脊髓性肌肉萎缩症分子病理机制的研究

Spinal muscular atrophy (SMA) is the most common genetic disease in young children, caused by lack of survival of motor neuron (SMN) protein. SMA is characterized by degeneration of spinal-cord α-motor neurons, which leads to progressive atrophy of voluntary muscles, and finally patients die from respiratory failure. So far, the molecular mechanisms of SMA pathogenesis remain elusive, and there is no effective treatment. It has been generally accepted that spinal cord motor neurons are the principal and directly affected cells in SMA; therefore, increasing SMN in the CNS, particularly the spinal cord motor neurons is at least required, if not sufficient, for SMA therapy. Surprisingly, we recently observed an efficient motor-neuron cell-nonautonomous rescue of severe SMA mice including a robust survival increase and great improvement of motor function, demonstrating that increase of SMN in peripheral tissues not only restores normal functions of peripheral cells and tissues but also preserves motor neurons and neuromuscular junctions. This raised a question: which peripheral tissues or organs are critical in premature death and motor neuron degeneration of SMA mice? We hypothesize that critical defects are present in peripheral tissues of SMA mice and the defects not only cause the premature death but also motor neuron degeneration by failing to supply essential neurotrophic factors or prevent the production of toxic factors that affect motor neuron maintenance. In this project we will systematically analyze histopathology, transcriptome and proteome of various tissues and organs of a severe SMA mouse model, explore the critical pathways downstream of SMN, and evaluate the importance of certain disrupted signaling pathways and potentially defective tissues in rescuing SMA mice.

脊髓性肌肉萎缩症（SMA）是儿童常见致死性遗传病，因缺乏SMN导致脊髓α运动神经元变性，继而引起骨骼肌萎缩，患者呼吸衰竭而亡。至今缺乏SMN引发SMA的分子机制仍不明了、也无治疗药物。传统观点认为提高脊髓运动神经元SMN水平是治疗SMA的必要条件。近期我们在《Nature》和《Genes & Development》报道，提高小鼠模型外周组织SMN水平能极大延长小鼠寿命，改善运动能力，表明外周SMN水平提高不仅维护外周组织功能也保护运动神经元。我们提出假设：SMA小鼠外周组织存在严重缺陷，是导致小鼠过早死和运动神经元变性的根本原因。本项目我们将对小鼠模型各组织进行全面系统的病理分析，找出关键病变组织，利用转录组和蛋白组分析寻找SMN下游的关键基因，探索异常表达基因和相关组织在疾病发展中的作用和治疗价值。本项目将不仅阐明SMA分子病理机制也为药物开发打下基础，因此具有重要理论意义和应用价值。

脊髓性肌萎缩症（SMA）是致死性遗传病，多发于儿童，患者SMN蛋白缺乏引发脊髓α-运动神经元退变和其支配的骨骼肌萎缩。至今，SMN缺乏是如何导致运动神经元退变的机制尚不清楚。近年来在严重型SMA模式小鼠的研究显示不仅脊髓运动神经元存在缺陷，外周组织也存在严重的病理变化，是一个多器官缺陷的疾病。本项目提出假设，SMN缺乏不仅靶向攻击脊髓运动神经元，也损伤其他细胞类型，同时外周组织可能释放有害因子进一步作用于脊髓运动神经元。项目对新生严重型模式小鼠各组织进行了系统性的病理筛查，发现心脏、小肠上皮存在严重结构和功能障碍，细胞增殖受阻并发生凋亡。转录组分析发现大量细胞周期相关基因表达紊乱，其中心脏Birc5刚出生就下调。Birc5表达的蛋白Survivin是心脏发育的关键蛋白，因此模式小鼠心肌存在多种结构和功能异常。小鼠肠道上皮增殖阻滞导致细胞松散、通透性增加、微生物入侵而引起全身性炎症，所有组织促炎性细胞因子表达增加，细菌释放的脂多糖抑制SMN2剪接使生成的SMN蛋白进一步减少。与细胞周期相关的大量基因是P53信号通路的下游靶基因，然而组织P53上调并不明显，因此我们构建了诱导SMN敲低的细胞株，当SMN表达下降，P53蛋白一天后即急剧增高，进一步发现其原因是泛素化减弱导致稳定性增强。对P53的蛋白修饰检测发现其N端磷酸化和C端的乙酰化程度明显增高，影响蛋白的稳定性和活性。对转录组ceRNA的研究发现miR-34a上调，其与P53相互激活，形成一个正反馈机制。我们得出结论，细胞SMN降低导致P53/miR-34a轴活性增强，引发下游基因表达紊乱，导致细胞增殖受阻或发生凋亡。在SMA模式小鼠中，脊髓运动神经元、心肌和小肠表皮易受到攻击，引起小肠通透性增加、心脏发育受阻和运动神经元退变；而各组织促炎性细胞因子和内毒素等加剧神经元的退变。本项目在体外和体内阐明了SMN下游一个重要信号通路，考虑到P53/miR-34a轴的重要功能，其在SMA病理中必然起着重要作用。本项目的发现对理解SMA发病机制具有重要的理论意义并具有潜在的应用价值。