Nestin Regulates Autophagy-Dependent Ferroptosis Mediated Skeletal Muscle Atrophy by Ubiquitinating MAP 1LC3B.

BACKGROUND: Programmed cell death plays a critical role in skeletal muscle atrophy. Ferroptosis, an iron-dependent form of programmed cell death driven by lipid peroxidation, has been implicated in various diseases, but its role in skeletal muscle atrophy remains unclear. METHODS: Ferroptosis in skeletal muscle atrophy was investigated using two models: dexamethasone (Dex)-induced atrophy (n = 6 independent cell cultures per group) and simulated microgravity (n = 6 mice per group). Conditional Nestin knockout (KO) mice were generated using CRISPR/Cas9 (n = 6-8 mice per group), with wild-type (WT) controls (n = 6-8). Phenotypic analyses included histopathology (HE staining), functional assessments (muscle strength, weight analysis, treadmill), and dystrophy evaluation (dystrophin staining). Molecular analyses involved flow cytometry, ELISA, transmission electron microscopy, PI staining, and IP/MS to delineate Nestin-regulated ferroptosis pathways in skeletal muscle atrophy. RESULTS: Ferroptosis was significantly activated in both atrophy models, with a 2.5-fold increase in lipid peroxidation (p < 0.01), a 2-fold accumulation of Fe(2+) (p < 0.01) and a 50% reduction in Nestin expression (p < 0.001). Nestin KO mice exhibited exacerbated muscle atrophy, showing a 40% decrease in muscle weight (p < 0.01) and a 30% reduction in muscle strength (p < 0.05) compared to WT mice. Nestin overexpression mitigated Dex-induced ferroptosis, reducing lipid peroxidation by 40%, decreasing Fe(2+) accumulation by 50% (p < 0.01), and improving muscle function by 30% (p < 0.05). Mechanistically, Nestin interacted with MAP 1LC3B (LC3B) to catalyse LC3B polyubiquitination at lysine-51, reducing LC3B availability for autophagy and inhibiting autophagy flux by 60% (p < 0.01), leading to a 50% reduction in ferroptosis (p < 0.001). CONCLUSIONS: Our study identifies Nestin as a critical regulator of ferroptosis-autophagy crosstalk in skeletal muscle atrophy. Targeting Nestin-LC3B ubiquitination may offer novel therapeutic strategies for preventing muscle wasting in diseases such as cachexia and sarcopenia.