H3K27 acetylation-induced FSTL1 upregulation by P300/RUNX1 co-activation exacerbated autophagy-mediated neuronal damage and NF-κB-stimulated inflammation in Alzheimer's disease

Abstract

Follistatin-like protein 1 (FSTL1) has been demonstrated to participate in the pathogenesis of several neurological diseases. The current study informed the role of H3K27 acetylation-induced FSTL1 upregulation in Alzheimer's disease (AD). Our investigation discovered the upregulated FSTL1 expression and enhanced autophagy activity in AD. FSTL1 knockdown successfully attenuated the injuries of Aβ_1-42-challenged SH-SY5Y cells through the inhibition of autophagy activity. Besides, FSTL1 deficiency suppresses the inflammatory response and NF-κB signaling in AD. Moreover, it was found that p300 was recruited by transcriptional factor RUNX1 to stimulate the H3K27 acetylation in FSTL1 promoter region, which caused the upregulation of FSTL1 in AD. To summarize, p300 acted as a co-activator of RUNX1 to trigger the activation of FSTL1 in AD, resulting in the exacerbated injuries and inflammatory responses of Aβ_1-42-induced SH-SY5Y cells.

Keywords: Alzheimer’s disease; Autophagy; FSTL1; H3K27 acetylation; Inflammation.

Fig. 1

FSTL1 expression and autophagy activity were increased in AD. A The mRNA expression of FSTL1 in serum samples were detected by RT-qPCR (Student’s t-test; **P < 0.01). B, C The mRNA and protein expression of FSTL1 were detected in Aβ₁₋₄₂-treated and untreated SH-SY5Y cells (Student’s t-test; **P < 0.01). D Protein expression of LC3 and P62 were detected in untreated (Control), Aβ₁₋₄₂-treated, or Aβ₁₋₄₂ + Chloroquine co-treated SH-SY5Y cells (One-way ANOVA; *P < 0.05, **P < 0.01)

Fig. 2

Effect of FSTL1 on Aβ₁₋₄₂-induced injuries in SH-SY5Y cells. Aβ₁₋₄₂-challenged SH-SY5Y cells were transfected with shNC or shFSTL1. A The mRNA level expression of FSTL1 by RT-qPCR (Student’s t-test; ***P < 0.001). B The protein expression of FSTL1 was detected by western blot (Student’s t-test; **P < 0.01). C Cell proliferation was observed via CCK-8 assay (One-way ANOVA; *P < 0.05, **P < 0.01). D Cell apoptosis was evaluated using TUNEL assay (One-way ANOVA; *P < 0.05, **P < 0.01). E Protein expression of cleaved caspase-3, and Bcl-2 was detected (One-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001). F LDH release was detected by a commercial LDH kit (One-way ANOVA; **P < 0.01, ***P < 0.001). G Intracellular ROS production was analyzed using flow cytometry (One-way ANOVA; *P < 0.05, **P < 0.01)

Fig. 3

FSTL1 induced autophagy to exacerbate Aβ₁₋₄₂-induced injuries. Aβ₁₋₄₂-challenged SH-SY5Y cells were treated with shNC, shFSTL1, or shFSTL1 + rapamycin. A The protein expression of LC3, Beclin-1, and P62 was detected. B–E The proliferation (B), apoptosis (C), LDH release (D), and ROS production (E) of treated cells were evaluated. (One-way ANOVA; *P < 0.05, **P < 0.01)

Fig. 4

FSTL1 activated the NF-κB signaling to promote the secretion of pro-inflammatory cytokines. SH-SY5Y cells divided into three groups: untreated control, Aβ₁₋₄₂, and Aβ₁₋₄₂ + shFSTL1. A ELISA assay detected the expression of TNF-α, IL-1β, and IL-6 in the cell supernatants B Western blot detected the expression of IκBα and p-IκBα with GAPDH used as control. C Cytoplasmic and nuclear NF-κB p65 were analyzed by western blot. (One-way ANOVA; *P < 0.05, **P < 0.01)

Fig. 5

p300 induces an upregulation of FSTL1 by modulating H3K27 acetylation. A UCSC database predicted the H3K27ac enrichment in the promoter region of FSTL1. B Western blot detected the protein level of FSTL1 in Aβ₁₋₄₂-challenged SH-SY5Y cells treated or untreated with Nabu (Student’s t-test; **P < 0.01). C ChIP assay assessed the H3K27ac enrichment in Aβ₁₋₄₂-challenged SH-SY5Y cells treated or untreated with Nabu (One-way ANOVA; ***P < 0.001). D H3K27ac enrichment in SH-SY5Y cells treated or untreated with Aβ₁₋₄₂ (One-way ANOVA; ***P < 0.001). E The mRNA and protein expression of FSTL1 in Aβ₁₋₄₂-challenged SH-SY5Y cells treated or untreated with A485 (Student’s t-test; **P < 0.01, ***P < 0.001). F The mRNA and protein expression of FSTL1 in Aβ₁₋₄₂-challenged SH-SY5Y cells treated or untreated with p300 overexpression plasmid (Student’s t-test; **P < 0.01). G, H Aβ₁₋₄₂-challenged SH-SY5Y cells were grouped into control and A485 (G) or control and pcDNA3.1/p300 (H) to detect H3K27ac enrichment in the promoter region of FSTL1 (Student’s t-test; **P < 0.01)

Fig. 6

RUNX1 recruited p300 to co-activate the transcription of FSTL1 in AD. A, B The protein level and transcription activation of FSTL1 were determined in Aβ₁₋₄₂-challenged SH-SY5Y cells transfected with pcDNA3.1/RUNX1 using western blot and luciferase reporter assay (Student’s t-test; **P < 0.01, ***P < 0.001). C Aβ₁₋₄₂-challenged SH-SY5Y cells were divided into four groups: control, oe-RUNX1, oe-p300, and oe-RUNX1 + oe-p300. Western blot detected the protein level of FSTL1 (One-way ANOVA; ***P < 0.001)