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_2,12 = 7.59, p < 0.01) and p62 (F_2,12 = 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人中很常见。受损的细胞质结构会随着衰老而聚集，尤其是在运动神经元等有丝分裂后细胞中。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。自噬通量可能由于起始、延长或降解阶段的缺陷而受损。每个自噬阶段所必需的核心蛋白表达水平的变化通过 Western blotting 在成年雄性和雌性小鼠的颈脊髓（对应于膈运动池的 C2-C6 段）中进行评估，分别为 6-、18-、和 24 个月大（分别反映 100%、90% 和 75% 的存活率）。没有证据表明年龄对自噬标记物 Beclin-1（Becn-1；起始）、ATG7 和 ATG5/12 复合物（延伸）或 LC3（延伸/降解）的表达有影响。与 24 个月相比，18 个月大的成年小鼠的颈脊髓中 p62 表达（退化标志物）明显减少。因此，在不同的动物中使用免疫荧光和共聚焦显微镜分析了运动神经元中 LC3 和 p62 的表达。LC3 和 p62 免疫反应性在灰质中很明显，在所有年龄组的白质中表达极少。使用以动物作为随机效应的混合线性模型来比较运动神经元中的相对 LC3 和 p62 表达与不同年龄组的灰质。LC3 和 p62 的表达在胆碱乙酰转移酶 (ChAT) 阳性运动神经元中更高（相对于灰质约 2-3 倍）。在不同年龄组中，LC3 的相对表达存在差异（F_2,12  = 7.59, p  < 0.01) 和 p62 (F _2,12 = 8.00，p < 0.01) 在颈椎运动神经元中。到 24 个月大时，雄性和雌性小鼠的运动神经元中的 LC3 表达增加了约 20%。与 6 个月相比，运动神经元中的 p62 表达在 18 个月时增加了约 70%，雄性小鼠在 24 个月大时没有进一步变化。雌性小鼠的 p62 表达在不同年龄组之间没有变化，比雄性小鼠高约 20%。我们的研究结果强调了自噬途径的重要变化，这些变化可能导致小鼠出现与衰老相关的神经肌肉功能障碍。在 18 个月大时，自噬体清除率增加（p62 表达减少）似乎是一种不局限于运动神经元的全局效应。到 24 个月大时，LC3 和 p62 的表达增加表明自噬受损，自噬体在颈部运动神经元中积聚。