ACSS2 phosphorylation at Ser659 attenuates neuronal apoptosis induced by cerebral ischemia-reperfusion by initiating autophagy

Acetyl-CoA synthetase 2 (ACSS2), as a key enzyme regulating the synthesis of acetyl-CoA, has been proven to be involved in various metabolic and epigenetic regulatory processes. However, the specific mechanisms by which ACSS2 functions in cerebral ischemia-reperfusion (CI/R) injury have not yet been fully elucidated. The middle cerebral artery occlusion (MCAO) model was established in C57BL/6 mice, and an in vitro model was generated using oxygen-glucose deprivation/reoxygenation (OGD/R).

The dynamic expression of ACSS2 mRNA and protein was assessed by RT-qPCR and Western blot, respectively. The subcellular localization of ACSS2 was analyzed by immunofluorescence staining and Western blot. TTC staining and TUNEL/NeuN double immunofluorescence were applied to assess cerebral infarct size and neuronal apoptosis, respectively.

To investigate the biological functions of ACSS2 and its Ser659 phosphorylation, we adopted pharmacological interventions with the ACSS2 inhibitor AD-8007 and the AMPK inhibitor Compound C, as well as the overexpression of wild-type ACSS2 (ACSS2-WT) and its Ser659 phosphorylation-deficient mutant (ACSS2-Mut). In the MCAO model, CI/R injury markedly upregulated ACSS2 expression and induced robust phosphorylation of ACSS2 at the Ser659 site. Subcellular localization analysis revealed that ischemic stress drove prominent nuclear translocation of total ACSS2 and p-ACSS2.

Pharmacological suppression of ACSS2 activity by AD-8007, or blockade of Ser659 phosphorylation by Compound C, obviously restrained I/R-triggered autophagy, accompanied by impaired LC3-II conversion and inhibited p62 degradation. This suppression further aggravated neuronal apoptosis and enlarged cerebral infarct volume. Consistent with in vivo findings, in vitro neuronal OGD/R challenge rapidly elevated ACSS2 Ser659 phosphorylation and nuclear translocation, and enhanced nuclear acetyl-CoA production, which subsequently increased H3K27 acetylation at the LC3B promoter.

Functional validation demonstrated that ACSS2-WT effectively activated autophagy and alleviated neuronal apoptosis under OGD/R, while the ACSS2-Mut lost such protective capacity. Moreover, Compound C administration abolished OGD/R-elicited ACSS2 phosphorylation and nuclear translocation, thereby repressing autophagic activation and worsening neuronal damage. ACSS2 confers neuroprotection against cerebral ischemia via Ser659 phosphorylation and subsequent nuclear translocation, which further activates autophagy-related gene expression.

In contrast, blockade of ACSS2 activity or its phosphorylation compromises protective autophagy and exacerbates ischemic brain damage. ACSS2 Ser659 phosphorylation is therefore essential for modulating autophagic activity in ischemic neurons, and targeting ACSS2 may provide a promising therapeutic approach for ischemic brain injury. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.