FADD磷酸化参与线粒体脑肌病糖代谢重编程及其调控机制研究

Mitochondrial encephalomyopathy is a group of metabolic neuromuscular diseases that are most common in adolescents. Abnormal mitochondrial proliferation are the most important pathological changes of these diseases. Fas-associated protein with death domain (FADD) is a critical adaptor protein for Fas induced apoptosis. Recent studies suggested that p-FADD mediated cell proliferation, energy metabolism and glucose metabolic reprogramming. Our previous studies found that 1) p-FADD mRNA lever was increased in muscle biopsy of patients with definite mitochondrial encephalomyopathy, and had a positive correlation with RRFs, which presented proliferated abnormal mitochondrion. 2) Functional magnetic resonance (1H-MRS) imaging showed elevated lactate in cerebral spinal fluid, which represented increased enhanced glycolysis；31P-MRS showed delayed recovery of energy reservation in skeletal muscle, which represented suppressed oxidative phosphorylation. 3) p-FADD regulated the expression of hexokinase(HK) in glycolysis. Base on the previous studies, we retrospectively analyzed the correlation between p-FADD and abnormal mitochondrial proliferation and its diagnostic value. Furthermore, cell culture and COX10 mutation mice were used to establish the function of p-FADD in mediating glycolysis and its downstream signal pathways and targets in mitochondrial encephalomyopathy in vivo and vitro.

线粒体脑肌病是青少年最常见的神经肌肉代谢病，异常线粒体增殖是影响其病情和预后的重要因素，其具体分子机制一直未能阐明。FADD是一种凋亡蛋白，最近研究发现磷酸化FADD（p-FADD）可调控细胞增殖、能量代谢和糖代谢重编程。本课题组在前期工作中发现1）线粒体病患者骨骼肌p-FADD高表达与代表线粒体异常增殖的破碎红纤维RRFs数量密切相关；2）患者功能磁共振氢谱显示代表糖酵解的脑脊液乳酸增高；磷谱显示代表三羧酸循环氧化磷酸化的骨骼肌能量储备受抑制；3）p-FADD可引起糖酵解限速酶己糖激酶HK上调。本项目拟在此基础上，从样本回顾性分析中总结p-FADD参与糖代谢重编程促进异常线粒体增殖的临床相关性及预后判断价值。同时在细胞和COX10突变的线粒体脑肌病小鼠模型中进一步深入探讨p-FADD在线粒体脑肌病中的作用方式及通过影响糖代谢重编程调控异常线粒体增殖的分子机制，解析下游效应途径及靶点。

1)p-FADD在线粒体脑肌病患者和COX10骨骼肌突变Ckmm-Cre鼠代表异常线粒体增殖的RRFs共定位和高表达，且与疾病进展呈正相关；p-FADD高表达与线粒体动力学蛋白Drp1和糖酵解关键酶HK呈正相关。制备慢病毒FADD shRNA，Ckmm股内及股外侧肌注射，行Affymetrix表达谱基因芯片检测和IPA平台分析，发现FADD shRNA鼠骨骼肌可能激活GP6信号通路，TGFB1激活后可能抑制DCN mRNA水平，同时CD44调控子可能通过ACKR3激活肌间结缔组织细胞交互运动。以上结果提示线粒体病与糖代谢重编程相关，p-FADD可能通过调控限速酶HK促进线粒体脑肌病无氧糖酵解，影响下游线粒体动力学蛋白Drp1，导致病态线粒体异常增殖，引起RRFs高表达和线粒体呼吸链COX酶活性降低，从而抑制线粒体氧化呼吸功能。.2)1H-MRS可监测线粒体脑肌病乳酸Lac、NAA等脑代谢物异常并早期诊断MELAS和CPEO。MELAS的线粒体细胞病（Lac和破碎红纤维RRFs均增高）与神经元活力呈正相关，与胶质增生无关；MELAS的骨骼肌COX阳性率与脑代谢Lac/Cr呈正相关，而与NAA/Cr呈负相关，表明线粒体神经病与神经元脆性和COX酶活性紊乱在MELAS反复卒中样发作和偏头痛机制中起重要作用。制备COX10脑神经突变 Nes-Cre鼠行缺血再灌注大脑中动脉梗死模型，第3天脑梗死面积和脑水肿体积均较野生鼠最重；Nes侧脑室注射FADD shRNA，梗死面积和水肿体积均显著下降，表明p-FADD可促进脑线粒体代谢紊乱加重缺血卒中脑损伤。.3)4W雄性肌营养不良DMD缺陷型mdx模型鼠腹腔注射生长激素释放肽ghrelin(100μg/kg/d)，结果表明Ghrelin可能通过下调JAK2-STAT3和p38 MAPK信号通路，抑制mdx骨骼肌和LPS诱导的原代肌细胞的炎性小体NLRP3及其分泌产物IL-1β活化，改善mdx骨骼肌功能，减轻mdx肌活检病理损伤并降低肌纤维间炎症细胞浸润。因此Ghrelin可能是DMD免疫治疗的潜在靶点。.4)对代谢性肌病行高精度mtDNA和高通量nDNA测序，发现罕见线粒体基因组单重结构性缺失突变~m.6352-13952和~m.5952-15977和核基因POLG复合突变，以及糖原累积症GAA和脂质代谢性肌病ETFDH罕见突变。