The axon-protective WLD(S) protein partially rescues mitochondrial respiration and glycolysis after axonal injury

Abstract

The axon-protective Wallerian degeneration slow (WLD(S)) protein can ameliorate the decline in axonal ATP levels after neurite transection. Here, we tested the hypothesis that this effect is associated with maintenance of mitochondrial respiration and/or glycolysis. We used isolated neurites of superior cervical ganglion (SCG) cultures in the Seahorse XF-24 Metabolic Flux Analyser to determine mitochondrial respiration and glycolysis under different conditions. We observed that both mitochondrial respiration and glycolysis declined significantly during the latent phase of Wallerian degeneration. WLD(S) partially reduced the decline both in glycolysis and in mitochondrial respiration. In addition, we found that depleting NAD levels in uncut cultures led to changes in mitochondrial respiration and glycolysis similar to those rescued by WLD(S) after cut, suggesting that the maintenance of NAD levels in Wld(S) neurites after axonal injury at least partially underlies the maintenance of ATP levels. However, by using another axon-protective mutation (Sarm1(-/-)), we could demonstrate that rescue of basal ECAR (and hence probably glycolysis) rather than basal OCR (mitochondrial respiration) may be part of the protective phenotype to delay Wallerian degeneration. These findings open new routes to study glycolysis and the connection between NAD and ATP levels in axon degeneration, which may help to eventually develop therapeutic strategies to treat neurodegenerative diseases.

Fig. 1

Mitochondrial respiration and glycolysis both decline after axonal injury. Oxygen consumption rate (OCR) in pmol/min (a) and extracellular acidification rate (ECAR) in pmH/min (b) of 6 days in vitro (DIV) mouse wild-type neurites measured early after cut (square) or late after cut (circle). Recordings took place under basal, oligomycin-inhibited (oligomy), FCCP-induced maximal and rotenone + antimycin A-inhibited (rot/antimy) conditions. *p < 0.05, **p < 0.01 two-way repeated measures ANOVA. Data are mean ± SEM of n = 5 independent experiments for both OCR and ECAR

Fig. 2

WLD^S partially rescues cellular energetics after axonal injury. OCRs in pmol/min (a) and ECARs in pmH/min (b) of six DIV mouse wild-type neurites (circle) and Wld ^S neurites (square) measured late after cut. Recordings took place under basal, oligomycin-inhibited (oligomy), FCCP-induced maximal and rotenone + antimycin A-inhibited (rot/antimy) conditions. *p < 0.05 two-way repeated measures ANOVA. Data are mean ± SEM of n = 6 independent experiments for both OCR and ECAR

Fig. 3

A reduction in NAD levels decreases cellular energetics. SCG explants were cultured for five DIV and treated with DMSO or 100 nM FK866 for 24 h as indicated. a NAD levels normalised to total protein (n = 3 per condition). ** p < 0.01 (Student’s t test). OCRs in pmol/min (b) and ECARs in pmH/min (c) of six DIV mouse wild-type SCG explants treated with DMSO (circle) or with 100 nM FK866 for 24 h (square). Recordings took place under basal, oligomycin-inhibited (oligomy), FCCP-induced maximal and rotenone + antimycin A-inhibited (rot/antimy) conditions. *p < 0.05 two-way repeated measures ANOVA. Data are mean ± SEM of n = 4 independent experiments for both OCR and ECAR. Higher OCR and ECAR readings were recorded as cell bodies were still present in these cultures

Fig. 4

Rescue of basal glycolysis rather than basal mitochondrial respiration may be part of the protective phenotype to delay Wallerian degeneration. OCRs in pmol/min (a) and ECARs in pmH/min (b) of six DIV mouse wild-type neurites (circle) and Sarm1 ^−/− neurites (square) measured late after cut. Recordings took place under basal, oligomycin-inhibited (oligomy), FCCP-induced maximal and rotenone + antimycin A-inhibited (rot/antimy) conditions. *p < 0.05, **p < 0.01 two-way repeated measures ANOVA. Data are mean ± SEM of n = 4 independent experiments for both OCR and ECAR