No abstract

Background Sarcopenia, the age-associated decline in skeletal muscle mass and strength, has long been considered a disease of muscle only, but accumulating evidence suggests that sarcopenia could originate from the neural components controlling muscles. To identify early molecular changes in nerves that may drive sarcopenia initiation, we performed a longitudinal transcriptomic analysis of the sciatic nerve, which governs lower limb muscles, in aging mice. Methods Sciatic nerve and gastrocnemius muscle were obtained from female C57BL/6JN mice aged 5, 18, 21 and 24 months old (n = 6 per age group). Sciatic nerve RNA was extracted and underwent RNA sequencing (RNA-seq). Differentially expressed genes (DEGs) were validated using quantitative reverse transcription PCR (qRT-PCR). Functional enrichment analysis of clusters of genes associated with patterns of gene expression across age groups (adjusted P-value < 0.05, likelihood ratio test [LRT]) was performed. Pathological skeletal muscle aging was confirmed between 21 and 24 months by a combination of molecular and pathological biomarkers. Myofiber denervation was confirmed with qRT-PCR of Chrnd, Chrng, Myog, Runx1 and Gadd45ɑ in gastrocnemius muscle. Changes in muscle mass, cross-sectional myofiber size and percentage of fibres with centralized nuclei were analysed in a separate cohort of mice from the same colony (n = 4-6 per age group). ResultsWe detected 51 significant DEGs in sciatic nerve of 18-month-old mice compared with 5-month-old mice (absolute value of fold change > 2; false discovery rate [FDR] < 0.05). Up-regulated DEGs included Dbp (log 2 fold change [LFC] = 2.63, FDR < 0.001) and Lmod2 (LFC = 7.52, FDR = 0.001). Down-regulated DEGs included Cdh6 (LFC = À21.38, FDR < 0.001) and Gbp1 (LFC = À21.78, FDR < 0.001). We validated RNA-seq findings with qRT-PCR of various up-and down-regulated genes including Dbp and Cdh6. Up-regulated genes (FDR < 0.1) were associated with the AMP-activated protein kinase signalling pathway (FDR = 0.02) and circadian rhythm (FDR = 0.02), whereas down-regulated DEGs were associated with biosynthesis and metabolic pathways (FDR < 0.05). We identified seven significant clusters of genes (FDR < 0.05, LRT) with similar expression patterns across groups. Functional enrichment analysis of these clusters revealed biological processes that may be implicated in age-related changes in skeletal muscles and/or sarcopenia initiation including extracellular matrix organization and an immune response (FDR < 0.05). Conclusions Gene expression changes in mouse peripheral nerve were detected prior to disturbances in myofiber innervation and sarcopenia onset. These early molecular changes we report shed a new light on biological processes that may be implicated in sarcopenia initiation and pathogenesis. Future studies are warranted to confirm the disease modifying and/or biomarker potential of the key changes we report here.

BackgroundSarcopenia, the age-associated decline in skeletal muscle mass and strength, has long been considered a disease of muscle only, but accumulating evidence suggests that sarcopenia could originate from the neural components controlling muscles. To identify early molecular changes in the efferent nerves that may drive sarcopenia initiation, we performed a longitudinal transcriptomic analysis of the sciatic nerve in aging mice.MethodsSciatic nerve and gastrocnemius muscle were obtained from young adult, middleaged, old, and sarcopenic (5,18, 21 and 24 months old, respectively) C57BL/6J female mice (n=6 per age group). Sciatic nerve RNA was extracted and subjected to RNA sequencing (RNA-seq), with real-time quantitative reverse transcription PCR (qRT-PCR) validation of differentially expressed genes (DEGs). Functional enrichment analysis of clusters of genes associated with patterns of gene expression across age groups was performed. Sarcopenia was confirmed with qRT-PCR of previously established markers of sarcopenia onset in gastrocnemius muscle.ResultsWe detected 33 significant DEGs in sciatic nerve of 18-month-old mice compared to 5-month-old mice (absolute value of fold change > 2; false discovery rate [FDR] < 0.05) which we validated with qRT-PCR of the three top up- and down-regulated genes. Up-regulated genes were associated with circadian rhythm and the AMPK signaling pathway, while down-regulated genes were associated with biosynthesis and metabolic pathways and circadian rhythm. Strikingly, we detected a significant increase in Myog expression (log2 fold change = 18.93, FDR q-value = 1.54×10−12) in sciatic nerve of 18-month-old mice, before up-regulation in muscle was observed. We identified seven clusters of genes with similar expression patterns across groups. Functional enrichment analysis of these clusters revealed biological processes that may be implicated in sarcopenia initiation including extracellular matrix organization and circadian regulation of gene expression.ConclusionsGene expression changes in mouse peripheral nerve can be detected prior to overt clinical onset of sarcopenia. These early molecular changes we report shed a new light on biological processes that may be implicated in sarcopenia initiation and pathogenesis. Future studies will validate which of the key changes we reported have disease modifying and/or biomarker potential.