Mitochondria‐Localized Nestin Protects Mesenchymal Stem Cells from Senescence by Maintaining Cristae Structure and Function

Abstract Nestin, a well‐characterized intermediate filament protein expressed in stem cells, is increasingly recognized for its non‐canonical roles in diverse subcellular compartments. Here, a novel mitochondrial localization of Nestin in human mesenchymal stem cells (hMSCs) is identified, where it functions as a critical protector against mitochondrial dysfunction and cellular senescence. It is demonstrated that Nestin is imported into the mitochondrial intermembrane space via its N‐terminal mitochondrial targeting sequence through Translocase of the Outer Mitochondrial Membrane 20 (TOM20)‐dependent machinery. Within mitochondria, Nestin directly interacts with Mic60 to maintain cristae architecture and sustain oxidative phosphorylation. Genetic ablation of mitochondrial Nestin triggers cristae disorganization, respiratory deficiency, and premature senescence in hMSCs. Strikingly, targeted restoration of the Mic60‐binding Tail3 domain of Nestin is sufficient to rescue cristae morphology, mitochondrial function, and senescence phenotypes. These findings establish a non‐filamentous role for Nestin in mitochondrial quality control and propose a new therapeutic strategy for age‐related disorders through modulation of mitochondrial Nestin‐Mic60 interactions.