Jujuboside A promotes Aβ clearance and ameliorates cognitive deficiency in Alzheimer's disease through activating Axl/HSP90/PPARγ pathway

Abstract

Rationale: It has been reported that peroxisome proliferator activated receptor γ (PPARγ) level decreases significantly in the brains of Alzheimer's disease (AD) patients and mice models, while the mechanism is unclear. This study aims to unravel the mechanism that amyloid β (Aβ) decreases PPARγ and attempted to discover lead compound that preserves PPARγ. Methods: In APP/PS1 transgenic mice and Aβ treated microglia, the interaction between HSP90 and PPARγ were analyzed by western blot. Using a PPRE (PPARγ responsive element) containing reporter cell line, compounds that activate PPARγ activity were identified. After genetic ablation or pharmacological inhibition of potential target pathways, the target of jujuboside A (JuA) was discovered through Axl/HSP90β. After oral administration or intrathecal injection, the anti-AD activity of JuA was evaluated by Morris water maze (MWM) test and object recognition test. Soluble Aβ42 levels and plaque numbers after JuA treatment were detected by thioflavin S staining, and the activation of microglia was assayed by immunofluorescence staining against Iba-1. Results: We found that Aβ stress decreased heat shock protein 90 β (HSP90β), subsequently reduced the abundance of PPARγ, and down-regulated Aβ clearance-related genes in BV2 cells and primary microglia. We identified that JuA stimulated the expression of HSP90β, strengthened the interaction between HSP90β and PPARγ, preserved PPARγ levels, and thus effectively promoted the clearance of Aβ42. We demonstrated that JuA increased HSP90β expression through Axl/ERK pathway. JuA significantly ameliorated cognitive deficiency in APP/PS1 transgenic mice, meanwhile, JuA significantly reduced the soluble Aβ42 levels and plaque numbers in the brain. Notably, the therapeutic effects of JuA were dampened by R428, an Axl inhibitor. Conclusions: This study suggests that the up-regulation of HSP90β by JuA through Axl is a potential therapeutic strategy to facilitate Aβ42 clearance and ameliorate cognitive deficiency in AD.

Keywords: Alzheimer's disease; Axl; HSP90β; Jujuboside A; PPARγ; amyloid β.

Figure 1

Aβ induces depletion of HSP90 and PPARγ. (A) Brain tissue from 8-month-old Wt or APP/PS1 transgenic mice was collected and subjected to Western blot analyses. BV2 cells (B) or primary microglia (C) were administrated Aβ42 (5 μM) for the indicated times. The detergent-soluble lysates from cells were extracted for Western blot assay. BV2 cells (D) or primary microglia (E) were administrated Aβ42 (5 μM) for 12 h. Total protein was extracted and subjected to IP study with indicated antibodies. (F) BV2 cells were incubated with Aβ42 (5 μM) for the indicated times and protein levels of HSP90α, HSP90β and PPARγ were detected. (G) BV2 cells were transfected with indicated siRNA for 48 h. (H) BV2 cells were transfected with p-HSP90β and administrated Aβ42 (5 μM) for 12 h. Whole cell proteins were collected for western blot assay. All Experiments were repeated three times. *p < 0.05, ***p < 0.001 vs. indicated control.

Figure 2

Aβ induces impaired PPARγ activity in microglia. (A) The structure and CAS number of JuA. (B) HEK293/PPRE-Luc cells were treated with Rog (10 μM) or indicated concentrations of JuA for 24 h, then cells were lysed and luciferase activity was measured. (C) BV2 cells were pretreated with JuA for 30 min, followed by administration of Aβ42 (5 μM) for 12 h. Cells were fixed and immunostained with PPARγ antibody. The representative images demonstrate the amount and localization of PPARγ (green). Nuclei were counterstained with DAPI (blue). Scale bar: 40 μm. BV2 cells (D) and primary microglia (E) were pretreated with JuA for 30 min, followed by administration of Aβ42 (5 μM) for 6 h. The expression of various genes was analyzed by Q-PCR. DMSO at 0.1% was used as control. BV2 cells (F) and primary microglia (G) were pretreated with JuA at indicated concentrations with or without GW9662 (2 μM) for 18 h, and then incubated with 2 mg/mL Aβ42 in the presence of vehicle (DMSO) or drug for an additional 24 h. The intracellular Aβ42 levels were measured using ELISA. Uptake of Aβ42 by BV2 (H) and primary microglia (I) was assessed by applying FITC-labeled Aβ42 to microglia after pretreatment with indicated compounds. The accumulation of the fluorophore was analyzed by flow cytometry. The results were normalized to total cellular protein. DMSO at 0.1% was used as control. All experiments were repeated three times. *p < 0.05, **p < 0.01, ***p < 0.001 vs. indicated control.

Figure 3

HSP90β is essential for maintaining PPARγ function in microglia. BV2 cells were pretreated with JuA at 1 μM, 5 μM or 25 μM for 30 min, followed by administration of Aβ42 (5 μM) for 12 h. Total protein was extracted and subjected to western blot (A) and IP study (B) with indicated antibodies. (C) BV2 cells were transfect with HSP90β siRNA for 48 h, and then pretreated with JuA for 30 min followed by administration of Aβ42 (5 μM) for 12 h. DSMO at 0.1% was used as Ctrl. Whole cell proteins were collected for western blot assay. All experiments were repeated three times. ###p < 0.001, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

JuA up-regulates HSP90β in an Axl/ERK-dependent manner. BV2 cells were treated with JuA (25 μM) for indicated periods of time (A), or treated with JuA at indicated concentrations for 0.5 h (B). (C) BV2 cells were pretreated with 0.1% DMSO (Ctrl), JuA (25 μM) or JuA (25 μM) + SCH772984 (10 μM) for 30 min, followed by administration of Aβ42 (5 μM) for 6 h. (D) BV2 cells were pretreated with 0.1% DMSO (Ctrl), JuA (25 μM) or JuA (25 μM) with the indicated antagonist of RTKs (Dovitinib at 1 μM, Gefinitib at 2.5 μM, Sunitinib at 2.5 μM and LDC1267 at 1 μM) for 30 min, followed by administration of Aβ42 (5 μM) for 12 h. (E) BV2 cells were pretreated with 0.1% DMSO (Ctrl), JuA (25 μM) or JuA (25 μM) with the indicated antagonist of TAM receptor (LDC1267 at 1 μM, UNC2250 at 5 μM, R428 at 5 μM) for 30 min, followed by administration of Aβ42 (5 μM) for 12 h. (F) BV2 cells were transiently transfected with non-target control siRNA or siAxl for 48 h. (G) BV2 cells were transfected with siAxl for 48 h, then pretreated with JuA (25 μM) followed by administration of Aβ42 (5 μM) for 12 h. The whole-cell proteins were subjected to western blot with the antibodies indicated. All experiments were repeated three times. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

JuA ameliorates the cognitive deficiency in APP/PS1 mice. (A) Experimental design for the animal study. 8-mon-old APP/PS1 mice were treated with saline (i.t.), Rog (0.5 mg/kg, i.t.) or JuA (0.5 mg/kg, 1.5 mg/kg, 5 mg/kg, i.t.) once daily for 7 days. (B) Escape latency and (C) swim speed during spatial acquisition training. (D) Representative motion track, (E) distance in the target quadrant, (F) time spent in the target quadrant and (G) the platform crossing number in the spatial probe test. (H) The procedure for the object recognition test. (I) Representative motion track and (J) the discrimination index in the object recognition test. After the MWM test and the object recognition test, animals were sacrificed and the brain tissues were collected. Aβ42 levels in the cortex (K) and hippocampus (L) were detected by ELISA. # p < 0.05, ###p < 0.001 vs. Wt group. * p < 0.05, **p < 0.01, ***p < 0.001 vs. APP/PS1 group.

Figure 6

JuA ameliorates the cognitive deficiency in APP/PS1 mice through Axl. 8-month-old APP/PS1 mice were treated with JuA (5 mg/kg, i.t.) or treated with JuA and R428 (3.5 mg/kg, i.t.) once daily for 7 days. (A) Escape latency during spatial acquisition training. (B) Representative motion track, (C) distance in the target quadrant, (D) time spent in the target quadrant, and (E) the platform crossing number in the spatial probe test. (F) Representative motion track and (G) discrimination index in the object recognition test. (H) After the MWM test and the object recognition test, animals were sacrificed and the brain tissues were collected. Aβ42 levels in the cortex and hippocampus were detected by ELISA. ** p < 0.01, ***p < 0.001.

Figure 7

JuA reduces plaques and relieves over-activation of microglia in the brain. 8-month-old APP/PS1 mice were treated with JuA (5 mg/kg, i.t.) or treated with JuA and R428 (3.5 mg/kg, i.t., 30 min before the JuA administration) once daily for 7 days. (A) Representative cortex and hippocampus sections stained with thioflavin S. Scale bar: 100 μm. (B) Brain tissues were fixed and stained with Iba-1 primary antibody, then imaged by immunofluorescence confocal microscopy (red). Nuclei were stained with DAPI (blue). Representative image demonstrating the morphology of microglia in the cortex and hippocampus. Scale bar: 100 μm. (C) Iba-1 levels in the cortex and hippocampus detected by Western blot. ** p < 0.01

Figure 8

Proposed mechanism of JuA-mediated Aβ clearance.