通过Mitophagy维持mtDNA稳定性在肥胖/T2DM加速脑老化中的作用及机制

Evidence has been indicated mutation or deletion of mitochondrial DNA (mtDNA) occurred at early, which prior to the phenotype of aging, suggestting mtDNA mutation/deletion initiated the aging. Clinical studies have shown the risk of brain aging increased in population suffered from the obesity/type 2 diabetes mellitus (T2DM). Based on the the type 1 and type 2 diabetic animal models, our previous studies have been proven that the diabetic encephalopathy is characterized by the acceleration of the brain aging, dysfunction of mitochondria and deficit of autophagy. Recently, works from different research teams indicated mitophagy plays important roles in maintaining the mitochondial homostasis through targeted sorting and cleaning the damaged mitochondria. Presumptively, abnormal mitophagy is involved in abberant metabolism in the brain following obesity/T2DM. Damaged mitochondria could not be sorted and cleaned promptly. And then, the mtDNA mutation/delation is aggravated and the brain aging is accelerated. Our present proposal aim to study the frequence and locus of the mtDNA mutation/deletion in the brain through the random mutation capture (RMC) with aging in obesity/T2DM rats. Accordingly, Different proteins were screened by mitochondrial proteomics, and the changes of mitophagy as well as its regulation were studied in this process. Additionally, the effects of caloric restriction or rapamycin-feeding to obesity/T2DM rats were observed. In vitro, nerve cells were treated with D-galactose. The mitophagy was up- or downregulated by target molecules. The changes of mtDNA mutation/deletion were measured. Performace of our present proposal is beneficial to explore the underlying mechanisms of obesity/T2DM accelerated brain aging.

研究表明线粒体DNA突变早于老化表型的出现，推测可能是老化的始作俑者。临床资料表明肥胖/糖尿病人群脑部衰退的风险大大增加，我们此前的研究也发现糖尿病脑损伤呈现脑老化加速的特征，伴随线粒体功能障碍、自噬异常。近年不同研究小组的工作均发现线粒体自噬具有严格的靶向性，是完成线粒体分选、清除、维持线粒体稳态的关键途径。我们推测肥胖/糖尿病由于代谢异常导致脑内线粒体自噬障碍，异常的线粒体不能被识别、清除，加剧了线粒体DNA突变，加速了脑老化。本项目在高脂饮食诱导的肥胖/糖尿病大鼠模型上，通过随机突变捕获技术分析神经细胞线粒体DNA突变频率及位置，利用线粒体蛋白组学方法筛选此过程差异性蛋白的表达，进而研究脑内线粒体自噬调节分子的变化，并观察限制饮食对此的影响。在D半乳糖诱导的神经细胞老化模型上，通过特异性上调或抑制线粒体自噬的靶向诱导分子，观察线粒体DNA的变化，揭示肥胖/糖尿病加速脑老化的分子机制。

肥胖、糖尿病等代谢性疾病常波及多个器官，包括心脏、肾脏、血管及脑，其中糖尿病导致的脑损伤也称之为糖尿病脑病。来自流行病学研究发现2型糖尿病人群罹患AD的风险是非糖尿病人群的2-3倍。年龄是神经退行性疾病和糖尿病的共同危险因素，因此探讨代谢异常状态下神经细胞老化以及老化的机制，尤其线粒体功能异常在其中的关键作用，有助于理解糖尿病脑病的发生发展机制。本项目基于多个动物模型（单纯高脂诱导的小鼠肥胖模型、高脂饮食联合STZ诱导的2型糖尿病小鼠模型、STZ诱导的1型糖尿病大鼠模型、脑小血管病大鼠模型、hIAPP转基因鼠2型糖尿病小鼠模型）研究了糖脂代谢异常与脑老化的关系及关键特征。重点研究了线粒体结构和功能变化在糖脂代谢异常所致脑老化中的关键作用及机制。在此基础上给予自噬诱导剂Spermidine观察了自噬信号、尤其线粒体相关自噬在其中的作用。细胞模型上，通过游离脂肪酸诱导的细胞损伤模型，D-Gal诱导的神经细胞老化模型，重点研究了神经细胞老化过程中线粒体稳态和自噬信号之间的关系。研究发现糖尿病导致脑老化加速，呈现AD样病理改变，脑内神经干细胞的增值及分化能力下降，与此对应的认知功能也明显下降。高脂饮食联合STZ注射，4个月后可见下丘脑出现神经细胞退变，自噬溶酶体降解途径障碍，自噬体聚集，其中部分自噬体内可见异常形态的线粒体。体外实验发现神经细胞老化伴随线粒体形态及功能异常，给予自噬诱导剂能够延缓老化表型，同时改善线粒体功能。这些研究结果表明糖脂代谢异常导致脑老化，早期表现为线粒体形态及功能的异常，尤其是线粒体DNA及mRNA稳定性的下降，自噬异常，给予自噬诱导剂Spermidine对糖脂代谢异常导致的神经细胞损伤有改善效应，有助于维持线粒体形态和结构的稳定性。