P300-Mediated ARRB1 Lactylation Promotes Mitochondrial Dysfunction and Neuronal Apoptosis in Subarachnoid Hemorrhage Via Upregulating S100A9.

Background: Lactylation, a novel lactate-derived posttranslational modification, has been demonstrated to be linked with brain function. The present research is intended to explore the role of β-arrestin1 (ARRB1) lactylation post-subarachnoid hemorrhage (SAH).

Methods: SAH models were established in mice via intravascular puncture and in primary neurons by oxyhemoglobin (oxyHb) stimulation. Lactylome analysis identified differentially lactylated proteins. Commercial kits measured lactate, mitochondrial membrane potential, reactive oxygen species (ROS), and ATP. Mitochondrial respiration was evaluated by detecting mitochondrial oxygen consumption rate. Cell viability and apoptosis were respectively determined by CCK-8 assay and flow cytometry/TUNEL assay. Protein interactions were assessed using co-immunoprecipitation and double-label immunofluorescence.

Results: Elevated lactate and ARRB1 lactylation were observed in the brain of SAH mice. In primary neurons, reducing lactate with oxamate reversed mitochondrial dysfunction and apoptosis induced by oxyHb. Overexpression of ARRB1 exacerbated oxyHb-induced neuronal injury, yet this effect was absent with the ARRB1-lysine (K) 195 arginine (R) mutant. E1A binding protein P300 (P300) promoted ARRB1 lactylation to upregulate its protein expression. P300 knockdown inhibited oxyHb-induced neuronal injury, but this inhibitory effect was counteracted by ARRB1 overexpression. In oxyHb-stimulated neurons, ARRB1 lactylation upregulated S100 calcium binding protein A9 (S100A9) protein. Additionally, ARRB1 knockdown prevented mitochondrial respiratory dysfunction in neurons induced by oxyHb, which was antagonized by recombinant S100A9. ARRB1 silencing mitigated SAH injury in mice via suppressing S100A9-mediated mitochondrial dysfunction.

Conclusion: P300 mediated ARRB1 lactylation, thereby increasing S100A9 to facilitate mitochondrial dysfunction and neuronal apoptosis in SAH. This study may provide prospective targets for improving SAH.