性机制。结果表明：（1）Chat的C端部分是新生神经元产生毒性所必需的。(2)Chat的C-末端部分具有Cas蛋白家族的结合结构域。然而，Cas蛋白（p130 Cas和NEDD 9）和Chat共转染到新生神经元中并没有加速神经元死亡。不与Cas蛋白结合的突变体Chat也加速了神经元死亡。这些结果表明，未知蛋白结合到Chat C-末端部分（Chat C）而不是Cas结合位点可能是诱导新生神经元毒性所必需的。最后，我们试图确定与Chat C结合并加速新生神经元死亡的蛋白质。构建并纯化了GST-Chat C融合蛋白。将来自培养的皮层神经元的裂解物与GST-Chat C融合蛋白一起孵育。对拉下的蛋白进行SDS-PAGE并用nanoLC-MS/MS分析。我们不能鉴定任何与GST-Chat C融合蛋白的结合蛋白。不能排除Chat C的均聚物形成会诱导新生神经元的神经毒性。

In Alzheimer's disease (AD), immature neuronal marker proteins increased in brain. However, the increased neurogenesis is not sufficient to repair the nervous system because progressive neuronal loss occurs in AD brain. Alteration of microenvironment in AD brain may affect the fate of immature neurons. Ss-amyloid (Ass) plays an important role in the early pathogenesis of AD. It is reasonable to speculate Ass alters the brain microenvironment to make it toxic to newborn neurons. To address these issues, we tried to identify the genes to inhibit survival of newborn neurons in Ass-induced genes. Among 7 Ass-induced genes, only Chat (Cas/HEF1-associated signal transducer) accelerated death of newborn neurons. Other 6 genes promoted neuronal survival or did not affect on neuronal survival. Next, we focused on Chat gene and tried to clarify toxic mechanism to newborn neurons. The results are follows :(1) C-terminal portion of Chat is necessary to induce toxicity in newborn neurons.(2) C-terminal portion of Chat have the binding domain to Cas protein family. However, co-transfection of Cas proteins (p130Cas and NEDD9) and Chat into newborn neurons did not accelerate neuronal death. The mutant Chat, which does not bind to Cas proteins, also accelerated neuronal death. These results suggests that the binding of unknown proteins to Chat C-terminal portion (Chat C) other than Cas binding site might be necessary to induce toxicity in newborn neurons. Finally we tried to identify the proteins that bind to Chat C and accelerate death of newborn neurons. A GST-Chat C fusion protein was constructed and purified. The lysate from cultured cortical neurons was incubated with a GST-Chat C fusion protein. Pull downed proteins were subjected to SDS-PAGE and analyzed with nanoLC-MS/MS. We could not identify any binding proteins to GST-Chat C fusion protein. It could not rule out that homopolymer formation of Chat C would induce neurotoxicity to newborn neurons.