FADD在线粒体脑肌病中的病理作用及其机制研究

Mitochondrial encephalomyopathy is a group of metabolic neuromuscular diseases that are mainly involved in the brain and skeletal muscle. Mitochondrial proliferation and apoptosis are the most important pathological changes of these diseases. Fas-associated protein with death domain (FADD) is a critical adaptor protein for Fas/FADD induced apoptosis. FADD protein phosphorylation could also regulate cycle progression and induce cell proliferation. Our previous studies found that p-FADD mRNA lever was increased in muscle biopsy of patients with definite mitochondrial encephalomyopathy, and FADD was colocalized with apoptosis myonuclei. Based on the previous work, Twinkle mutation mice and patients skeletal muscle biopsies were used to establish mitochondrial function defects in vivo and vitro. We are going to perform further experiments at tissue, cellular and molecular levels and focus on the following two questions: 1) to prove that FADD could induce mitochondrial proliferation and cell apoptosis, and establish its importance in brain and muscle pathology in mitochondrial encephalomyopathy. 2) to find out the relevant mechanisms through which FADD induce mitochondrial proliferation and cell apoptosis in mitochondrial encephalomyopathy. To be specifically, we want to explore the way through which FADD phosphorylation affect the cell cycle to promote mitochondrial DNA replication and FADD induce neuron and muscle cell apoptosis. Our study could provide new targets for therapeutic strategies of mitochondrial encephalomyopathy.

线粒体脑肌病是一种主要累及脑和骨骼肌的代谢性神经肌肉病，线粒体增殖与细胞凋亡是该病重要的病理改变。FADD是Fas死亡相关结构域蛋白，Fas/FADD通路是诱导细胞凋亡的主要途径，同时磷酸化的FADD亦可调控细胞周期促进细胞增殖。本项目在前期研究发现线粒体脑肌病患者活检肌肉中FADD磷酸化表达增高，FADD与凋亡肌细胞共表达的基础上，采用体内外线粒体缺陷模型及患者肌活检标本，利用分子生物学、细胞培养等技术，从整体、细胞及分子水平进行以下两方面研究：1）证实FADD在线粒体增殖和细胞凋亡中的重要作用，并确立其与线粒体脑肌病脑损伤与肌肉病变的密切相关性。2）研究FADD介导线粒体脑肌病线粒体增殖和细胞凋亡的可能机制，具体探讨FADD磷酸化影响细胞周期促进线粒体DNA复制，及FADD诱导神经元和肌细胞肌核凋亡的分子机制。本研究结果将为线粒体脑肌病的发病机制、诊断和治疗提供新的思路和科学线索。

线粒体脑肌病是一种累及全身多系统的代谢性神经肌肉病，其骨骼肌受损尤为重要，肌纤维线粒体增殖与细胞凋亡是该病重要的病理改变。研究证实磷酸化FADD可通过调控细胞周期促进细胞增殖，亦可通过Fas/FADD通路诱导细胞凋亡。本项目在标书计划及前期研究结果基础上，结合本领域国内外最新报道进行了一系列实验，主要探讨了FADD对线粒体脑肌病骨骼肌线粒体病态增殖和细胞凋亡的病理作用，并进一步探讨了其下游的信号通路和分子机制。.1）通过不断收集临床线粒体脑肌病病例、扩大检测样本量进一步证实了异常线粒体增殖和细胞凋亡是线粒体脑肌病的重要病理改变。进一步研究发现线粒体脑肌病患者线粒体动力蛋白DRP1,OPA1和MFN2的mRNA和蛋白表达水平升高。免疫荧光观察发现p-FADD与线粒体动力蛋白的共表达。我们的研究结果说明p-FADD升高与线粒体异常增殖产生的破碎红纤维RRFs密切相关，同时RRFs升高与线粒体动力蛋白表达增高也密切相关，且p-FADD蛋白与线粒体动力蛋白存可共表达。因此我们推测p-FADD异常增高可能通过诱导线粒体动力蛋白表达增加从而引起异常线粒体增殖产生大量RRFs，引起线粒体脑肌病患者的肌无力、运动不耐受症状。.2）利用磁共振磷谱31P-MRS分析线粒体脑肌病伴高乳酸血症和卒中样发作（MELAS综合症）患者骨骼肌的能量代谢与线粒体氧化磷酸化呼吸链酶活性情况，从而探讨线粒体呼吸链复合物COX（细胞色素C氧化酶）在MELAS发病中的作用。.3）从国外Jackson Lab公司引进COX10基因敲除鼠（J024697 B6.129X1 - Cox10 tm1Ctm/J），并在南京大学生物模式动物研究所保种并和Cre小鼠交配繁育，制备COX10基因敲除的模型小鼠，模拟人的线粒体脑肌病发病过程，进一步探讨FADD在线粒体脑肌病中的病理作用及其分子机制。