Department of Anesthesiology & Perioperative Medicine, Mayo Clinic, College of Medicine and Science, 200 First Street SW, Rochester, MN 55905, USA.
Department of Physiology & Biomedical Engineering, Mayo Clinic, College of Medicine and Science, 200 First Street SW, Rochester, MN 55905, USA.
Exp Gerontol. 2021 Feb;144:111193. doi: 10.1016/j.exger.2020.111193. Epub 2020 Dec 5.
Neuromuscular dysfunction is common in old age. Damaged cytoplasmic structures aggregate with aging, especially in post-mitotic cells like motor neurons. Autophagy is a ubiquitous cell process that aids in the clearance of damaged aggregates. Accordingly, we hypothesized that autophagy is impaired in old age, contributing to neuromuscular dysfunction via an effect in motor neurons. Autophagy flux may be impaired as a result of deficits in the initiation, elongation or degradation phases. Changes in the expression levels of core proteins necessary for each of the autophagy phases were evaluated by Western blotting in the cervical spinal cord (segments C2-C6 corresponding to the phrenic motor pool) of adult male and female mice at 6-, 18-, and 24-months of age (reflecting 100%, 90% and 75% survival, respectively). There was no evidence of an effect of age on the expression of the autophagy markers Beclin-1 (Becn-1; initiation), ATG7 and ATG5/12 complex (elongation) or LC3 (elongation/degradation). Reduced p62 expression (a marker of degradation) was evident in the cervical spinal cord of adult mice at 18-months compared to 24-months. Accordingly, expression of LC3 and p62 in motor neurons was analyzed using immunofluorescence and confocal microscopy in separate animals. LC3 and p62 immunoreactivity was evident in the gray matter with minimal expression in the white matter across all age groups. A mixed linear model with animal as a random effect was used to compare relative LC3 and p62 expression in motor neurons to gray matter across age groups. Expression of both LC3 and p62 was higher in choline acetyl transferase (ChAT)-positive motor neurons (~2-3 fold vs. gray matter). Across age groups, there were differences in the relative expression of LC3 (F = 7.59, p < 0.01) and p62 (F = 8.00, p < 0.01) in cervical motor neurons. LC3 expression in motor neurons increased ~20% by 24-months of age in both male and female mice. p62 expression in motor neurons increased ~70% by 18-months compared to 6-months with no further changes by 24-months of age in male mice. p62 expression did not change across age groups in female mice, and was ~20% higher than in males. Our findings highlight important changes in autophagy pathways that likely contribute to the development of aging-related neuromuscular dysfunction in mice. At 18-months of age, increased autophagosome clearance (reduced p62 expression) appears to be a global effect not restricted to motor neurons. By 24-months of age, increased expression of LC3 and p62 indicates impaired autophagy with autophagosome accumulation in cervical motor neurons.
神经肌肉功能障碍在老年中很常见。细胞质结构随着年龄的增长而聚集，尤其是在后有丝分裂细胞（如运动神经元）中。自噬是一种普遍存在的细胞过程，有助于清除受损的聚集体。因此，我们假设自噬在老年时受损，通过对运动神经元的影响导致神经肌肉功能障碍。自噬流可能会因起始、延伸或降解阶段的缺陷而受损。通过 Western blot 评估了成年雄性和雌性小鼠颈椎脊髓（对应于膈神经运动池的 C2-C6 节段）中自噬各阶段核心蛋白表达水平的变化，这些小鼠的年龄分别为 6 个月、18 个月和 24 个月（分别反映 100%、90%和 75%的存活率）。年龄对自噬标志物 Beclin-1（起始）、ATG7 和 ATG5/12 复合物（延伸）或 LC3（延伸/降解）的表达没有影响。与 24 个月相比，18 个月时成年小鼠颈椎脊髓中 p62 表达（降解标志物）减少。因此，使用免疫荧光和共聚焦显微镜在单独的动物中分析了运动神经元中的 LC3 和 p62 的表达。LC3 和 p62 免疫反应性在灰质中明显，在所有年龄组的白质中表达最小。使用具有动物作为随机效应的混合线性模型比较了各组年龄的运动神经元中 LC3 和 p62 的相对表达。LC3 和 p62 在胆碱乙酰转移酶（ChAT）阳性运动神经元中的表达较高（~2-3 倍于灰质）。在各组年龄中，LC3（F=7.59，p<0.01）和 p62（F=8.00，p<0.01）在颈椎运动神经元中的相对表达存在差异。在雄性和雌性小鼠中，24 个月时 LC3 在运动神经元中的表达增加了约 20%。与 6 个月相比，18 个月时 p62 在运动神经元中的表达增加了约 70%，到 24 个月时没有进一步变化。在雌性小鼠中，p62 的表达没有随年龄变化，比雄性高约 20%。我们的研究结果强调了自噬途径的重要变化，这些变化可能导致与衰老相关的神经肌肉功能障碍在小鼠中发生。在 18 个月时，自噬体清除增加（p62 表达减少）似乎是一种普遍的影响，而不仅仅局限于运动神经元。到 24 个月时，LC3 和 p62 的表达增加表明自噬受损，颈椎运动神经元中自噬体积累。
