A Class I HDAC Inhibitor Rescues Synaptic Damage and Neuron Loss in APP-Transfected Cells and APP/PS1 Mice through the GRIP1/AMPA Pathway

Abstract

As a neurodegenerative disease, Alzheimer's disease (AD) seriously affects the health of older people. Changes in synapses occur first over the course of the disease, perhaps even before the formation of Aβ plaques. Histone deacetylase (HDAC) mediates the damage of Aβ oligomers to dendritic spines. Therefore, we examined the relationship between HDAC activity and synaptic defects using an HDAC inhibitor (HDACI), BG45, in the human neuroblastoma SH-SY5Y cell line with stable overexpression of Swedish mutant APP (APPsw) and in APP/PS1 transgenic mice during this study. The cells were treated with 15 μM BG45 and the APP/PS1 mice were treated with 30 mg/kg BG45. We detected the levels of synapse-related proteins, HDACs, tau phosphorylation, and amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors using Western blotting and immunohistochemistry. We also measured the expression of cytoskeletal proteins in the cell model. The mRNA levels of the glutamate ion receptor alginate subunit 2 (GRIK2), sodium voltage-gated channel beta subunit (SCN3B), synaptophysin (SYP), Grm2 (the gene encoding glutamate receptor subunit 2 (GluR2)), Grid2IP, glutamate receptor interacting protein 1 (GRIP1), and GRIP2 were detected to explore the effects of the HDACI on regulating the expression of synaptic proteins and AMPA receptors. According to our studies, the expressions of HDAC1, HDAC2, and HDAC3 were increased, which were accompanied by the downregulation of the synapse-related proteins SYP, postsynaptic dendritic protein (PSD-95), and spinophilin as early as 24 h after transfection with the APPsw gene. BG45 upregulated the expression of synapse-related proteins and repaired cytoskeletal damage. In vivo, BG45 alleviated the apoptosis-mediated loss of hippocampal neurons, upregulated synapse-related proteins, reduced Aβ deposition and phosphorylation of tau, and increased the levels of the synapse-related genes GRIK2, SCN3B, SYP, Grm2, and Grid2IP. BG45 increased the expression of the AMPA receptor subunits GluA1, GluA2, and GluA3 on APPsw-transfected cells and increased GRIP1 and GRIP2 expression and AMPA receptor phosphorylation in vivo. Based on these results, HDACs are involved in the early process of synaptic defects in AD models, and BG45 may rescue synaptic damage and the loss of hippocampal neurons by specifically inhibiting HDAC1, HDAC2, and HDAC3, thereby modulating AMPA receptor transduction, increasing synapse-related gene expression, and finally enhancing the function of excitatory synapses. BG45 may be considered a potential drug for the treatment of early AD in further studies.

Keywords: AMPA receptor; Alzheimer’s disease; HDAC inhibitor; synapse; β-amyloid.

Figure 1

SH-SY5Y cells transfected with the APPsw gene were established as an AD cell model. (A) The transfection efficiency was observed in SH-SY5Y cells. (B) The expression of the APPsw gene was detected in different SH-SY5Y cell clones using RT-PCR. (C) Immunoblot analysis of APP levels in GFP- and APPsw-positive cells. Quantification of APP levels normalized to β-actin levels, and the results are presented as a % of the control and showed significant differences between SH-SY5Y cells and APPsw-positive cells (p < 0.01). (D) Immunoblot analysis of tau and p-tau levels in GFP- and APPsw-positive cells. Quantification of p-tau levels normalized to tau levels, and the results are presented as a % of control and showed significant differences between SH-SY5Y cells and APPsw-positive cells (p < 0.05). (E,G) Immunofluorescence staining for APP in GFP- and APPsw-positive cells (p < 0.05). (F,H) Immunofluorescence staining for Aβ in GFP- and APP-positive cells (p < 0.05). Scale bar = 50 μm (A,E,F). Significant differences were determined using Student’s t-test. All values are presented as the means ± SD from three experiments. * p < 0.05, ** p < 0.01.

Figure 2

PSD-95 expression in APPsw-positive cells at different time points. (A) Immunoblot analysis of PSD-95 levels in SH-SY5Y cells at 60 h and in APPsw-positive cells at different time points (24 h, 36 h, 48 h, and 60 h). (B) Quantification of PSD-95 levels normalized to β-actin levels, and the results are presented as a % of the control and showed significant differences between SH-SY5Y cells and APPsw-positive cells at 36 h (p < 0.001). Significant differences were determined using repeated measures ANOVA. All values are presented as the means ± SD from three independent experiments. * p < 0.05, *** p < 0.001.

Figure 3

The effects of BG45 on Class I HDACs (HDAC1, 2, and 3). (A) The effect of BG45 on GFP-positive and APPsw-positive cell viability. (B) Immunoblot analysis of HDAC1, 2, and 3 levels in GFP- and APPsw-positive cells treated with vehicle or BG45 (15 μM) for different periods. (C) Quantification of HDAC1 levels. (D) Quantification of HDAC2 levels. (E) Quantification of HDAC3 levels. Significant differences were determined using Student’s t-test. All values are presented as the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01.

Figure 4

Effects of BG45 on the levels of APP. (A) Immunoblot analysis of APP levels in GFP- and APPsw-positive cells treated with vehicle or BG45 (15 μM). (B) Quantification of APP levels. Significant differences were determined using Student’s t-test. All values are presented as the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01.

Figure 5

The effects of BG45 on the levels of synapse-related proteins and F-actin. (A) Immunoblot analysis of SYP, PSD-95, and spinophilin levels in GFP- and APPsw-positive cells treated with vehicle or BG45 (15 μM) for different periods (24 h, 36 h, and 48 h). (B) Quantification of SYP levels. (C) Quantification of PSD-95 levels. (D) Quantification of spinophilin levels. (E) Immunofluorescence staining for spinophilin in GFP- and APPsw-positive cells treated with vehicle or BG45 (15 μM) for 36 h. (F) Immunofluorescence staining for phalloidin/F-actin in GFP- and APPsw-positive cells treated with vehicle or BG45 (15 μM) for 36 h. (G) Quantification of spinophilin levels. (H) Quantification of F-actin levels. Scale bar = 50 μm (E,F). Significant differences were determined using Student’s t-test. All values are presented as the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 6

BG45 increased the cell surface levels of AMPA receptor subunits. (A) Immunostaining for cell surface GluA1, GluA2, and GluA3 receptors on GFP- and APPsw-positive cells treated with vehicle or BG45 (15 μM). The lower images show the positive cells magnified in the white box. (B) Quantification of the cell surface GluA1 levels. (C) Quantification of the cell surface GluA2 levels. (D) Quantification of the cell surface GluA3 levels. Scale bar = 50 μm (A). Significant differences were determined using Student’s t-test. All values are presented as the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01.

Figure 7

Protective effect of BG45 on hippocampal neurons in APP/PS1 transgenic mice (n = 5). (A) Immunofluorescence staining for the caspase-3 and MAP2 proteins in hippocampal neurons from animals treated with vehicle or BG45. (B) Quantification of the caspase-3 protein levels. (C) Quantification of the MAP2 protein levels. (D) Percentage of MAP2+ cells. Scale bar = 50 μm (A). Significant differences were determined using one-way ANOVA. All values are presented as the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001, n = 5 mice per group.

Figure 8

BG45 decreased Aβ deposition in hippocampal neurons and the level of the phosphorylated tau protein in APP/PS1 transgenic mice (n = 5). (A) Immunohistochemistry showing Aβ deposition in hippocampal neurons from mice treated with vehicle or BG45. The lower right corner of the image shows a magnified image that clearly depicts Aβ deposition. (B) Quantification of Aβ deposition. (C) Western blot showing p-tau/tau levels in hippocampal neurons from mice treated with vehicle or BG45. (D) Quantification of the level of the phosphorylated tau protein. Scale bar = 100 μm (A). Significant differences were determined using one-way ANOVA. All values are presented as the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01, n = 5 mice per group.

Figure 9

BG45 reduced HDAC1 and HDAC2 protein expression in hippocampal neurons from APP/PS1 mice (n = 5). (A) Immunoblot analysis of levels of the HDAC1 and HDAC2 proteins in the hippocampus of mice treated with vehicle or BG45. (B) Quantification of the HDAC1 levels. (C) Quantification of the HDAC2 levels. Significant differences were determined using one-way ANOVA. All values are presented as the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01, n = 5 mice per group.

Figure 10

BG45 upregulates the expression of synapse-related proteins in hippocampal neurons of APP/PS1 mice (n = 5). (A) Immunohistochemical staining for the spinophilin protein in hippocampal neurons from mice treated with vehicle or BG45. (B) Immunoblot analyses showing levels of the spinophilin, PSD-95, and synaptophysin proteins in the hippocampus of mice treated with vehicle or BG45. (C) Quantification of spinophilin levels in the hippocampus. (D–F) Quantification of levels of synapse-related proteins. Scale bar = 100 μm (A). Significant differences were determined using one-way ANOVA. All values are presented as the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001, n = 5 mice per group.

Figure 11

BG45 increased the levels of synapse-related genes (GRIK2, SCN3B, SYP, Grm2, and Grid2IP) in APP/PS1 mice (n = 5). (A–E). GRIK2, SCN3B, SYP, Grm2, and Grid2IP mRNA expression levels. Significant differences were determined using one-way ANOVA. All values are presented as the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001, n = 5 mice per group.

Figure 12

Effects of BG45 on the expression levels of glutamate receptor proteins and related genes. (A,B). GRIP1 and GRIP2 mRNA expression levels. (C) Immunoblot analysis of p-GluR2 (s880) and Glu2/3/4 levels in the hippocampus of mice treated with vehicle or BG45. (D) Quantification of p-GluR2 levels in the hippocampus. Significant differences were determined using one-way ANOVA. All values are presented as the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001, n = 5 mice per group.