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

IntroductionPeripheral 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. MethodsMass 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. ResultsEx 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. ConclusionThese 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. Plain English summaryAge-related peripheral neuropathies are common disorders that can cause pain, numbness, weakness and reduced mobility, affecting millions of people worldwide. They become more common from around the age of 50 and affect men and women differently. These conditions are thought to result from age-related changes in the structure and function of peripheral nerves, which reduce their ability to repair themselves after injury. In this study, we used advanced protein analysis (proteomics) to investigate how ageing affects peripheral nerves in male and female mice. We also used an ex vivo model, in which nerves are studied outside the body, to examine how age and sex influence the molecular changes that occur during nerve degeneration. We found that degeneration caused widespread changes in the proteins present in the sciatic nerve in both young and old mice. Most of these changes were similar in males and females, but some important differences emerged. Male nerves showed stronger signs of inflammation, whereas female nerves showed increased activity of metabolic pathways, including those involved in folate metabolism. Ageing nerves also appeared less able to remove damaged material and showed signs of activating degeneration-related processes even before injury. Overall, the ex vivo model reproduced many of the molecular changes seen after nerve injury in living animals, although it did not fully capture the inflammatory response, suggesting that signals from the rest of the body, including factors carried in the blood, also contribute to nerve degeneration. HighlightsO_LIEx vivo nerve degeneration caused major protein changes in young and old mouse sciatic nerves. C_LIO_LIMost degeneration-related protein changes were shared between males and females. C_LIO_LIAgeing altered the nerve proteome, with stronger ageing-related shifts in males. C_LIO_LIMale nerves showed stronger inflammatory and immune-related signatures. C_LIO_LIFemale nerves showed enrichment of metabolic pathways, including folate biosynthesis, and ex vivo degeneration did not fully reproduce the inflammatory response seen after injury in vivo. C_LI