抄録
We previously found that diosgenin, a constituent of Dioscorea Rhizoma, restored Aβ-induced axonal degeneration and improved memory function in a mouse model of Alzheimer's disease (AD), 5XFAD. In this study, we aimed to investigate the downstream signaling of diosgenin, which are involved in axonal regrowth and memory recovery.
Vehicle solution or diosgenin (0.1 µmol/kg/day, p.o.) was administered to wild-type or 5XFAD mice (male, 24-27 weeks old) for 15 days. The diosgenin-administered 5XFAD mice showed significant improvement in object recognition memory. After the behavioral test, protein expressions in cortical lysates were compared on 2D-PAGE. We focused several proteins showing drastic changes in the expression level and analyzed those by MALDI-TOF/MS. Heat shock cognate 70 (HSC70) was identified as the protein reduced by diosgenin administration in 5XFAD. Next, cultured cortical neurons (ddY, E14) were treated by Aβ25-35. Aβ25-35 treatment for 3 days increased the expression level of HSC70 and decreased the axonal density. Post treatment by diosgenin (0.1 and 1 µM) significantly decreased HSC70 expression and increased density of axons. Next, we clarified the relationships between HSC70 and axonal degeneration. HSC70 binds to other proteins (client proteins) to work as a molecular chaperone; therefore, we explored associated molecules with HSC70 in neurons. By co-immunoprecipitation and nano-LC/MS analysis, α-tubulin was identified as an associated molecule with HSC70. After Aβ25-35 treatment, α-tubulin expression was greatly reduced in the degenerated axons. However, diosgenin treatment significantly increased the α-tubulin level and promoted axonal regrowth. Therefore, we speculated that HSC70-induced degradation of α-tubulin may be suppressed by diosgenin. A specific inhibitor of HSC70, VER-155008 (0.05, 0.5 and 5 µM) significantly restored Aβ-induced axonal atrophy in neurons. VER-155008 administration (10 µmol/kg/day, i.p.) significantly improved memory deficits in the 5XFAD mice.
Our study suggests that Aβ25-35-induced increase in HSC70 may promote degradation of α-tubulin, which results in axonal degeneration. Besides, diosgenin-elicited reduction of HSC70 may suppress the degradation of α-tubulin, and promote axonal regrowth. We also showed that specific inhibition of HSC70 restored axonal degeneration and improved memory function in 5XFAD mice. This study indicates that inhibiting HSC70 function is a new therapeutic target in AD.
