Neuromuscular Unit, Dino Ferrari Centre, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Foundation Ca' Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy.
Neurology Unit, Neuroscience Section, Department of Pathophysiology and Transplantation, Dino Ferrari Centre, IRCCS Foundation Ca' Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy.
JAMA Neurol. 2015 Jun;72(6):666-75. doi: 10.1001/jamaneurol.2015.0178.
The important depletion of mitochondrial DNA (mtDNA) and the general depression of mitochondrial respiratory chain complex levels (including complex II) have been confirmed, implying an increasing paucity of mitochondria in the muscle from patients with types I, II, and III spinal muscular atrophy (SMA-I, -II, and -III, respectively).
To investigate mitochondrial dysfunction in a large series of muscle biopsy samples from patients with SMA.
DESIGN, SETTING, AND PARTICIPANTS: We studied quadriceps muscle samples from 24 patients with genetically documented SMA and paraspinal muscle samples from 3 patients with SMA-II undergoing surgery for scoliosis correction. Postmortem muscle samples were obtained from 1 additional patient. Age-matched controls consisted of muscle biopsy specimens from healthy children aged 1 to 3 years who had undergone analysis for suspected myopathy. Analyses were performed at the Neuromuscular Unit, Istituto di Ricovero e Cura a Carattere Scientifico Foundation Ca' Granda Ospedale Maggiore Policlinico-Milano, from April 2011 through January 2015.
We used histochemical, biochemical, and molecular techniques to examine the muscle samples.
Respiratory chain activity and mitochondrial content.
Results of histochemical analysis revealed that cytochrome-c oxidase (COX) deficiency was more evident in muscle samples from patients with SMA-I and SMA-II. Residual activities for complexes I, II, and IV in muscles from patients with SMA-I were 41%, 27%, and 30%, respectively, compared with control samples (P < .005). Muscle mtDNA content and cytrate synthase activity were also reduced in all 3 SMA types (P < .05). We linked these alterations to downregulation of peroxisome proliferator-activated receptor coactivator 1α, the transcriptional activators nuclear respiratory factor 1 and nuclear respiratory factor 2, mitochondrial transcription factor A, and their downstream targets, implying depression of the entire mitochondrial biogenesis. Results of Western blot analysis confirmed the reduced levels of the respiratory chain subunits that included mitochondrially encoded COX1 (47.5%; P = .004), COX2 (32.4%; P < .001), COX4 (26.6%; P < .001), and succinate dehydrogenase complex subunit A (65.8%; P = .03) as well as the structural outer membrane mitochondrial porin (33.1%; P < .001). Conversely, the levels of expression of 3 myogenic regulatory factors-muscle-specific myogenic factor 5, myoblast determination 1, and myogenin-were higher in muscles from patients with SMA compared with muscles from age-matched controls (P < .05).
Our results strongly support the conclusion that an altered regulation of myogenesis and a downregulated mitochondrial biogenesis contribute to pathologic change in the muscle of patients with SMA. Therapeutic strategies should aim at counteracting these changes.
已证实线粒体 DNA（mtDNA）大量损耗和线粒体呼吸链复合物水平（包括复合物 II）普遍降低，这意味着从 I、II 和 III 型脊肌萎缩症（分别为 SMA-I、-II 和 -III）患者的肌肉中，线粒体的数量越来越少。
研究大量 SMA 患者肌肉活检样本中的线粒体功能障碍。
设计、地点和参与者：我们研究了 24 名经基因证实的 SMA 患者的股四头肌样本和 3 名 SMA-II 患者的脊柱侧凸矫正手术中的脊旁肌样本。另外还从 1 名患者获得了尸检肌肉样本。年龄匹配的对照组由年龄在 1 至 3 岁之间的健康儿童的肌肉活检标本组成，这些标本是因疑似肌病而进行分析的。分析于 2011 年 4 月至 2015 年 1 月在米兰的 Ca' Granda Ospedale Maggiore Policlinico-Milano 的神经肌肉科进行。
我们使用组织化学、生化和分子技术检查肌肉样本。
呼吸链活性和线粒体含量。
组织化学分析结果表明，SMA-I 和 SMA-II 患者的肌肉样本 COX 缺陷更为明显。与对照样本相比，SMA-I 患者肌肉中的复合物 I、II 和 IV 的残余活性分别为 41%、27%和 30%（P <.005）。所有 3 种 SMA 类型的肌肉 mtDNA 含量和柠檬酸合酶活性均降低（P <.05）。我们将这些改变与过氧化物酶体增殖物激活受体共激活因子 1α、核呼吸因子 1 和核呼吸因子 2、线粒体转录因子 A 及其下游靶标的下调联系起来，暗示整个线粒体生物发生受到抑制。Western blot 分析结果证实了呼吸链亚基的水平降低，包括线粒体编码的 COX1（47.5%；P =.004）、COX2（32.4%；P <.001）、COX4（26.6%；P <.001）和琥珀酸脱氢酶复合物亚基 A（65.8%；P =.03）以及结构外膜线粒体孔蛋白（33.1%；P <.001）。相反，SMA 患者肌肉中 3 种肌源性调节因子-肌肉特异性肌生成因子 5、成肌决定因子 1 和肌生成素的表达水平高于年龄匹配的对照组（P <.05）。
我们的结果强烈支持这样的结论，即肌肉发生的调节改变和下调的线粒体生物发生导致 SMA 患者肌肉的病理变化。治疗策略应旨在对抗这些变化。
