Belosludtseva Natalia V, Ilzorkina Anna I, Dubinin Mikhail V, Mikheeva Irina B, Belosludtsev Konstantin N
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, 142290 Pushchino, Russia.
Department of Biochemistry, Cell Biology and Microbiology, Mari State University, 424001 Yoshkar-Ola, Russia.
Front Biosci (Landmark Ed). 2025 Mar 18;30(3):28260. doi: 10.31083/FBL28260.
Amyotrophic lateral sclerosis (ALS) is a progressive multisystem disease characterized by limb and trunk muscle weakness that is attributed, in part, to abnormalities in mitochondrial ultrastructure and impaired mitochondrial functions. This study investigated the time course of structural and functional rearrangements in skeletal muscle mitochondria in combination with motor impairments in Tg (copper-zinc superoxide dismutase enzyme (SOD1) G93A) dl1/GurJ (referred to as SOD1-G93A/low) male mice, a familial ALS model, as compared with non-transgenic littermates.
The neurological status and motor functions were assessed weekly using the paw grip endurance method and the grid suspension test with two-limb and four-limb suspension tasks. Transmission electron microscopy followed by quantitative analysis was performed to study ultrastructural alterations in the quadriceps femoris. Functional analysis of skeletal muscle mitochondria was performed using high-resolution Oxygraph-2k (O2K) respirometry and methods for assessing the calcium retention capacity index and the content of lipid peroxidation products in freshly isolated preparations.
Based on the behavioral phenotyping data, specific age groups were identified: postnatal day 56 (P56) ( = 10-11), 84 (P84) ( = 10-11), and 156 (P154) ( = 10-12), representing the pre-symptomatic, early-symptomatic and late-symptomatic stages of ALS progression in SOD1-G93A/low mice, respectively. Electron microscopy showed mosaic destructive changes in subsarcolemmal mitochondria in fibers of the quadriceps femoris from 84-day-old SOD1-G93A/low mice. Morphometric analysis revealed an elevation in the mean size of the mitochondria in SOD1-G93A mice at P84 and P154. In addition, the P154 transgenic group demonstrated a decrease in sarcomere width and the number of mitochondria per unit area. At the symptomatic stage, SOD1-G93A mice exhibited a decreased respiratory control ratio, ADP-stimulated, and uncoupled respiration rates of mitochondria isolated from the quadriceps femoris muscle, as measured by high-resolution respirometry. In parallel, the mitochondria showed lower calcium retention capacity and increased levels of lipid peroxidation products compared with the control.
Taken together, these results indicate stage-dependent changes in skeletal muscle mitochondrial ultrastructure and functions associated with defective oxidative phosphorylation, impaired calcium homeostasis, and oxidative damage in the SOD1-G93A/low mouse model, which appears to be a promising direction for the development of combination therapies for ALS.
肌萎缩侧索硬化症（ALS）是一种进行性多系统疾病，其特征为肢体和躯干肌肉无力，部分原因是线粒体超微结构异常和线粒体功能受损。本研究调查了家族性ALS模型Tg（铜锌超氧化物歧化酶（SOD1）G93A）dl1/GurJ（简称SOD1-G93A/低）雄性小鼠与非转基因同窝小鼠相比，骨骼肌线粒体结构和功能重排的时间进程以及运动功能障碍情况。
每周使用握力耐力法和网格悬挂试验（包括双肢和四肢悬挂任务）评估神经状态和运动功能。进行透射电子显微镜检查并随后进行定量分析，以研究股四头肌的超微结构改变。使用高分辨率Oxygraph-2k（O2K）呼吸测定法以及评估新鲜分离制剂中钙保留能力指数和脂质过氧化产物含量的方法，对骨骼肌线粒体进行功能分析。
根据行为表型数据，确定了特定的年龄组：出生后第56天（P56）（n = 10 - 11）、84天（P84）（n = 10 - 11）和156天（P154）（n = 10 - 12），分别代表SOD1-G93A/低小鼠ALS进展的症状前、早期症状和晚期症状阶段。电子显微镜显示，84日龄SOD1-G93A/低小鼠股四头肌纤维的肌膜下线粒体出现镶嵌性破坏变化。形态计量分析显示，P84和P154时SOD1-G93A小鼠线粒体的平均大小增加。此外，P154转基因组的肌节宽度和单位面积线粒体数量减少。在症状期，通过高分辨率呼吸测定法测量，SOD1-G93A小鼠股四头肌分离的线粒体的呼吸控制率、ADP刺激的呼吸率和解偶联呼吸率均降低。同时，与对照组相比，线粒体显示出较低的钙保留能力和脂质过氧化产物水平升高。
综上所述，这些结果表明，在SOD1-G93A/低小鼠模型中，骨骼肌线粒体超微结构和功能存在阶段依赖性变化，与氧化磷酸化缺陷、钙稳态受损和氧化损伤相关，这似乎是开发ALS联合治疗方法的一个有前景的方向。
