Salidroside reduces tau hyperphosphorylation via up-regulating GSK-3β phosphorylation in a tau transgenic Drosophila model of Alzheimer’s disease
- Bei Zhang†1, 2,
- Qiongqiong Li†1,
- Xingkun Chu2,
- Suya Sun1Email author and
- Shengdi Chen1, 2Email author
© The Author(s). 2016
Received: 5 November 2016
Accepted: 18 November 2016
Published: 29 November 2016
Alzheimer’s disease (AD) is an age-related and progressive neurodegenerative disease that causes substantial public health care burdens. Intensive efforts have been made to find effective and safe treatment against AD. Salidroside (Sal) is the main effective component of Rhodiola rosea L., which has several pharmacological activities.
The objective of this study was to investigate the efficacy of Sal in the treatment of AD transgenic Drosophila and the associated mechanisms.
We used tau transgenic Drosophila line (TAU) in which tau protein is expressed in the central nervous system and eyes by the Gal4/UAS system. After feeding flies with Sal, the lifespan and locomotor activity were recorded. We further examined the appearance of vacuoles in the mushroom body using immunohistochemistry, and detected the levels of total glycogen synthase kinase 3β (t-GSK-3β), phosphorylated GSK-3β (p-GSK-3β), t-tau and p-tau in the brain by western blot analysis.
Our results showed that the longevity was improved in salidroside-fed Drosophila groups as well as the locomotor activity. We also observed less vacuoles in the mushroom body, upregulated level of p-GSK-3β and downregulated p-tau following Sal treatment.
Our data presented the evidence that Sal was capable of reducing the neurodegeneration in tau transgenic Drosophila and inhibiting neuronal loss. The neuroprotective effects of Sal were associated with its up-regulation of the p-GSK-3β and down-regulation of the p-tau.
Alzheimer’s disease (AD) is a progressive and fatal brain disorder, and affects approximately 36 million people worldwide. This number is expected to double during the next 20 years . Neuropathologically, it is characterized by accumulation of extracellular senile plaques consisting of deposits of beta-amyloid (Aβ) and intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein, which ultimately lead to neuronal loss and brain atrophy [2, 3].
In fact, the tau hypothesis suggests that neurofibrillary tangles in the brain represent a major component of the pathophysiology of Alzheimer’s disease , which is attributable to an abnormal phosphorylation of tau protein in the brains of AD patients. Under normal circumstances, tau protein is a neuronal microtubule-associated protein that has a crucial role in assemblage and stabilization of microtubules on neuronal axons and the inhibition of apoptosis [5, 6]. However, when tau is abnormally hyperphosphorylated, it destabilizes microtubules by decreasing the binding affinity of tau, and consequently leads to microtubule destabilization, disruption of the axonal transport system, and ultimately, the formation of intracellular neurofibrillary tangles (NFTs). NFT formation spreads to various brain areas during AD progression, ultimately causing neuronal death [7–13]. Previous studies have shown that increasing tau phosphorylation occurs early in the development of AD [14, 15], and that Aβ associated clinical cognitive decline is identified only following such elevated tau phosphorylation [14, 16]. It is expected that intervening the formation of these toxic assemblies would attenuate the appearance and development of the symptoms of AD. Although many researches have discovered a great deal of pharmaceutical treatments for AD, no effective compound has been found so far for this debilitating neurodegenerative disease.
Over the past decades, drug therapies for AD primarily aim at slowing down the cognitive decline and ameliorating the behavioral symptoms, but the pharmacological effects of these drugs remain unsatisfactory. Salidroside (Sal), as one of the active ingredients extracted from the root of Rhodiola rosea L, which is extensively used in traditional folk medicine in Asian and European countries and has been reported to exhibit various strong pharmacological activities. The main effects of Sal are described as anti-oxidative, anti-apoptosis, anti-inflammatory, anti-cancer, and anti-fatigue effects [17–23]. Additional studies have shown that Sal exerts a neuroprotective effect. For example, Sal is able to protect neurons from apoptosis induced by various factors [24–26]. It remains undemonstrated whether Sal exerts neuroprotection against tau-induced toxicity in AD.
In the present study, we investigated the therapeutic potential of Sal in tau transgenic AD model. We found that Sal treatment could improve locomotor functions and prolong lifespan of AD transgenic Drosophila. Moreover, we demonstrated that Sal could protect neurons against tau-induced toxicity, which might be associated with regulation of GSK-3β.
Salidroside (Sal, Purity > 99.7%) was obtained from the Green Valley Pharmaceutical Corporation (Shanghai, China). It was dissolved in PBS to a stock concentration of 100 mM and stored at − 20 °C. Donepezil was supplied by Eisai Pharmaceutical Co., Ltd. (Tokyo, Japan).
The following antibodies were used: Phospho-GSK-3β antibody, GSK-3β antibody, Mouse monoclonal Phospho-tau (ser396) antibody and tau (Cell Signaling Technology), Mouse monoclonal anti-β-actin antibody (Sigma–Aldrich, Clone AC-15), HRP-conjugated goat anti-mouse IgG (Jackson Immuno Research Laboratories, PA, USA). All chemicals were purchased from Sigma-Aldrich except those noted otherwise.
All Drosophila stocks were maintained at 25 °C under a 12:12 h light: dark cycle at constant 65% humidity as previously described . The flies were raised in 50 ml plastic vials containing standard Drosophila medium. Transgenic upstream activating sequence (UAS) carrying human tau was obtained from Drosophila Stock Center (Institute of Biochemistry and Cell Biology, Shanghai).
New flies were collected within 24 h after eclosion for the experiment. At least 100 flies of each genotype were collected and divided into fresh food vials of 20 flies. Food vials were changed every 2–3 days, and the number of dead flies was counted at that time. The survival times described were given as median standard error of the median. Survival curves were analyzed using Kaplan-Meier estimation and log-rank statistical analysis.
For immunostaining analysis, flies (n = 10 for each group) were fixed in freshly prepared 4% paraformaldehyde, processed to embed in paraffin blocks, and sectioned at a thickness of 5 μm. Sections were placed on slides, stained with hematoxylin and eosin, and examined by bright field illumination using a Leica DM 2500 microscope at the magnification of 60×. The areas of the vacuoles in the cell body or neuropil regions were captured.
Western blot analysis
After treatment, fly heads (n = 50 for each group) were homogenized in lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitor cocktail (Roche, Basle, Switzerland) and 1 mM phenylmethyl sulfonyl-fluoride (PMSF) for 30 min on ice. Total extracts were centrifuged at 14,000 × g for 30 min and boiled in 4× SDS loading buffer for 5 min. The samples were subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). The membranes were blocked using 5% skim milk in TBST for 1 h then incubated at 4 °C overnight with respective primary antibodies to t-GSK-3β(1:1000), p-GSK-3β(1:1000), t-tau (1:1000), p-tau (1:1000) and β-actin (1:5000). After being washed three times with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse antibody (1:10000) for 2 h at room temperature. Visualized with the indicated antibodies using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and analyzed by ImageJ (National Institutes of Health) software. All the experiments were performed at least three times and the most representative results were shown.
All statistical analysis was performed using SPSS software 19.0(SPSS Inc., Chicago, IL). The Kaplan–Meier test was used to assess the difference in the lifespan curves. Two-group comparisons were analyzed using Student t-test. A comparison of three or more groups was performed using one-way ANOVA followed by Tukey’s test. All experiments were carried out in triplicate (n = 3) and results were expressed as the mean ± standard error of the mean (SEM). Calculated comparisons were at confidence interval (CI) 95%. A P-value < 0.05 was considered statistically significant.
Sal prolonged the lifespan of AD transgenic flies
Sal treatment improved locomotor activity in AD flies
Effects of Sal on neuronal loss in AD flies
Sal regulated GSK-3β phosphorylation
During the last decade, Drosophila has emerged and been recognized as a powerful model to study human neurodegenerative diseases including AD. Although this model can not detect memory and cognitive function, the short generation time and short lifespan make it particularly amenable to study such age-related disorders [30, 35–37]. In the present study, we showed that Sal treatment prolonged the lifespan and improved locomotor abilities in a tau-expressing transgenic Drosophila model. Furthermore, we demonstrated that Sal could dramatically attenuate the neuronal loss in the brains. As far as we know, this is the first evidence for Sal play an important protective role in neurons through up-regulatingGSK-3β phosphorylation in transgenic flies. As Sal was reported with property of non-toxic and mitigated neurotoxicity , our study provides a potential promising drug candidate for AD therapy.
In the last two decades, drug discovery and development efforts for AD have been dominated by the “amyloid cascade hypothesis,” focusing on targets defined by this hypothesis and proposing amyloid as the main cause of neural death and dementia. Unfortunately, several clinical trials with anti-Aβ agents failed, thus challenging the hypothesis that Aβ accumulation is the initiating event in the pathological cascade of AD, so we need to explore some novel therapeutic approaches and targets . In recent years, tau-based treatments for AD have become a point of increasing focus and future investigational therapies . Inhibition of the toxicity of tau in the brain may offer significant promise for the treatment of this disease. Our experiments in tau-expressing transgenic Drosophila showed that Sal attenuated tau-induced cytotoxicity effectively, suggesting a novel effect of Sal through inhibiting the tau phosphorylation in AD brain.
GSK-3β is a ubiquitously expressed serine/threonine kinase that plays a key role in the pathogenesis of AD. GSK-3β phosphorylates tau in most serine and threonine residues hyperphosphorylated in paired helical filaments . The effect of Sal in the flies increased GSK-3β phosphorylation significantly, while inhibiting tau phosphorylation simultaneously. These results suggest a possible causal relationship for Sal effect between tau hyperphosphorylation and the regulation of GSK-3β phosphorylation. Taken together, the findings of these experiments support the proposition that Sal plays an important role in providing the neuroprotection for AD by regulating tau phosphorylation.
In summary, we demonstrated that the treatment with Sal relieved the behavioral and pathological changes in a tau transgenic Drosophila model, and the mechanism was associated with its reducing tau hyperphosphorylation via up-regulating GSK-3β phosphorylation. These findings suggest that the Sal may protect neurons from degeneration in brains of AD models, and provide a potential approach in prevention and treatment of AD models. Although Sal has been prescribed to patients with cardiovascular disease and exhibited various pharmacological activities, further multiple studies should be carried out to evaluate the efficacies of Sal against AD.
We thank Dr. Nan-Jie Xu for helpful comments on the manuscript, and Dr. Gang Pei for providing transgenic Drosophila.
This work was supported by grants from the National Natural Science Fund (91332107, 81430022, 81371407). All founding were used for the design, collection, analysis and interpretation of data and in writing in the manuscript.
Availability of data and material
The data will not be shared with the reason that we decided to do further research.
All authors read and approved the final manuscript. BZ summarized the background. QL, XC and BZ performed the experiment. BZ and QL conceived, designed, and performed the paper. SC and SS revised the paper.
The authors declare that they have no competing interest.
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