Neuroprotective effects of salidroside through PI3K/Akt pathway activation in Alzheimer's disease models

Abstract

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by deposits of aggregated amyloid-β (Aβ) peptide and neurofibrillary tangles in the brain parenchyma. Despite considerable research to elucidate the pathological mechanisms and identify therapeutic strategies for AD, effective treatments are still lacking. In the present study, we found that salidroside (Sal), a phenylpropanoid glycoside isolated from Rhodiola rosea L., can protect against Aβ-induced neurotoxicity in four transgenic Drosophila AD models. Both longevity and locomotor activity were improved in Sal-fed Drosophila. Sal also decreased Aβ levels and Aβ deposition in brain and ameliorated toxicity in Aβ-treated primary neuronal culture. The neuroprotective effect of Sal was associated with upregulated phosphatidylinositide 3-kinase (PI3K)/Akt signaling. Our findings identify a compound that may possess potential therapeutic benefits for AD and other forms of neurodegeneration.

Keywords: Alzheimer’s disease; Drosophila; amyloid-β; neuroprotective effect; salidroside.

Figure 1

Sal treatment increases the life span of a transgenic AD Drosophila model. Notes: (A) Survival analysis of AD transgenic flies. (B) Effect of Sal on fly longevity. (C) Sal prolonged APP/BACE fly survival. (D) Sal prolonged EAβ fly survival. Kaplan–Meier cumulative survival analysis. Data are presented as mean ± SEM of three independent experiments. *P<0.05, **P<0.01, and ***P<0.001. Abbreviations: APP, amyloid precursor protein; BACE, beta-secretase; CS, Canton-S; Ctrl, control; Sal, salidroside; AD, Alzheimer’s disease; SEM, standard error of the mean.

Figure 2

Salidroside increases locomotor activity. Notes: (A) The climbing abilities of EAPP/BACE, EAβ₄₂, EAβ₄₂; Aβ₄₂, and Aβ_{42 E22G} transgenic flies were assessed after eclosion. Climbing deficits in these fly lines were compared with the control flies (CS). (B) The percentage of flies climbing up 8 cm in 10 seconds increased after salidroside or Aricept treatment in EAPP/BACE and EAβ lines at day 30. Mean ± SEM. *P<0.05, **P<0.01, and ***P<0.001 compared to the control group with one-way ANOVA analysis followed by Tukey’s test. Abbreviations: APP, amyloid precursor protein; BACE, beta-secretase; CS, Canton-S; ANOVA, analysis of variance; Ctrl, control; Sal, salidroside; Aβ, amyloid-β; SEM, standard error of the mean.

Figure 3

Effect of Sal on Aβ accumulation in vivo and Aβ induced neurotoxicity in vitro. Notes: (A) Micrographs of Aβ peptide expression in intact transgenic fly brains. CS, EAPP/BACE and EAβ; Aβ flies showed spot-like staining for Aβ-antibody 6E10 staining. Scale bar in insets represent 50 µm. Arrowheads indicate 6E10-antibody positivity. (B) Immunofluorescence staining shows the level of Aβ deposition in EAPP/BACE flies after Sal or Aricept treatment for 30 days. Arrowheads indicate 6E10-antibody positivity. Scale bar, 50 μm. The number of amyloid plaques was quantified. *P<0.05, **P<0.01. (C) Aβ₄₀ and Aβ₄₂ levels were measured by ELISA at day 30. The values are mean ± SEM. Each value represents the mean of three experiments. *P<0.05 and **P<0.01 versus the control group. (D) Primary cultured neurons were transfected with tdTomato and APP plasmids and treated with Sal for 24 hours. The axonal lengths (arrowheads) were measured and quantified. Scale bar, 100 μm. *P<0.05 and **P<0.01 by one-way ANOVA followed by Tukey’s test. Abbreviations: APP, amyloid precursor protein; BACE, beta-secretase; ELISA, enzyme-linked immunosorbent assay; h, hours; ANOVA, analysis of variance; CS, Canton-S; WT, wild type; Sal, salidroside; Ctrl, control; Aβ, amyloid-β; SEM, standard error of the mean; Con, normal control.

Figure 3

Effect of Sal on Aβ accumulation in vivo and Aβ induced neurotoxicity in vitro. Notes: (A) Micrographs of Aβ peptide expression in intact transgenic fly brains. CS, EAPP/BACE and EAβ; Aβ flies showed spot-like staining for Aβ-antibody 6E10 staining. Scale bar in insets represent 50 µm. Arrowheads indicate 6E10-antibody positivity. (B) Immunofluorescence staining shows the level of Aβ deposition in EAPP/BACE flies after Sal or Aricept treatment for 30 days. Arrowheads indicate 6E10-antibody positivity. Scale bar, 50 μm. The number of amyloid plaques was quantified. *P<0.05, **P<0.01. (C) Aβ₄₀ and Aβ₄₂ levels were measured by ELISA at day 30. The values are mean ± SEM. Each value represents the mean of three experiments. *P<0.05 and **P<0.01 versus the control group. (D) Primary cultured neurons were transfected with tdTomato and APP plasmids and treated with Sal for 24 hours. The axonal lengths (arrowheads) were measured and quantified. Scale bar, 100 μm. *P<0.05 and **P<0.01 by one-way ANOVA followed by Tukey’s test. Abbreviations: APP, amyloid precursor protein; BACE, beta-secretase; ELISA, enzyme-linked immunosorbent assay; h, hours; ANOVA, analysis of variance; CS, Canton-S; WT, wild type; Sal, salidroside; Ctrl, control; Aβ, amyloid-β; SEM, standard error of the mean; Con, normal control.

Figure 4

Sal inhibits Aβ-induced neurotoxicity by activating the Akt/mTOR/p70S6K pathway in primary cultured cortical neurons. Notes: (A) APP overexpressed primary cultured cortical neurons were treated with Sal or Aricept for 24 hours. Levels of Akt and phosphorylated Akt were detected and compared with the Aβ treatment group. (B) The expression levels of mTOR, p70S6K, and their phosphorylated forms were detected. All the asterisks indicate quantitatively significant differences compared with the Aβ group. All data are presented as mean ± SEM. *P<0.05 and **P<0.01 (one-way ANOVA and Tukey’s test). Abbreviations: Sal, salidroside; mTOR, mammalian target of rapamycin; p70S6K, ribosomal protein S6 kinase; APP, amyloid precursor protein; ANOVA, analysis of variance; Aβ, amyloid-β; SEM, standard error of the mean; Con, normal control.