Sarcopenia involves a progressive loss of skeletal muscle force, quality and mass during ageing, which results in increased inability and death; however, no cure has been established thus far. Growth differentiation factor 5 (GDF5) has been described to modulate muscle mass maintenance in various contexts. For our proof of concept, we overexpressed GDF5 by AAV vector injection in Tibialis Anterior (TA) muscle of adult aged (20 months) mice and performed molecular and functional analysis of skeletal muscle. We analysed human Vastus Lateralis muscle biopsies from adult young (21-42 years) and aged (77-80 years) donors, quantifying the molecular markers modified by GDF5 overexpression (OE) in mouse muscle. We validated the major effects of GDF5 overexpression using human immortalized myotubes and Schwann Cells (SCs). We established a pre-clinical study by treating chronically (for 4 months) aged mice using recombinant GDF5 protein (rGDF5) in systemic administration and evaluated the long-term effect of this treatment on muscle mass and function. Here, we demonstrated that GDF5 OE in the old TAs promoted an increase of 16.5% of muscle weight (P = 0.0471) associated with a higher percentage of 5000-6000 µm2 large fibres (P = 0.0211), without the induction of muscle regeneration. Muscle mass gain was associated with an amelioration of 26.8% of rate of force generation (P = 0.0330) and a better neuromuscular connectivity (P = 0.0098). Moreover, GDF5 OE preserved neuromuscular junction (NMJ) morphology (38.5% of nerve terminal area increase, P < 0.0001) and stimulated the expression of re-innervation-related genes, in particular markers of SCs (fold change 3.19 for S100b gene expression, P = 0.0101). To further characterize the molecular events induced by GDF5 OE during ageing, we performed a genome-wide transcriptomic analysis of treated muscles and showed that this factor leads to a “rejuvenating” transcriptomic signature in aged mice, as 42% of the transcripts dysregulated by ageing reverted to youthful expression levels upon GDF5 OE (P < 0.05). Towards a pre-clinical approach, we performed a long-term systemic treatment using rGDF5 and showed its effectiveness in counteracting age-related muscle wasting, improving muscle function (17,8% of absolute maximal force increase, P = 0.0079), ensuring neuromuscular connectivity and preventing NMJ degeneration (7,96% of AchR area increase, P = 0.0125). In addition, in human muscle biopsies, we found the same age-related alterations than those observed in mice and improved by GDF5 and reproduced its major effects on human cells, suggesting this treatment as efficient in humans. Overall, these data provide a foundation to examine the curative potential of GDF5 drug in clinical trials for sarcopenia and, eventually, other neuromuscular diseases.

中文翻译：

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GDF5 作为年龄相关神经肌肉衰竭的恢复治疗方法

肌肉减少症涉及衰老过程中骨骼肌力量、质量和质量的逐渐丧失，从而导致丧失能力和死亡的增加；然而，迄今为止尚未找到治愈方法。生长分化因子 5 (GDF5) 已被描述为在各种情况下调节肌肉质量维持。为了证明我们的概念，我们通过 AAV 载体注射到成年（20 个月）小鼠的胫骨前肌 (TA) 中过度表达 GDF5，并对骨骼肌进行分子和功能分析。我们分析了来自成年年轻（21-42 岁）和老年（77-80 岁）供体的人类股外侧肌活检，量化了小鼠肌肉中 GDF5 过表达 (OE) 修饰的分子标记。我们使用人类永生化肌管和雪旺细胞 (SC) 验证了 GDF5 过度表达的主要影响。我们通过使用重组 GDF5 蛋白 (rGDF5) 全身给药来治疗长期（4 个月）衰老的小鼠，建立了一项临床前研究，并评估了这种治疗对肌肉质量和功能的长期影响。在这里，我们证明旧 TA 中的 GDF5 OE 促进肌肉重量增加 16.5% (P = 0.0471)，与更高比例的 5000-6000 µm2 大纤维 (P = 0.0211) 相关，而不诱导肌肉再生。肌肉质量增加与力量产生率改善 26.8% (P = 0.0330) 和更好的神经肌肉连接性 (P = 0.0098) 相关。此外，GDF5 OE 保留了神经肌肉接头 (NMJ) 形态（神经末梢面积增加 38.5%，P < 0.0001）并刺激重新神经支配相关基因的表达，特别是 SC 标记物（S100b 基因表达的倍数变化 3.19） ，P = 0.0101）。为了进一步表征衰老过程中 GDF5 OE 诱导的分子事件，我们对接受治疗的肌肉进行了全基因组转录组分析，结果表明，该因子导致衰老小鼠出现“返老还童”转录组特征，因为 42% 的转录本因衰老而失调GDF5 OE后恢复到年轻的表达水平(P＜0.05)。针对临床前方法，我们使用 rGDF5 进行了长期全身治疗，并证明了其在抵消与年龄相关的肌肉萎缩、改善肌肉功能（绝对最大力量增加的 17.8%，P = 0.0079）方面的有效性，确保神经肌肉连接性并防止 NMJ 变性（AchR 面积增加 7.96%，P = 0.0125）。此外，在人类肌肉活检中，我们发现了与在小鼠中观察到的相同的与年龄相关的改变，并通过 GDF5 得到改善，并重现了其对人类细胞的主要影响，表明这种治疗对人类有效。总体而言，这些数据为检验 GDF5 药物在肌肉减少症以及最终其他神经肌肉疾病的临床试验中的治疗潜力奠定了基础。