OBJECTIONS: Nestin,a class Ⅵ intermediate filament (IF) protein,is widely accepted as the classical neural stem/progenitor cells (NSPCs) marker.Our previous study has demonstrated Nestin could influence the mitochondrial dynamics and reactive oxygen species (ROS) generation in gastrointestinal stromal tumor (GIST) cells,but it is unknown whether Nestin plays important roles in maintaining the NSPCs stemness by regulating the mitochondrial metabolism.As a result,we investigated the effects of Nestin on NSPCs self-renewal and mitochondrial regulation.METHODS: The effects of Nestin knockdown in NSPCs compared with that in non-transfected control (NTC) cells were determined by clone-forming assays,Ki67 immunostaining,as well as measuring the differentiation tendency.Mitochondrial morphology alterations were detected in NSPCs treated with non-directed differentiation medium or Nestin RNA interference (RNAi),respectively,by using immunofluorescence.Metabolic changes were reflected by oxygen consume ratio (OCR),lactate production,glucose uptake and ATP production tests.Immunofluorescence and co-immunoprecipitation assays were used to study the interactions of Nestin with mitochondria and mitochondrial fission mediator Dynamin-related protein 1 (Drp1).RESULTS: Nestin knockdown triggers the mitochondrial network remodeling and enhances oxidative phosphorylation (OXPHOS) in NSPCs,with repressing their self-renewal and enhancing their tendency toward differentiation.Mitochondrial morphology is dynamically controlled by the balance between fission and fusion mediators;one of these mediators,the pro-fission factor,Drp1,showed decreased activation in Nestin-knockdown cells.Transfection of a gain-of-function pDrp1S592E in Nestin knockdown NSPCs could induce the mitochondria fragmentation and enhance self-renewal.CONCLUSIONS: Together,our results identify Nestin as a novel metabolic regulator in NSPCs,whose impact on Drp1 activation is crucial for NSPCs stemness maintenance.