Sex-Dependent Proteomic Remodelling During ex vivo Degeneration of Young and Aged Murine Peripheral Nerves

Introduction: Peripheral nerve ageing leads to profound proteomic remodelling, with shifts in metabolic and inflammatory signalling pathways resembling changes that occur during nerve degeneration and regeneration following injury. Moreover, aged nerves exhibit impaired degeneration and regeneration, contributing to age-related peripheral neuropathies that show sex-specific differences in prevalence. However, it remains unclear whether these alterations arise from intrinsic nerve changes or an altered systemic environment. Therefore, we investigated the impact of sex on age-related proteome changes and nerve-intrinsic proteomic responses in young and aged male and female nerves using an ex vivo degeneration model. Methods: Mass spectrometry-based proteomics were performed on young and old nerves from male and female animals, as well as on contralateral nerves after seven days of ex vivo nerve degeneration. A comparative bioinformatic analysis was then used to identify changes during ageing and ex vivo nerve degeneration that were independent of sex, as well as changes that were sex-specific. Results: Ex vivo nerve degeneration induced extensive proteome remodelling in mouse sciatic nerves that was largely independent of age and sex. Principal component and clustering analyses clearly separated intact from degenerated nerves, while revealing only subtle age- and sex-related effects, with more pronounced ageing-associated changes in males. Approximately 20% of age-regulated proteins and 7-10% of degeneration-regulated proteins exhibited sex-specific expression patterns. Degeneration was characterised by increased abundance of lysosomal and repair-associated proteins alongside reduced myelin and axonal proteins, consistent with active tissue remodelling. In aged nerves, impaired protein clearance and partial pre-activation of degeneration-associated pathways suggested altered injury responses. Comparative analyses demonstrated positive correlations of protein abundance changes between ex vivo and in vivo degeneration datasets, although the temporal dynamics were altered in aged nerves. Pathway enrichment analyses identified coordinated regulation of metabolic, RNA-processing and vesicular transport pathways, while ageing was associated with enhanced immune signalling and reduced lipid metabolism. Sex-specific analyses revealed stronger inflammatory signatures in males, whereas females exhibited enrichment of metabolic pathways, including folate biosynthesis. Conclusion: These findings reveal distinct sex-specific molecular features of peripheral nerve ageing, characterised by enhanced inflammatory signalling in males and metabolic adaptations that may confer resilience in females. Our datasets provide a comprehensive molecular resource of sex-dependent changes in peripheral nerve ageing and nerve-intrinsic injury responses, offering a foundation for identifying therapeutic strategies to promote healthy peripheral nerve ageing.