Expression data in muscle cells (myotubes) of motor neuron disorders (ALS, SBMA, SMA-IV) and healthy controls

Despite the discovery of many genetic risk factors, the cause of the motor neuron death that drives terminal pathology in Amyotrophic Lateral Sclerosis (ALS) remains unknown. We report that the skeletal muscle of ALS patients secretes exosomal vesicles that are specifically toxic to motor neurons. This could not be attributed to a trivial down-stream consequence of muscle denervation. In a study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) from 67 human subjects, including healthy and disease controls, ALS myotubes had a consistent signature of disrupted exosome biogenesis and RNA-processing, and their exosomes induced shortened, less branched, neurites, greater death, and disrupted localization of RNA and RNA-processing proteins in motor neurons. Toxicity was dependent on presence of the FUS protein, which is highly expressed in recipient motor neurons. As part of this work, we carried out gene expression analysis of myotubes (differentiated myoblasts) comparing ALS against two other motor neuron disorders as disease controls (SBMA, Spinal and bulbar muscular atrophy; and Spinal Muscular Atrophy Type 4, SMA-IV) and healthy controls. Primary muscle stem cells (myoblasts) were extracted from deltoid muscle biopsies of a total of 23 subjects: 6 ALS, 5 SMA-IV, 6 SBMA, and 6 Healthy controls. Myoblasts were differentiated for 3 days to form myotubes, before RNA extraction and microarray analysis using Affymetrix Human exon array 1.0 ST v2.

尽管已发现诸多遗传风险因子，但驱动肌萎缩侧索硬化（Amyotrophic Lateral Sclerosis, ALS）终末期病理的运动神经元死亡病因仍不明晰。我们在此报道，ALS患者的骨骼肌会分泌对运动神经元具有特异性毒性的外泌体囊泡，且该毒性并非源于肌肉去神经支配的次要下游效应。

在针对67名人类受试者（涵盖健康对照与疾病对照）的肌肉活检样本，以及活检来源的未经历去神经支配的分化肌肉干细胞（肌管）的研究中，ALS来源的肌管呈现出外泌体生物发生与RNA加工过程紊乱的一致特征谱；其分泌的外泌体可诱导运动神经元的神经突变短、分支减少，细胞死亡率升高，并破坏RNA及RNA加工蛋白在运动神经元内的定位。

该毒性依赖于FUS蛋白的存在，而FUS蛋白在受体运动神经元中呈高表达。

作为本研究的一部分，我们对肌管（分化的成肌细胞）开展了基因表达分析，将ALS与另外两种运动神经元疾病作为疾病对照——脊髓延髓肌萎缩症（Spinal and bulbar muscular atrophy, SBMA）、脊髓性肌萎缩症4型（Spinal Muscular Atrophy Type 4, SMA-IV）——及健康对照进行比较。

研究共从23名受试者的三角肌活检样本中提取原代肌肉干细胞（成肌细胞），其中ALS患者6名、SMA-IV患者5名、SBMA患者6名、健康对照6名。成肌细胞经3天诱导分化形成肌管后，进行RNA提取，并采用Affymetrix人类外显子阵列1.0 ST v2芯片完成微阵列分析。