Increases of iASPP-Keap1 interaction mediated by syringin enhance synaptic plasticity and rescue cognitive impairments via stabilizing Nrf2 in Alzheimer's models

Abstract

Oxidative stress is an important pathogenic manifestation of Alzheimer's disease (AD) that contributes to synaptic dysfunction, which precedes Aβ accumulation and neurofibrillary tangle formation. However, the molecular machineries that govern the decline of antioxidative defence in AD remains to be elucidated, and effective candidate for AD treatment is limited. Here, we showed that the decreases in the inhibitor of apoptosis-stimulating protein of p53 (iASPP) was associated with the vulnerability to oxidative stress in the amyloid precursor protein (APP)/presenilin 1 (PS1) mouse brain. Treatment with an antioxidant, syringin, could ameliorate AD-related pathologic and behavioural impairments. Interestingly, syringin treatment resulted in an upregulation of iASPP and the increase in the interaction of iASPP with Kelchlike ECH-associating protein 1 (Keap1). Syringin reduced neuronal apoptosis independently of p53. We confirmed that syringin-induced enhancement of antioxidant defenses involved the stabilization of Nrf2 in overexpressing human Swedish mutant APP (APPswe) cells in vitro. Syringin-mediated Nrf2 nuclear translocation facilitated the activation of the Nrf2 downstream genes via iASPP/Nrf2 axis. Our results demonstrate that syringin-mediated increases of iASPP-Keap1 interaction restore cellular redox balance. Further study on the syringin-iASPP interactions may help in understanding the regulatory mechanism and designing novel potent modulators for AD treatment.

Keywords: Alzheimer's disease; Amyloid pathology; Oxidative stress; Synaptic plasticity; Syringin; iASPP/Nrf2 axis.

Fig. 1

Syringin treatment alleviates the cognitive impairment of APP/PS1 mice. Syringin (Syr) at dosages of 20 mg/kg or 60 mg/kg body weight was given by oral gavage to APP/PS1 transgenic (Tg) or age-matched wild-type (WT) C57BL/6 mice (starting from 4 months old) for 5 months. Mice treated with vehicle were used as the control group (Con). (A-B) A novel object recognition (NOR) test was performed to analyse the recognition memory of the mice. Short-term memory (STM) (5-min retention interval) and long-term memory (LTM) (24-h retention interval) analyses were presented. The mice exhibited comparable total exploration time and distance. (C) The discrimination index (calculated as the percentage of time spent exploring the new object/total exploration time) of Tg mice was significantly less than WT mice. Syr treatment ameliorates the discrimination index versus vehicle-treated group in the APP/PS1mice (***p < 0.001 relative to the WT group; ^###p < 0.001 relative to the vehicle group). Representative traces showing the exploration to the familiar (red circle) and the novel (green circle) object in the STM (D) and LTM (E) trial. Green dot indicates the location of the mouse when the test started, and the red dot represents the location of the animal when the test ended. (F) The Morris water maze (MWM) test was performed to assess the long-term and spatial memory of the mice. No significant differences in the escape latency were observed among the groups in the 2 days of the visible platform trial. During the following navigation test in which the mice searched for the hidden platform, Tg mice showed a longer escape latency than WT mice on the 5th, 6th and 7th day (**p < 0.01, WT versus Tg mice; ^##p < 0.01, Syr-treated Tg mice versus vehicle-treated Tg group). (G) Representative path graph showing the mice performance in hidden platform trial on the 7th day. (H) Quantification of probe trials on the 8th day (no platform) in the MWM test showed the number of times that the mice crossed through the location in which the platform had been previously placed. Tg mice exhibited fewer passing times than the WT mice. Syr treatment increased the number of times that the Tg mice passed across the location (***p < 0.001 relative to the WT group; ^##p < 0.01 relative to the vehicle group). (I) Representative path graph recording the performance of the mice in the probe trials (without platform) of the MWM test. Blue dot represents the location of the animal when the test started, and the red dot indicates the location of the mouse when the test ended. All data are presented as the mean ± S.E.M. Repeated measures ANOVA with post hoc Fisher's PLSD tests were applied to estimate the statistical significance. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Syringin administration ameliorates the hippocampal synaptic plasticity in the APP/PS1 mice. Brain tissues were collected from APP/PS1 transgenic (Tg) and age-matched wild-type (WT) C57BL/6 mice given syringin (Syr, 20 mg/kg or 60 mg/kg body weight) or vehicle (Con) by oral gavage. (A) Basal synaptic transmission was estimated using acute brain slices containing the hippocampus. Input/output curves were calculated according to the field excitatory postsynaptic potentials (fEPSPs) in the CA1 stratum radiatum. (B) Paired pulse ratio was recorded to assess the presynaptic function (**p < 0.01, Tg versus WT group; ^##p < 0.01 Syr-treated Tg versus vehicle-treated Tg group). (C) Comparative analysis of the minimum (100 μA) and maximum (350 μA) output. (D) Shown are the time course of long-term potentiation (LTP) in Tg and WT CA1 pyramidal neurons after high-frequency stimuli (HFS). Top panels showing the sample traces before (light traces) and after (heavy traces) the HFS. Calibration bar: 1 mV/10 ms. (E) Comparison of the LTP magnitude between WT and Tg mice; replotted from (D). (F) Representative immunoblot images showing the protein expression of postsynaptic density 95 (PSD-95, postsynaptic components), synaptophysin (SYN, presynaptic components) and synapsin 1 (related to synaptic vesicles) in the hippocampus of the mouse brains (***p < 0.001 relative to the WT group; #p < 0.05, ##p < 0.01, ###p < 0.001 relative to the vehicle group). (G) The morphology of dendrites and spines (red) in the cortex of the mouse brains was displayed by immunostaining using an antibody against unphosphorylated neurofilament heavy chain (SMI-32). The nuclei (blue) were labelled with 4′,6-diamidino-2-phenylindole (DAPI). Asterisks indicate the losses of dendrites and spines in the APP/PS1 mice. Scale bars: 100 μm. Quantification showing the density and areas of the spines and the total dendrite areas (*p < 0.05, ***p < 0.001 relative to the WT group; ##p < 0.01, ###p < 0.001 relative to the vehicle group). Two-way ANOVA with post hoc Fisher's PLSD tests were applied to assess the statistical significance. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Syringin management reduces Aβ accumulation and neuronal apoptosis in the brains of APP/PS1 mice. AD-related pathology in the mouse brains of APP/PS1 transgenic with syringin (Syr, 20 mg/kg or 60 mg/kg body weight) or vehicle treatment (Con) were detected. (A) Soluble and insoluble human Aβ1-42 and human Aβ1-40 levels were determined by ELISA. Quantification showing the ratio of Aβ1-42 to Aβ1-40. (B) Representative images of immunofluorescence staining with anti-Aβ antibody showing Aβ deposition (green) in the APP/PS1 mouse brain. DAPI was used to label nuclei (blue). High-magnification images of the cortex and hippocampus (Hippo) were depicted in the right panels. Scale bars: 200 μm. Relative percentages of Aβ plaques with various diameters were quantified. (C) Cresyl violet stains showing the Nissl body in the neuronal cells of the hippocampal CA1 and CA3 regions. Scale bars: 50 μm. The numbers of Nissl body indicate the surviving neurons. (D) Apoptosis levels of neuronal cells from the mouse brains were measured using Annexin V-FITC/PI staining by flow cytometry. (E) Representative Western blot images showing the protein expression of a disintegrin and metalloproteinase 10 (ADAM10), β-secretase 1 (BACE1) and γ-secretase, presenilin 1 (PS1), Nicastrin, APH-1 and Pen 2. (F) The β- and γ-secretase activities are shown. One-way ANOVA with post hoc Fisher's PLSD tests were adopted to estimate the statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 relative to the vehicle-treated controls). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Effects of syringin treatment on regulating p53 and the members of the apoptosis-stimulating protein of p53 (ASPP) family in the APP/PS1 mouse brains. The assessment of p53 in the nucleus (N-p53) and cytosol (C-p53), nuclear iASPP (N-iASP), cytosolic iASPP (C- iASPP), ASPP1 and ASPP2 in the mouse brains of APP/PS1 transgenic (Tg) and age-matched wild-type (WT) C57BL/6 mice given syringin (Syr, 20 mg/kg or 60 mg/kg body weight) or vehicle treatment (Con) were performed. Representative images of Western blot assays showing the protein levels (A). (B) mRNA levels of p53, iASPP, ASPP1 and ASPP2 were determined by real-time PCR. (C) Quantification showing the p53 DNA-binding activities. (D) The p53-iASPP interactions, the binding of p53 with ASPP1 and the p53-ASPP2 protein complex were measured by co-immunoprecipitation (CO-IP). (E) Chromatin immunoprecipitation (ChIP) analyses were performed to estimate p53-mediated gene transcription of PUMA and Bax. Two-way ANOVA with post hoc Fisher's PLSD tests (*p < 0.05, **p < 0.01, ***p < 0.001 relative to the WT group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 relative to the vehicle-treated group).

Fig. 5

Syringin administration amends the antioxidant defense in APP/PS1 mouse brains. (A) ELISA assays was performed to measure the levels of 8-isoprostane in the blood serum of APP/PS1 transgenic (Tg) and age-matched wild-type (WT) mice given syringin (20 mg/kg or 60 mg/kg body weight) or vehicle (Con) treatment. (B) ROS contents in the mouse brains were determined using CM-H2DCFDA fluorescence probe. (C) The contents of reduced glutathione (GSH) and oxidative glutathione (GSSG) in the brain tissues were assessed. Quantification showing the GSH/GSSG ratio. (D) The activities of glutathione peroxidase (GPX) and glutathione reductase (GR) were determined by colorimetric and ultraviolet colorimetric methods. (E) MitoSOX red assays by flow cytometry showing the levels of mitochondrial superoxide in the mouse brain. (F) Oxidized dihydroethidium (DHE) signal (red) showing the superoxide levels in the hippocampus of APP/PS1 and age-matched WT mice. Scale bars: 300 μm. High-magnification images of the CA3 and CA1 regions in the square boxes are depicted in the right panels. The high burden area of Aβ plques by Thioflavine (Thio S) labelled (green) also showing the significant positive staining of oxidized DHE. Scale bars: 60 μm. Two-way ANOVA with post hoc Fisher's PLSD tests (*p < 0.05, **p < 0.01 relative to the WT group; ^##p < 0.01, ^###p < 0.001 relative to the vehicle-treated group). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Syringin management enhances the cytosolic iASPP-Keap1 interaction and the nuclear Nrf2 DNA-binding activity in the APP/PS1 mouse brain. (A) Western blot assays showing the protein expression of Nrf2 in the cytosol and nuclei of the brains in the APP/PS1 transgenic (Tg) and age-matched wild-type (WT) mice given syringin (20 mg/kg or 60 mg/kg body weight) or vehicle (Con) treatment. (B) mRNA levels of Nrf2 were examined by real-time PCR. (C) DNA-binding activities of Nrf2 were determined by ELISA assays. (D) The binding of Keap1 with iASPP was assessed by co-immunoprecipitation (Co-IP) assays. (E) Representative immunofluorescence images of iASPP (red) and Thioflavine (Thio S, green) labelling in the hippocampal sections of APP/PS1 and age-matched WT mouse brains. White arrows indicate the iASPP-immunoreactive cells around the Thio S-positive plaques. Scale bars: 200 μm. High-magnification images (the bottom three panels) show the localization of iASPP (indicated by white arrowheads) and Aβ-containing plaques. Scale bar = 60 μm. (F) Immunostaining with anti-Nrf2 antibody showing the cytosolic and nuclear localization of Nrf2 in the CA3 and CA1 regions of the hippocampus. Black arrowheads indicate the representative Nrf2-immunoreactive stains in the neuronal nuclei. Scale bars: 60 μm. Square boxes showing the high magnification. Two-way ANOVA with post hoc Fisher's PLSD tests (*p < 0.05, **p < 0.01 relative to the WT group; ##p < 0.01, ###p < 0.001 relative to the vehicle-treated group). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

IASPP increases mediated by syringin exert antioxidant activity and reduce apoptosis by stabilizing Nrf2. (A) ROS contents are significantly increased under H₂O₂ (50 μM, 24 h) treatment in N2a cells or N2a cells transfected with the human beta-amyloid precursor protein Swedish mutation (APPswe). N2a and APPswe cells with vehicle treatment were used as controls (Con). CM-H2DCFDA staining by flow cytometry assays showing that the ROS levels are markedly reduced under incubating with 1-mM of syringin (Syr) for 6 h followed by 50 μM H₂O₂ addition and up to 24 h in both N2a and APPsw cells. (B) Cell cycle of N2a and APPswe cells were evaluated. Quantification showing the propidium iodide (PI) uptake of cells. (C) Annexin V-FITC/PI staining showing the protective effects of syringin against H₂O₂-induced apoptosis in N2a and APPswe cells. (D) Soluble Aβ1-40 and Aβ1-42 levels secreted by the cells were measured by ELISA. (E) Representative Western blot images showing the protein expression of iASPP and Nrf2 in the nucleus (N-Nrf2) and the cytosol (C-Nrf2). (F) mRNA levels of Nrf2 are shown. (G) Nrf2 DNA-binding activity in the N2a and APPswe cells was measured. (H) The iASPP-Keap1 interactions in the N2a and APPswe cells were determined by Co-immunoprecipitation (Co-IP). The small interfering RNA (siRNA)-mediated inhibition of iASPP (si-iASPP, 50 nM) abolished syringin-triggered increases of Nrf2 levels in the cytoplasm and nuclei (I) and abrogated the syringin-induced ROS reduction (J) in the N2a and APPswe cells. (K) Western blot analysis showed that upregulation of NQO1 and γGCL-C triggered by iASPP lentiviral gene transfer (LV-iASPP) or Syr treatment were blocked by transfected with 60 nM of Nrf2 siRNA (si-Nrf2) or Nrf2 inhibitor, trigonelline (0.5 μM, 24h), in the APPswe cells. Statistical significance: *p < 0.05, **p < 0.01 relative to N2a controls; ^##p < 0.01 relative to the vehicle-treated group; ^$$p < 0.01 relative to H₂O₂ treatment group. Data were analysed with multivariate ANOVA with post hoc Fisher's PLSD tests (A–H). *p < 0.05, **p < 0.01 relative to N2a controls; ^##p < 0.01 relative to the vehicle-treated group; ^$$p < 0.01 relative to Syr treatment group. Data were analysed with multivariate ANOVA with post hoc Fisher's PLSD tests (I–J); **p < 0.01 compared with the vehicle-treated group; ^##p < 0.01 compared with LV-iASPP management cells; ^$$p < 0.01 relative to Syr-treated group. Data were analysed with one-way ANOVA with post hoc Fisher's PLSD tests (K). Values are represented at least three independent experiments.

Fig. 8

Graphical abstract illustrates the possible neuroprotective mechanisms of syringin against AD-related neurodegeneration via iASPP/Nrf2 axis. Under physiological conditions, nuclear factor-erythroid 2-related factor 2 (Nrf2) Nrf2 interacts with Kelch-like ECH-associated protein 1 (KEAP1) in the cytoplasm and undergoes degradation by the proteasome through ubiquitination. Mild reactive oxygen species (ROS) production could induce Nrf2 release from Keap1 and facilitate it nuclear translocation, initiating transcription of antioxidative genes. (A) In Alzheimer's disease (AD) brain, Nrf2 signalling is decline. Chronic oxidative stress is related to the excessive generation of ROS, which facilitate progressive production and accumulation of β-amyloid (Aβ). The processing of Aβ aggregation further deteriorates oxidative imbalance. Higher proteasomal degradation of Nrf2 might worsen the poor antioxidant levels in AD brain. (B) Syringin (Syr) interacts with inhibitor of apoptosis-stimulating protein of p53 (iASPP), and increases the expression and activity of iASPP. IASPP competes with Nrf2 for Keap1, leading to Nrf2 dissociated from Keap1. Nrf2 stabilization in cytosol facilitates it translocate into nucleus. In the nucleus, Nrf2 interacts with small musculoaponeurotic fibrosarcoma (sMaf) proteins, binding to the antioxidant response elements (ARE). The activation of Nrf2 initiates the gene expression such as NAD (P) H: quinone oxidoreductase 1 (NQO1), glutamyl cysteine ligase (GCL), glutathione reductase (GR), determination of glutathione peroxidase (GPX), etc., playing the neuroprotective roles in ameliorating the AD-related pathology.