去乙酰化酶SIRT3保护阿尔茨海默病神经细胞损伤的机制研究

Mitochondrial dysfunction and metabolic abnormalities are considered to be the main features in early-onset Alzheimer’s disease. Abnormalities of mitochondrial function and neuronal cell death induced by amyloid β (Aβ), including decreased mitochondrial respiratory chain enzymatic activities, increased reaction oxygen species (ROS) and hypometabolism, but the underlying mechanism remains unclear. SIRT3 plays an important role to maintain mitochondrial function by deacetylating its target proteins. In the present study, we explored SIRT3 pathway by using Aβ1-42 treated cultured primary mice cortical neurons in vitro and cerebral cortex and hippocampus of mAPP mice in vivo. The ratio of NAD+/NADH, expression levels and deacetylation levels of SIRT3, CypD, FOXO3α, PGC1α were detected in vitro and in vivo. The cortical neurons or mAPP mice cerebral cortex mitochondria were isolated for determining activities of mitochondrial respiratory chain enzymes and deacetylased activity of SIRT3. The ultrastructure of mitochondria was observed under transmission electron microscope (TEM). Moreover, mitochondrial membrane potential (MMP) and ROS production were tested by staining the specific probe. In addition, we overexpressed SIRT3 in primary cultured cortical neurons or 3, 6 months old mAPP mice through lentivirus infection to verify the neuroprotective effects of SIRT3 in Aβ rich environment. Taken together, regulation of SIRT3 may be a novel target for preventing and treating AD in future.

β淀粉样蛋白（Aβ）在阿尔茨海默病（AD）神经细胞线粒体损伤和细胞死亡中扮演重要的作用，但其机制还需进一步探索。线粒体去乙酰化酶SIRT3能够降低线粒体蛋白的乙酰化水平，维持线粒体功能。本研究拟用Aβ处理的小鼠原代大脑皮质神经细胞和mAPP转基因小鼠分别作为AD细胞模型和AD动物模型，测定原代神经细胞和3、6月龄的mAPP小鼠大脑皮质、海马组织中NAD+/NADH的比值，SIRT3的表达与活性、靶蛋白的表达和乙酰化水平， ATP生成，线粒体呼吸链复合酶活性和线粒体膜电位，阐明SIRT3在Aβ诱导的神经细胞损伤中的作用；用携载SIRT3的慢病毒感染AD细胞和mAPP小鼠的海马CA1区，增加SIRT3的表达，验证SIRT3抵抗Aβ神经毒性的效果。本研究从细胞和动物两个水平模拟临床AD患者的发病和神经元损伤，探索SIRT3在Aβ丰富环境对神经细胞的保护作用，为临床预防和治疗AD寻求新的药物靶点。

Aβ诱导的神经细胞线粒体损伤和能量代谢紊乱被认为是早期阿尔茨海默病（AD）的主要特征，但具体机制还不清楚。本研究旨在探索SIRT3抵抗Aβ神经毒性，改善AD认知障碍的机制。首先，用Aβ1-42处理小鼠大脑皮质神经细胞，发现细胞反应性氧化产物（ROS）、线粒体ROS水平升高，细胞ATP水平显著下降，线粒体膜电位（MMP）显著下降，细胞色素C氧化酶（CcO）活性显著下降；同时，细胞的NAD+/NADH比值和SIRT3的表达和活性降低。使用Sirt3基因或突变型Sirt3基因过表达慢病毒感染神经细胞，结果发现过表达Sirt3不仅能够显著逆转Aβ1-42导致的神经细胞NAD+/NADH比值下降以及SIRT3去乙酰化酶活性减低，还能减少Aβ1-42导致的细胞ROS水平上升，逆转细胞ATP水平下降和MMP降低，过表达突变型Sirt3则无此效应。为进一步探索SIRT3改善AD认知障碍的机制，我们运用脑立体定位技术将Sirt3基因或突变型Sirt3基因过表达慢病毒注入3月龄AD转基因小鼠（mAPP小鼠）海马CA1区，检测7月龄小鼠的行为学和海马神经细胞的变化。结果发现，与mAPP小鼠相比，过表达Sirt3基因的mAPP小鼠空间学习记忆功能得到明显改善；电镜检测结果发现过表达Sirt3基因的mAPP小鼠海马神经细胞的细胞核核膜清晰，线粒体形态大小正常，双层膜结构完整，线粒体嵴清晰可见；生化实验则提示过表达Sirt3基因的mAPP小鼠海马CA1区神经细胞内ROS和线粒体ROS的水平减少，海马组织ATP水平、NAD+/NADH的比值和SIRT3酶活性显著升高，乙酰化CypD、乙酰化FOXO3α水平下降，磷酸化PGC1α水平上升，CypD的表达减少，PGC1α和FOXO3α的表达明显增加，而过表达突变型Sirt3基因则无上述作用。本研究从细胞和动物两个水平模拟临床AD患者的发病和神经细胞损伤，揭示SIRT3抵抗Aβ毒性的神经细胞保护作用机制。本研究结果为以SIRT3为靶点研发临床预防和治疗AD的药物提供新的思路和实验证据。