m6A demethylase ALKBH5 drives denervation‐induced muscle atrophy by targeting HDAC4 to activate FoxO3 signalling

Skeletal muscle atrophy is a common clinical manifestation of various neurotrauma and neurological diseases. In addition to the treatment of primary neuropathies, it is a clinical condition that should be investigated. FoxO3 activation is an indispensable mechanism in denervation-induced muscle atrophy; however, upstream factors that control FoxO3 expression and activity have not been fully elucidated. N6 -methyladenosine (m6 A) methylation is a novel mode of epitranscriptional gene regulation that affects several cellular processes. However, the biological significance of m6 A modification in FoxO3-dependent atrophy is unknown.We performed gain-of-function and loss-of-function experiments and used denervation-induced muscle atrophy mouse model to evaluate the effects of m6 A modification on muscle mass control and FoxO3 activation. m6 A-sequencing and mass spectrometry analyses were used to establish whether histone deacetylase 4 (HDAC4) is a mediator of m6 A demethylase ALKBH5 regulation of FoxO3. A series of cellular and molecular biological experiments (western blot, immunoprecipitation, half-life assay, m6 A-MeRIP-qPCR, and luciferase reporter assays among others) were performed to investigate regulatory relationships among ALKBH5, HDAC4, and FoxO3.In skeletal muscles, denervation was associated with a 20.7-31.9% decrease in m6 A levels (P < 0.01) and a 35.6-115.2% increase in demethylase ALKBH5 protein levels (P < 0.05). Overexpressed ALKBH5 reduced m6 A levels, activated FoxO3 signalling, and induced excess loss in muscle wet weight (-10.3% for innervation and -11.4% for denervation, P < 0.05) as well as a decrease in myofibre cross-sectional areas (-35.8% for innervation and -33.3% for denervation, P < 0.05) during innervation and denervation. Specific deletion of Alkbh5 in the skeletal muscles prevented FoxO3 activation and protected mice from denervation-induced muscle atrophy, as evidenced by increased muscle mass (+16.0%, P < 0.05), size (+50.0%, P < 0.05) and MyHC expression (+32.6%, P < 0.05). Mechanistically, HDAC4 was established to be a crucial central mediator for ALKBH5 in enhancing FoxO3 signalling in denervated muscles. ALKBH5 demethylates and stabilizes Hdac4 mRNA. HDAC4 interacts with and deacetylates FoxO3, resulting in a significant increase in FoxO3 expression (+61.3-82.5%, P < 0.01) and activity (+51.6-122.0%, P < 0.001).Our findings elucidate on the roles and mechanisms of ALKBH5-mediated m6 A demethylation in the control of muscle mass during denervation and activation of FoxO3 signalling by targeting HDAC4. These results suggest that ALKBH5 is a potential therapeutic target for neurogenic muscle atrophy.