N⁶-Methyladenosine-Driven Nuclear Export of circKCNN2 Impairs GRP75 Function to Promote Neuronal Apoptosis

Emerging evidence implicates circular RNAs (circRNAs) in Alzheimer's disease (AD) pathogenesis. Notably, circKCNN2 correlates with clinical dementia severity in AD patients; however, its biological functions and regulatory mechanisms in neuronal systems remain poorly understood. Here, we elucidated the molecular mechanism by which circKCNN2 regulated neuronal apoptosis and delineated the underlying post-transcriptional regulatory axis. Using SH-SY5Y cells as an in vitro neuronal model, we manipulated circKCNN2 expression through overexpression and siRNA-mediated knockdown. Apoptosis was assessed by flow cytometry and TUNEL assays. Methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), MS2-based RNA pull-down coupled with mass spectrometry, fluorescence in situ hybridization, and nuclear/cytoplasmic fractionation were employed to characterize the underlying mechanism. circKCNN2 overexpression significantly induced apoptosis, whereas its knockdown attenuated apoptosis. circKCNN2 underwent N6-methyladenosine (m⁶A) modification. Mechanistically, METTL3-catalyzed m⁶A modification enabled YTHDC2 binding and facilitated the nuclear export of circKCNN2. Both METTL3 depletion and YTHDC2 overexpression caused nuclear retention and attenuated apoptosis. Furthermore, MS2-based RNA pull-down coupled with mass spectrometry identified GRP75 as a cytoplasmic interactor of circKCNN2. Functionally, GRP75 overexpression rescued circKCNN2-induced apoptosis. Our findings revealed a novel "modification-localization-function" cascade wherein the METTL3-YTHDC2-m⁶A axis dictates circKCNN2 nuclear-cytoplasmic trafficking and the subsequent inhibition of GRP75, thereby driving neuronal apoptosis. This mechanism established circKCNN2 as a pathogenic driver in AD and highlighted the m⁶A regulatory machinery as a potential target for further investigation.