Osmotin attenuates amyloid beta-induced memory impairment, tau phosphorylation and neurodegeneration in the mouse hippocampus

Abstract

The pathological hallmarks of Alzheimer's disease (AD) include amyloid beta (Aβ) accumulation, neurofibrillary tangle formation, synaptic dysfunction and neuronal loss. In this study, we investigated the neuroprotection of novel osmotin, a plant protein extracted from Nicotiana tabacum that has been considered to be a homolog of mammalian adiponectin. Here, we observed that treatment with osmotin (15 μg/g, intraperitoneally, 4 hr) at 3 and 40 days post-intracerebroventricular injection of Aβ1-42 significantly ameliorated Aβ1-42-induced memory impairment in mice. These results revealed that osmotin reverses Aβ1-42 injection-induced synaptic deficits, Aβ accumulation and BACE-1 expression. Treatment with osmotin also alleviated the Aβ1-42-induced hyperphosphorylation of the tau protein at serine 413 through the regulation of the aberrant phosphorylation of p-PI3K, p-Akt (serine 473) and p-GSK3β (serine 9). Moreover, our western blots and immunohistochemical results indicated that osmotin prevented Aβ1-42-induced apoptosis and neurodegeneration in the Aβ1-42-treated mice. Furthermore, osmotin attenuated Aβ1-42-induced neurotoxicity in vitro.To our knowledge, this study is the first to investigate the neuroprotective effect of a novel osmotin against Aβ1-42-induced neurotoxicity. Our results demonstrated that this ubiquitous plant protein could potentially serve as a novel, promising, and accessible neuroprotective agent against progressive neurodegenerative diseases such as AD.

Figure 1. Effect of osmotin on spontaneous alternation behavior.

The mice were treated with Aβ_1-42 (3 μl/mouse, i.c.v.) or vehicle (control) and maintained for 3 or 40 days, represented by Aβ_1-42 (3 days), Aβ_1-42 (40 days) and control. Osmotin (15 μg/g, i.p., 4 hr) was administered to the mice on days 3 and 40 post-injection of Aβ_1-42, represented by Aβ_1-42 (3 days) +Os and Aβ_1-42 (40 days) +Os, respectively. The spontaneous alternation behavior percentages were measured for 8 min using the Y-maze task in the respective groups after 4 hr of osmotin and saline administration The columns represent the means ± SEM; n = 15 for each experimental group. #significantly different from the vehicle-treated control mice; *significantly different from the Aβ_1-42-treated mice.

Figure 2. Osmotin reduced Aβ_1-42-induced synaptotoxicity.

(A) Western blot analysis of the mouse hippocampus using anti-synaptophysin and anti-SNAP-25 antibodies. The cropped bands were quantified using Sigma Gel software, and the differences are represented in the histogram. An anti-β-actin antibody was used as a loading control. The band density values are expressed in arbitrary units (A.U.) as the means ± SEM for the indicated proteins (n = 10 mice/group). (B) Representative images showing the results of immunofluorescence reactivity for Aβ (D-11) (FITC-labeled, green) and synaptophysin (TRITC-labeled, red). The 40-day post-Aβ_1-42-treated mice exhibited decreased synaptic strength based on a reduction in synaptophysin immunoreactivity compared with the control mice. Osmotin treatment prevented the Aβ_1-42-induced reduction in immunofluorescence reactivity for synaptophysin. #significantly different from the vehicle-treated control mice; *significantly different from the Aβ_1-42-treated mice. n = 5 mice/group, n = 3 experiment. Magnification 40x; scale bar = 50 μm. (C) Western blot analysis of the mouse hippocampus using anti-p-GluR1 (Ser845), anti-total GluR1 and anti-PSD95 antibodies. The cropped bands were quantified using Sigma Gel software, and the differences are represented in the histogram. An anti-β-actin antibody was used as a loading control. The band density values are expressed in A.U. as the means ± SEM for the indicated proteins (n = 10 mice/group). #significantly different from the vehicle-treated control mice; *significantly different from the Aβ_1-42-treated mice.

Figure 3. Osmotin attenuated the expression levels of Aβ and BACE-1.

(A) Western blot analysis of Aβ (D-11) and BACE-1 expression in the mouse hippocampus. The cropped bands were quantified using Sigma Gel software, and the differences are represented in the graphs. β-actin was used as a loading control. The density values are expressed in A.U. as the means ± SEM for the indicated proteins (n = 10 mice/group). (B) Thioflavin S staining demonstrating the formation of Aβ plaques at 40 days post-Aβ_1-42 injection. Treatment with osmotin significantly reduced the plaque number and burden (%) compared with Aβ_1-42 treatment alone. n = 5 mice/group, n = 3 experiment. Magnification 10x and 40x. Scale bar = 100 μm and 20 μm. #significantly different from the vehicle-treated control mice; *significantly different from the Aβ_1-42-treated mice.

Figure 4. Osmotin treatment prevents Aβ-induced tau hyperphosphorylation via the regulation of the PI3K/Akt/GSK-3β signaling pathway.

(A) Western blot analysis of the mouse hippocampus using anti-p-PI3K, anti-total PI3K, anti-p-Akt (Ser473), anti-total Akt, anti-p-GSK-3β (Ser9), anti-total GSK3β, anti-p-Tau (Ser413) and anti-total tau antibodies. The cropped bands were quantified using Sigma Gel software, and the differences are represented by the histogram. An anti-β-actin antibody was used as a loading control. The band density values are expressed in A.U. as the means ± SEM for the indicated proteins (n = 10 mice/group). #significantly different from the vehicle-treated control mice; *significantly different from the Aβ_1-42-treated mice. (B) Immunohistochemistry for p-GSK3β (Ser9) showing that p-GSK3β (Ser9) immunoreactivity was decreased in the 40-day post-Aβ_1-42-treated mice. Treatment with osmotin significantly increased the expression of p-GSK3β (Ser9) compared with Aβ_1-42 treatment alone in the DG, CA1 and CA3 regions of the hippocampus. n = 5 mice/group, n = 3 experiment. Scale bar = 100 μm. #significantly different from the vehicle-treated control mice; *significantly different from the Aβ_1-42-treated mice.

Figure 5

(A,B) Representative images showing immunofluorescence using the anti-Aβ (D-11) (FITC-labeled, green) and anti-p-tau (Ser413) (TRITC-labeled, red) antibodies. The mice treated 40 days post-Aβ_1-42exhibited increased Aβ (green FITC-labeled) and p-Tau (Ser413) (red TRITC-labeled) immunofluorescence reactivity in the CA3 and DG regions of the hippocampus. Treatment with osmotin ameliorated the effects of Aβ_1-42 and significantly decreased the immunoreactivity for p-tau (Ser413) and Aβ (D-11). n = 5 mice/group, n = 3 experiment. Magnification 40x; scale bar = 50 μm. #significantly different from the vehicle-treated control mice; *significantly different from the Aβ_1-42-treated mice.

Figure 6. Osmotin prevents Aβ_1-42-induced apoptosis and neurodegeneration.

(A) Western blot analysis of the mouse hippocampus using anti-p53, anti-caspase-9, anti-cleaved caspase-3 and anti-PARP-1 antibodies. The cropped bands were quantified using Sigma Gel software, and the differences are represented by the histogram. An anti-β-actin antibody was used as a loading control. The band density values are expressed in A.U. as the means ± SEM for the indicated hippocampal proteins (n = 10 mice/group). #significantly different from the vehicle-treated control mice; *significantly different from the Aβ_1-42-treated mice. (B) The cells that were immunoreactive to the anti-activated caspase-3 antibody were examined in the DG, CA3 and CA1 regions of the hippocampus of the mice treated 40 days post-Aβ_1-42. The number of caspase-3-positive cells was increased in the Aβ_1-42-treated mice compared with the control mice. Treatment with osmotin significantly ameliorated the Aβ-induced increase in the number of caspase-3-positive cells. n = 5 mice/group, n = 3 experiment. Scale bar = 200 μm. #significantly different from the vehicle-treated control mice; *significantly different from the Aβ_1-42-treated mice.

Figure 7. Representative photomicrograph of Nissl staining in the DG, CA3 and CA1 regions of the mouse hippocampus.

n = 5 mice/group, n = 3 experiment. Scale bar = 200 μm. #significantly different from the vehicle-treated control mice; *significantly different from the Aβ_1-42-treated mice.

Figure 8. Osmotin attenuated the deleterious effects of Aβ_1-42 in vitro.

(A). The ApoTox-Glo^TM assay in neuronal HT22 cells. Cell viability was decreased but cytotoxicity and caspase-^3/7 activation were increased after treatment with Aβ_1-42 (5 μM) compared with the control treatment. Treatment with osmotin at three different concentrations (0.1, 0.2, or 0.4 μM) significantly reduced the effects of Aβ_1-42, thereby increasing cell viability and decreasing cytotoxicity and caspase-^3/7 activation. (B) The ApoTox-Glo^TM assay in primary hippocampal neuron cultures from GD 17.5 rat fetuses. Cell viability was decreased but cytotoxicity and caspase-^3/7 activation were increased after Aβ_1-42 (5 μM) treatment compared with the control treatment. Treatment with osmotin at three different concentrations (0.1, 0.2, or 0.4 μM) significantly reduced the effects of Aβ_1-42, thereby increasing cell viability and decreasing cytotoxicity and caspase-^3/7 activation. #significantly different from the control; *significantly different from the Aβ_1-42-treated mice. Ns = not significant compared with the Aβ_1-42-treated mice. n = 3 per experiment.