Echinacoside Suppresses Amyloidogenesis and Modulates F-actin Remodeling by Targeting the ER Stress Sensor PERK in a Mouse Model of Alzheimer's Disease

Abstract

Endoplasmic reticulum stress (ERS) plays a vital and pathogenic role in the onset and progression of Alzheimer's disease (AD). Phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) induced by ERS depresses the interaction between actin-binding protein filamin-A (FLNA) and PERK, which promotes F-actin accumulation and reduces ER-plasma membrane (PM) communication. Echinacoside (ECH), a pharmacologically active component purified from Cistanche tubulosa, exhibits multiple neuroprotective activities, but the effects of ECH on ERS and F-actin remodeling remain elusive. Here, we found ECH could inhibit the phosphorylation of PERK. Firstly ECH can promote PERK-FLNA combination and modulate F-actin remodeling. Secondly, ECH dramatically decreased cerebral Aβ production and accumulation by inhibiting the translation of BACE1, and significantly ameliorated memory impairment in 2 × Tg-AD mice. Furthermore, ECH exhibited high affinity to either mouse PERK or human PERK. These findings provide novel insights into the neuroprotective actions of ECH against AD, indicating that ECH is a potential therapeutic agent for halting and preventing the progression of AD.

Keywords: Alzheimer’s disease; PERK; amyloid β; eIF2α; echinacoside; endoplasmic reticulum stress; f-actin; filamin-A.

FIGURE 1

Chemical structure of Echinacoside (ECH).

FIGURE 2

ECH treatment improves spatial learning and memory in 10-month-old 2 × Tg-AD mice in the Morris water maze test. (A) In the place navigation test, the vehicle-treated 2 × Tg-AD mice showed significantly longer escape latencies compared with vehicle-treated Non-Tg mice (^##P < 0.01, ^###P < 0.001, vehicle-treated 2 × Tg-AD mice vs. vehicle-treated Non-Tg mice), and ECH treatment reduced escape latency in 2 × Tg-AD mice remarkably (*P < 0.05, **P < 0.01, and ***P < 0.001, ECH-treated 2 × Tg-AD mice vs. vehicle-treated 2 × Tg-AD mice). (B) No significant differences were detected in the average swimming speed among the 4 groups during the place navigation test (P = 0.516). During the spatial probe test, (C) the number of crossings of the area of the removed platform and (D) time spent in the target vs. opposite quadrant were recorded; vehicle-treated 2 × Tg-AD mice showed significantly inferior performance than ECH-treated 2 × Tg-AD mice and vehicle- and ECH-treated Non-Tg mice. (E) Representative images of the route of travel during the spatial probe test were recorded. No significant differences were detected in (F) the escape latency (P = 0.379) or (G) the average swimming speed (P = 0.725) among the 4 experimental groups during the visible-platform test. All data are presented as the mean ± SEM (n = 18 mice per group). Non-Tg, non-transgenic littermates; 2 × Tg-AD, APPswe/PS1dE9 mice; Veh, vehicle (normal saline); ECH, echinacoside; NS, no significant difference.

FIGURE 3

ECH reduces Aβ-positive plaque load and Aβ production in the hippocampus and cortex of 2 × Tg-AD mice. Mouse brain sections were stained with anti-Aβ antibody 6E10. (A) Images of Aβ immunoreactivity in hippocampus and cortex of the indicated groups of mice were photographed with an IX71 microscope. Scale bar = 200 μm. Aβ-positive plaque load in the (B) hippocampus and (C) cortex of mice were analyzed with Image-Pro Plus 6.0 software, and the plaque load was defined as the percentage of the sum of Aβ deposit areas compared with the total section area (Non-Tg Veh, n = 5; Non-Tg ECH, n = 6; 2 × Tg-AD Veh, n = 5; 2 × Tg-AD ECH, n = 5). The levels of (D) total Aβ, (E) Aβ_1–42, and (F) Aβ_1–40 in brain homogenates of 2 × Tg-AD mice administered vehicle or ECH were measured by enzyme-linked immunosorbent assay (ELISA), and the values are presented as nanogram per milligram of brain tissue. All data are presented as the mean ± SEM (n = 6 mice per group). Non-Tg, non-transgenic littermates; 2 × Tg-AD, APPswe/PS1dE9 mice; Veh, vehicle (normal saline); ECH, echinacoside; NS, no significant difference.

FIGURE 4

ECH downregulates the protein level and enzymatic activity of BACE1 in 2 × Tg-AD mice. (A) The protein level of BACE1 in brain homogenates of mice was assayed by Western blot. (B) BACE1 activity was fluorimetrically monitored as described under section “Materials and Methods” in the brain homogenates of the indicated groups of mice. (C) The effects of ECH on BACE1 gene transcription were evaluated by real-time RT-PCR. Effects of ECH on (D) Protein levels of the soluble APP β fragments (sAPPβ), (E) ubiquitination degradation of BACE1 and (F) full-length APP (flAPP) level were determined by Western blot. All data are presented as the mean ± SEM (n = 6 mice per group). Non-Tg, non-transgenic littermates; 2 × Tg-AD, APPswe/PS1dE9 mice; Veh, vehicle (normal saline); ECH, echinacoside; NS, no significant difference.

FIGURE 5

Effects of ECH on α- and γ-secretase in 2 × Tg-AD mice. The levels of (A) A-disintegrin and metalloproteinase 10 (ADAM10) and (B) presenilin 1 (PS1) from the brain of mice were determined by Western blot. The enzymatic activity of (C)α-secretase and (D)γ-secretase in the brains of the indicated groups of mice were fluorimetrically monitored as described under section “Materials and Methods.” All data are expressed as the percentage of vehicle-treated Non-Tg mice and are presented as the mean ± SEM (n = 6–8 mice per group). Non-Tg, non-transgenic littermates; 2 × Tg-AD, APPswe/PS1dE9 mice; Veh, vehicle (normal saline); ECH, echinacoside; NS, no significant difference.

FIGURE 6

ECH depresses ERS via PERK/eIF2α-mediated pathway in 2 × Tg-AD mice. (A) Hippocampal tissues were observed under the transmission electron microscope (TEM), and the morphological changes in neuronal ER are indicated with thick arrows. Scale bar = 500 nm. The levels of GRP78 (B), phosphorylated, and total eIF2α (C) and PERK (D) from brain homogenates of the indicated groups of mice were detected by Western blot. The relative levels of phosphorylated proteins were normalized to the total protein content and are expressed as the percentage of Non-Tg Veh mice. All data are presented as the mean ± SEM (n = 6–8 mice per group). Non-Tg, non-transgenic littermates; 2 × Tg-AD, APPswe/PS1dE9 mice; Veh, vehicle (normal saline); ECH, echinacoside; NS, no significant difference; N, nucleus.

FIGURE 7

ECH promotes the combination of PERK and FLNA and ER-PM contacts. (A) Aβ_1–42-treated SH-SY5Y cells were incubated with or without ECH. Immunofluorescence assay showed the colocalization of FLNA (red) and PERK (green), scale bar = 50 μm. (B) Mander’s overlap colocalization analysis of data represented in (A) (mean ± SEM; n = 3, 30 cells were analyzed per condition). (C) Representative electron micrographs of hippocampal tissues of Non-Tg mice and 2 × Tg-AD mice treated with or without ECH. Black arrows denote F-actin, and hollow arrow denoted ER-PM contact sites, scale bar = 1.0 μm. FLNA, filamin-A; Non-Tg, non-transgenic littermates; 2 × Tg-AD, APPswe/PS1dE9 mice; Veh, vehicle (normal saline); ECH, echinacoside; NS, no significant difference.

FIGURE 8

ECH modulates the ratio of F-actin and G-actin. (A) representative images of F-actin and G-actin in the hippocampus of Non-Tg mice and 2 × Tg-AD mice administrated with or without ECH. Immunofluorescence assay showed the colocalization of F-actin (red) and G-actin (green), scale bar = 50 μm (Upper three-row panel) and scale bar = 20 μm (Fourth-row panel). (B) Mander’s overlap colocalization analysis of data represented in (A). (mean ± SEM; n = 3, 35 cells were analyzed per condition). (C) Cerebral F-actin/G-actin ratio in Non-Tg mice and 2 × Tg-AD mice administrated with or without ECH. Non-Tg, non-transgenic littermates; 2 × Tg-AD, APPswe/PS1dE9 mice; Veh, vehicle (normal saline); ECH, echinacoside; NS, no significant difference.

FIGURE 9

MST measurement and binding mode of ECH and mPERK or hPERK. MST binding cure (n = 3) and MST curve (inset) of ECH binding to mPERK (A) or hPERK (B). (C) Two-dimensional and (D) three-dimensional binding model between mouse PERK (EIF2AK3, PDB ID: 3DQ2) and ECH. (E) Two-dimensional and (F) three-dimensional binding models between human PERK (EIF2AK3, PDB ID: 4X7J) and ECH. The black dotted lines represent the hydrogen bond. The blue sticks in the three-dimensional binding model (D,F) represent ECH.

FIGURE 10

Diagram illustrating the mechanism of ECH regulating F-actin remodeling and decreasing Aβ accumulation by depressing PERK phosphorylation. PERK phosphorylation inhibits the combination of PERK dimer and FLAN, thus accelerates F-actin accumulation, then interferes with the formation of ER-PM contacts. The perturbed ER-PM contacts further deteriorate ERS. During the ERS, PERK is activated by phosphorylation, which in turn activates eIF2α, resulting in the upregulation of BACE1 translation and increased production of Aβ, and excessive accumulation of Aβ leads to ERS. The above process forms a vicious cycle that causes cognitive impairment. ECH can prevent the phosphorylation of PERK and the subsequent F-actin accumulation and Aβ overproduction, thus blocking the vicious cycle and ameliorating the cognitive impairments in AD.