Altered Redox Signalling in both Muscle and Nerve are Required for Neuromuscular Breakdown
Authors: Pollock Natalie , Hemmings Kay , Staunton Caroline A. , Owen Euan D. , Scalabrin Mattia , Macpherson Peter C. , Vasilaki Aphrodite , Van Remmen Holly , Richardson Arlan , Brooks Susan V. , McArdle Anne , Jackson Malcolm J. 
Published: April 2020

Our previous work has demonstrated that loss of superoxide dismutase1 (Sod1) in whole body KO mice (Sod1KO) results in an accelerated loss of muscle mass and function that is associated with a breakdown of neuromuscular junctions (NMJ). Deletion of Sod1 specifically in muscle or nerve caused a milder phenotype. These observations led us to hypothesise that altered redox signalling in muscle or nerve alone is not sufficient to induce sarcopenic changes but requires defects in both tissues. Inducible motor neuron Sod1 KO (i‐mnSod1KO) mice were created in order to examine whether loss of Sod1 in nerves in adult‐life affected age‐associated muscle wasting and weakness.

This current work has examined changes in the neuromuscular system of Sod1KO, nerve rescue (SynTgSod1KO), i‐mnSod1KO mice and WT mice with advancing age.

Hind limb skeletal muscles and sciatic nerves from adult (8–10 months), mid age (16–18 months) and old WT (24–27 months) mice, mid‐age and old i‐mnSod1KO mice, adult Sod1KO and adult SynTgSod1KO were used. Fixed and stained EDL muscles were used for NMJ analysis. Alterations in NMJ structure were quantified including pre‐and post‐synaptic overlap, fragmentation, complexity and area. Schwann cells were stained with fluorescently tagged s100 antibody. Western blot analysis of the sciatic nerves was performed to compare levels of oxidative damage assessed by the contents of 3‐NT and protein carbonylation. Electron paramagnetic resonance (EPR) analysis was carried out by infusing mice with a CPH spin probe and excising the gastrocnemius (GTN) muscle and sciatic nerve to examine the level of oxidised adducts. Samples were placed in Krebs buffer, frozen in liquid nitrogen and scanned on Bruker e‐scan EPR.

A decline in NMJ structure was seen with advancing age. Despite some structural alterations, mid age mice showed no significant changes in NMJ structure in either WT or i‐mnSod1KO mice. There were significant alterations in all mice at 24 months and older. NMJs were substantially more disrupted in old i‐mnSod1Ko mice compared with old WT mice. Overlap and complexity of NMJs were both significantly reduced, but fragmentation was similar. The pattern of s100 staining was also altered with advancing age.

The concentration of the 3‐carboxy‐proxyl radical (CP) measured by EPR in the sciatic nerve or GTN of adult WT, i‐mnSod1KO and SynTgSod1KO mice was not significantly different, although Sod1KO mice demonstrated a significant increase in CP concentration compared with adult WT.

Data suggest that an accumulation of age‐related changes in the muscle alongside altered redox signalling in the nerve due to lack of Sod1 is needed to bring about a significant breakdown of the NMJ.

Support or Funding Information

With thanks to National Institute of Ageing (AG051442) and University of Liverpool for their support.