Novel osmotin inhibits SREBP2 via the AdipoR1/AMPK/SIRT1 pathway to improve Alzheimer's disease neuropathological deficits

Abstract

Extensive evidence has indicated that a high rate of cholesterol biogenesis and abnormal neuronal energy metabolism play key roles in Alzheimer's disease (AD) pathogenesis. Here, for we believe the first time, we used osmotin, a plant protein homolog of mammalian adiponectin, to determine its therapeutic efficacy in different AD models. Our results reveal that osmotin treatment modulated adiponectin receptor 1 (AdipoR1), significantly induced AMP-activated protein kinase (AMPK)/Sirtuin 1 (SIRT1) activation and reduced SREBP2 (sterol regulatory element-binding protein 2) expression in both in vitro and in vivo AD models and in Adipo^-/- mice. Via the AdipoR1/AMPK/SIRT1/SREBP2 signaling pathway, osmotin significantly diminished amyloidogenic Aβ production, abundance and aggregation, accompanied by improved pre- and post-synaptic dysfunction, cognitive impairment, memory deficits and, most importantly, reversed the suppression of long-term potentiation in AD mice. Interestingly, AdipoR1, AMPK and SIRT1 silencing not only abolished osmotin capability but also further enhanced AD pathology by increasing SREBP2, amyloid precursor protein (APP) and β-secretase (BACE1) expression and the levels of toxic Aβ production. However, the opposite was true for SREBP2 when silenced using small interfering RNA in APPswe/ind-transfected SH-SY5Y cells. Similarly, osmotin treatment also enhanced the non-amyloidogenic pathway by activating the α-secretase gene that is, ADAM10, in an AMPK/SIRT1-dependent manner. These results suggest that osmotin or osmotin-based therapeutic agents might be potential candidates for AD treatment.

Figure 1

Osmotin increased phosphorylated AMP-activated protein kinase (p-AMPK) and sirtuin 1 (SIRT1) levels and their activities in vitro and in vivo. (a) Western blot analysis of p-AMPK and SIRT1 in lysates of SH-SY5Y cells after treatment with Aβ_1–42 (5 μM), osmotin (Os; 0.2 μM), compound C (C; 20 μM) and EX527 (80 μM) for 24 h and (b, c) their activity histograms. (d–f) Western blot analysis with the respective density histograms of p-AMPK and SIRT1 in lysates of APPswe/ind-transfected (for 72 h) SH-SY5Y cells subjected to the small interfering RNA (siRNA)-induced silencing of AMPK and SIRT1 for 48 h, respectively, after 24 h of treatment with Os (0.2 μM). (g) Western blot analyses of p-AMPK and SIRT1 levels in hippocampal extracts from 5-, 9- and 12-month-old wild-type (WT) and vehicle (Veh)- or Os-treated (for a short time) amyloid precursor protein/presenilin 1 (APP/PS1) and APP_SW mice and (h) AMPK and SIRT activity histograms in brain homogenates of Adipo^−/− and APP/PS1 mice. The assays were performed three times with similar results, and representative data from one experiment with triplicate samples are shown. The bands were quantified using Sigma Gel System (SPSS, Chicago, IL, USA), and their differences are presented as histograms derived from a one-way analysis of variance (ANOVA) followed by a t-test and as the mean±s.e.m. for the respective indicated proteins (n=5/group). After developing of respective antibodies, membranes were reprobed for the β-actin signal, shown as the loading control. Histograms depict analyses of band density relative to the density of WT on the same blot. Significance: *P<0.05, **P<0.01 and ***P<0.001.

Figure 2

Osmotin reduced amyloid burden by inhibiting SREBP2 (sterol regulatory element-binding protein 2) expression in amyloid precursor protein/presenilin 1 (APP/PS1) mice. (a) Western blot analysis of SREBP2, BACE1, APP and amyloid-β (Aβ) levels in hippocampal and cortex extracts from 5-, 9- and 12-month-old wild-type (WT) and vehicle (Veh)- or osmotin (Os)-treated (for a short time) APP/PS1 mice. Representative blots and histograms depicting analyses of band density relative to WT bands are shown. The membranes were reprobed for β-actin as a housekeeping control. The bars indicate the mean±s.e.m. (n=5/group). (b, c) Enzyme-linked immunosorbent assay (ELISA) histograms (relative) of soluble (b) Aβ_1–42 and (c) Aβ_1–40 in cortex and hippocampal brain homogenates of APP/PS1 mice with or without osmotin treatment. All of the methods and procedures recommended by the manufacturer were followed, and experiments were performed in triplicate. (d, e) Fluorescence images with relative density histograms of cortex and hippocampal regions of 12- and 16-month-old Veh- and Os-treated (4 weeks) APP/PS1 mice, indicating the localization of (d) Aβ plaques and (e) Aβ (6E10) aggregates. Significance: *P<0.05, **P<0.01 and ***P<0.001.

Figure 3

Osmotin (Os) reduced amyloid-β (Aβ) production in APPswe/ind-transfected SH-SY5Y cells in an AMPK/SIRT1/SREBP2-dependent manner. Western blot analysis and respective density histograms of (a) amyloid precursor protein (APP) and Aβ, (b) APP and (c, d) BACE1 and SREBP2 (sterol regulatory element-binding protein 2) in APPswe/ind-transfected SH-SY5Y cells subjected to the small interfering RNA (siRNA)-induced silencing of AMP-activated protein kinase (AMPK), sirtuin 1 (SIRT1) and SREBP2, respectively. Cells were transfected for 72 h followed by siRNA treatment for 48 h and osmotin treatment for 24 h. The β-actin signal is shown as a loading control. (e) Osmotin treatment increased the levels of sAPP-α, ADAM10 and ADAM17 protein expression in brain tissues homogenates from 9-month-old APP/presenilin 1 (PS1) mice compared with vehicle-treated mice. (f–h) ADAM10 western blot analyses and respective density histograms of APPswe/ind-transfected SH-SY5Y cells subjected to the siRNA-induced silencing of AMPK and SIRT1. All the respective details are described in the Materials and methods section. Significance: *P<0.05, **P<0.01 and ***P<0.001.

Figure 4

Osmotin (Os) augmented the number of functional synapses without affecting synaptic strength and rescued long-term potentiation (LTP) deficits in the CA1 region of hippocampal slices from Alzheimer's disease (AD) mice. (aa1) Representative traces of spontaneous unitary excitatory postsynaptic currents (EPSCs) recorded in the CA1 region of hippocampal slices from wild-type (WT; left trace), amyloid precursor protein/presenilin 1 (APP/PS1) and osmotin-treated APP/PS1 (right trace) mice. (a2, left) Cumulative miniature EPSC (mEPSC) amplitude distributions from all recorded neurons for the three mouse groups (control (black circle), APP/PS1 (red trace) and osmotin-treated APP/PS1 (blue circle) groups). (a2, right) Summary of mEPSC amplitudes for the three groups. (a3, left) Cumulative mEPSC frequency distributions from all recorded neurons for the three groups. (a3, right) Summary of mEPSC amplitudes for the three groups. Summary of mEPSC frequencies in control APP/PS1 and osmotin-treated APP/PS1 mice. (b1, 2, 3, upper trace) A representative trace of LTP induction in the CA1 region of the hippocampi of control mice, APP/PS1 mice and osmotin-treated APP/PS1 mice, respectively. (b1, 2, 3, lower trace) Average time course of LTP induction in the CA1 region of the hippocampi of control, APP/PS1 mice and osmotin-treated APP/PS1 mice, respectively. (b4) Summary of the effects of osmotin on LTP induction in APP/PS1 mice. The bars represent the mean±s.e.m. (n=8). Significance: *P<0.05.

Figure 5

Osmotin (Os) diminished hippocampus-dependent spatial memory deficits in amyloid precursor protein/presenilin 1 (APP/PS1) mice. Escape latency (a) before osmotin treatment during the training period (submerged platform) and (b) after treatment in the Morris water maze (MWM) test. The escape latency of the training days was determined using repeated-measures analysis of variance (ANOVA). The data represent mean±s.e.m. (n=10/group). (c) The time spent in the target quadrant during the MWM probe test (platform removed) is expressed as the percentage of alternation. (d) The histogram shows the results of the Y-maze spontaneous alternation test performed to assess the effect of osmotin treatment on short-term memory in double-transgenic APP/PS1 Alzheimer's disease (AD) model mice. The data represent mean±s.e.m. (n=10/group). Significance: *P<0.05, **P<0.01 and ***P<0.001.