[DOT1L controls neuronal amyloid precursor protein expres-sion via the p38 MAPK-mediated mitochondrial dynamics...

To investigate the regulatory role of epigenetic regulator disruptor of telomeric silencing 1-like (DOT1L) and its mediated histone H3 lysine 79 (H3K79) methylation in modulating neuronal amyloid precursor protein (APP) expression, and to elucidate the underlying mechanisms involving mitochondrial dynamics homeo-stasis and the upstream p38 mitogen-activated protein kinase (p38 MAPK).

Alzheimer's disease (AD) models were established using APP/presenilin-1 (APP/PS1) double-transgenic mice and N2a cells overexpressing the human Swedish mutant APP (N2a-APPswe). Immunofluorescence staining was employed to assess DOT1L expression and localization in mouse brain tissues. N2a-APPswe cells were treated with the DOT1L-specific inhibitor EPZ5676 and divided into four groups: blank control, solvent control, DOT1L inhibitor, and DOT1L inhibitor plus p38 agonist (Gynostemma pentaphyllum extract). Western blotting was performed to measure the phosphorylation levels of DRP1 at Ser616 and Ser637, the levels of autophagy-related proteins p62 and the LC3-Ⅱ/LC3-Ⅰ ratio, the phosphorylation level of p38 MAPK, as well as the expression of APP and APP-processing proteins BACE1 and PS1. Reverse transcription quantitative polymerase chain reaction was used to detect mRNA levels of APP and genes involved in mitochondrial fission and fusion. Proteomics data were systematically analyzed through Gene Ontology analysis, WikiPathways enrichment analysis, and STRING protein-protein interaction network analysis to identify key signaling pathways. Mitochondrial network morphology was evaluated by Mito-Tracker fluorescence staining to measure average branch length.

DOT1L expression was significantly reduced in neurons of APP/PS1 mice compared to wild-type controls. DOT1L inhibition led to decreased H3K79 dimethylation levels (P<0.01), accompanied by a marked increase in APP protein expression (P<0.01), although APP mRNA levels were reduced (P<0.01). Proteomics analysis revealed that differentially expressed proteins were highly enriched in the mitochondrial electron transport chain. Compared with the solvent control, the DOT1L inhibitor group showed inhibited mitochondrial fission, as evidenced by decreased p-DRP1 (Ser616), increased p-DRP1 (Ser637), downregulated MIEF1 mRNA, upregu-lated MFN1 mRNA (all P<0.05), and increased average mitochondrial branch length (P<0.05), along with reduced phosphorylation level of p38 MAPK (P<0.05). Co-administration of the p38 agonist significantly reversed these mitochondrial dynamics abnormalities (all P<0.05) and attenuated the abnormally elevated protein levels of APP, BACE1, and PS1 (all P<0.05) compared to the DOT1L inhibitor group.

DOT1L maintains normal mitochondrial fission and functional homeostasis through regulation of the p38 MAPK mediated signaling pathway, thereby modulating APP expression.