神经元内apoE4(1-272)诱发内质网应激致线粒体形态功能紊乱的分子机制研究

It is well known that apoE4 hydrolysis fragments (1-272) is the key molecular marker of neurodegeneration in Alzheimer's disease (AD), which can lead to neurotoxicities in neurons，but the molecular mechanism is not clear. Our previous results showed that apoE4 (1-272) fragments could induce endoplasmic reticulum stress(ER Sress) and mitochondrial dysfunction while the abnormal expression and modification of mitochondria fusion and fission proteins were detected in neurons. ER stress was also found before occuring mitochondrial dysfunction. Thus, apoE4(1-272) fragments through triggering ER stress -induced mitochondrial fragmentation may promote the damage of mitochondrial function, lead to neurodegeneration.By using of the pre-established neuron-specific apoE4 (1-272) expression transgenic mice , tetracycline controlling apoE4 (1-272) expression cell model and the multiple research tools such as ultrastructural pathology, morphology, biology, physics, biochemistry, molecular biology technology, we will verify the above assumptions. Our results will for the first time explain the mechanism of apoE4 (1-272) inducing mitochondria fragmentation, leading to neurodegeneration and seeking for the effective target.

国内外研究和我们前期结果均显示神经元内apoE4(1-272)是致阿尔茨海默病(AD)病变的主要apoE4分子，但机制尚不清。我们前期研究发现神经元内apoE4(1-272)可诱发内质网应激(ERS)和线粒体形态功能紊乱，同时伴有线粒体分裂、融合蛋白表达和修饰异常；且线粒体形态功能紊乱发生在ERS之后；采用ERS保护剂4-PBA干预后可显著改善线粒体形态功能。因此提出神经元内apoE4(1-272)可能通过诱发ERS，损害线粒体融合分裂动态，促使线粒体出现形态及功能紊乱，导致神经细胞变性。本项目拟利用前期建立的apoE4(1-272)转基因鼠和四环素可调控的apoE4(1-272)表达细胞模型，深入探讨神经元内apoE4(1-272)致线粒体形态功能紊乱的可能分子机制，验证ERS是apoE4(1-272)致线粒体动态失衡的关键环节，为apoE4致AD发病的机制提供新的科学依据。

apoE4基因是最常见的AD易感基因，众多国内外研究均提示神经元内ApoE4蛋白的水解片段ApoE4(1-272)可能是真正的致AD病变分子，但机制仍不清楚。近来发现线粒体片段化发生于AD神经元纤维缠结早期。N2a细胞中无ApoE4基因，因此我们通过建立Dox可调控apoE4(1-272)表达的N2a细胞系—N2a/apoE4(1-272)和神经元特异apoE4(1-272)转基因小鼠，证实了ApoE4(1-272)片段在细胞内的累积可诱发N2a细胞发生线粒体片段化以及线粒体形态和功能发生紊乱；并检测到线粒体融合蛋白MFN-2表达下降；扫描电镜观察到线粒体破碎现象；同时发现神经元丢失现象。结果分析表明：线粒体片段化以及形态功能紊乱发生于神经元丢失之前。这些结果提示ApoE4(1-272)片段可通过下调MFN-2表达触发线粒体分裂增加进而导致线粒体片段化。进一步的实验表明：ApoE4(1-272)片段的堆积触发内质网应激，导致N2a细胞中MFN-2蛋白含量显著下调。内质网应激保护剂PBA可显著抑制线粒体片段化及缓解神经元丢失。综合上述结果，我们认为：神经元细胞内apoE4(1-272)的堆积，可通过触发内质网应激，水解线粒体融合蛋白MFN-2，诱导线粒体发生破碎，导致线粒体功能紊乱，进而导致神经元变性。这些研究结果为理解apoE4(1-272)导致神经元变性的机制提供了新信息，为减缓AD毒性提供了新的分子靶标。