糖尿病影响自噬体与溶酶体的融合减弱Aβ清除的分子机制

Diabetes mellitus (DM) has been regarded as an important risk factor for Alzheimer’s disease (AD), and diabetic patients and animals have shown cognitive dysfunction. More research has shown that the amyloid-β (Aβ), which is a hallmark of AD, was found deposited in the hippocampus of diabetic rats.Further evidence shows that Aβ generation and failure of Aβ clearance are symptoms of both AD and DM. All evidence suggests that Aβ metabolism is abnormal in DM patients and animal models. Autophagy-lysosome is a cellular pathway involved in protein and organelle degradation. Recent studies have shown that the stimulation of autophagy can reduce Aβ accumulation and alleviate memory deficits in transgenic AD mice. Our previous findings indicate that DM activated autophagy, but lysosome function was impaired. Autophagy-lysosome dysfunction may be involved in the Aβ deposition in diabetic cognitive impairment. But the specific mechanism is not very clear. We conjecture that diabetes could affect the fusion of autophagososme and lysosome, then lysosome degradation dysfunction, to block the autopahgy afflux, then Aβ is accumulated. We plan to use the STZ-induced diabetic rats and the hippocampal neurons with high glucose by primary culture as the research models, to observe the changing of fluorescence of mRFP-GFP-LC3 in the hippocampal neurons to make sure the fusion of autophagososme and lysosome by fluorescence microscopy, to measure the expression of fusion related protein Rab7 by western blooting, immunofluorescence staining and real-time PCR to confirm wheather the fusion of autophagososme and lysosome was normal or not. The further to measure the expression and the activity of lysosome related protein Cath L,Cath D by molecular biological technique, to test the specific activity and lysosome acid phosphatase activity by ELISA, to determine lysosome degradation function during the process of fusion. At last to examine the changing of the above under the intervening by the signaling pathway inhibitor for mTOR, class Ⅲ PI3K/PKB and SIRT1, to confirm the signaling pathways of diabetes regulating autophagy fusion. This project is aming to confirm the partial mechanism of diabetic cognitive dysfunction, to clear the paitical mechanism of Aβ deposion in the diabetic rats' brain, to provide new theoretical evidences for preventing DM round into AD.

糖尿病被视为阿尔茨海默病（AD）的独立危险因素，可促进脑内Aβ的沉积。自噬参与Aβ沉积的过程，自噬体与溶酶体的融合是自噬完成的关键。我们前期研究发现，STZ诱导的糖尿病大鼠成模12周时记忆损害，海马神经元自噬活性改变，自噬流中断，Aβ沉积，但具体机制不清楚。结合文献我们推测糖尿病影响自噬体与溶酶体的融合，致溶酶体降解障碍是自噬流中断、Aβ沉积的可能原因。我们拟以STZ诱导的糖尿病大鼠和高糖干预的原代海马神经元为研究对象，荧光显微镜观察mRFP-GFP-LC3示踪自噬体与溶酶体的融合现象，分子生物学方法检测融合相关蛋白Rab7的表达，证实自噬体与溶酶体的融合存在异常；进而检测溶酶体相关蛋白Cath L/D的表达水平、活性及溶酶体特异酸性磷酸酶的活性,确定融合过程中溶酶体降解障碍;其次在信号通路抑制剂干预下观察上述变化，明确糖尿病调控自噬融合的信号通路，为预防糖尿病进展为AD提供新的理论依据。

糖尿病被视为阿尔茨海默病（AD）的独立危险因素，可促进脑内Aβ的沉积。自噬参与Aβ沉积的过程，自噬体与溶酶体的融合是自噬完成的关键。我们前期研究发现，STZ诱导的糖尿病大鼠成模12周时记忆损害，海马神经元自噬活性改变，自噬流中断，Aβ沉积，但具体机制不清楚。我们在本项目中，通过在体和离体实验的研究，明确了糖尿病对大鼠记忆能力的影响，对大鼠海马神经元及高糖对体外培养的海马神经细胞超微结构以及自噬形态的影响；明确了糖尿病对自噬体-溶酶体融合的影响以及对Aβ 清除的影响；明确了糖尿病对溶酶体降解功能的影响和糖尿病调节自噬-溶酶体途径的信号通路。结果显示：1.STZ诱导的糖尿病大鼠在成模第12周表现出空间记忆障碍，逃避潜伏期延长，穿越中心区域的次数明显减少（P<0.05）；2.糖尿病大鼠海马部位Aβ表达明显升高（P<0.05）,CTSD, CTSL表达下降（P<0.05）; 3.高糖组SH-SY5Y细胞的自噬小体较正常组增加，提示自噬激活；转染自噬双标腺病毒mRFP-GFP-LC3的结果显示：高糖组SH-SY5Y细胞自噬流增强; 4.流式检测结果发现高糖组SH-SY5Y细胞的凋亡率增加，Rap和Nico的干预组降低了高糖培养的SH-SY5Y细胞的凋亡率（P<0.05）; 5. 高糖组的SH-SY5Y细胞溶酶体较正常组增加，自噬-溶酶体的融合减少，当加入自噬激活剂3MA后，溶酶体的红色荧光明显减少；加入Rap和Nico后，自噬-溶酶体的融合明显减少，溶酶体增加，提示高糖影响了自噬小体与溶酶体的融合; 6.高糖组ACP2活性较正常对照组下降（P<0.01）,HG+Rap组和HG+Nico组的ACP2活性较高糖组明显升高（P<0.01）. 以上研究表明：糖尿病大鼠脑内存在自噬-溶酶体融合障碍，高糖状态下影响溶酶体酸性磷酸酶的活性，导致自噬体与溶酶体融合障碍；溶酶体功能异常，溶酶体降解障碍，从而影响Aβ的清除，沉积增加，神经元损伤，细胞凋亡增加，影响认知功能。这一结果表明自噬-溶酶体融合障碍参与了糖尿病大鼠脑内Aβ的沉积过程，造成了认知损害。通过本研究说明自噬-溶酶体融合障碍参与了Aβ清除的过程，进一步丰富了糖尿病促进AD进展的理论机制，为临床预防AD的发生提供依据。