Placenta-derived mesenchymal stem cells improve memory dysfunction in an Aβ1-42-infused mouse model of Alzheimer's disease

Abstract

Mesenchymal stem cells (MSCs) promote functional recoveries in pathological experimental models of central nervous system (CNS) and are currently being tested in clinical trials for neurological disorders, but preventive mechanisms of placenta-derived MSCs (PD-MSCs) for Alzheimer's disease are poorly understood. Herein, we investigated the inhibitory effect of PD-MSCs on neuronal cell death and memory impairment in Aβ1-42-infused mice. After intracerebroventrical (ICV) infusion of Aβ1-42 for 14 days, the cognitive function was assessed by the Morris water maze test and passive avoidance test. Our results showed that the transplantation of PD-MSCs into Aβ1-42-infused mice significantly improved cognitive impairment, and behavioral changes attenuated the expression of APP, BACE1, and Aβ, as well as the activity of β-secretase and γ-secretase. In addition, the activation of glia cells and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were inhibited by the transplantation of PD-MSCs. Furthermore, we also found that PD-MSCs downregulated the release of inflammatory cytokines as well as prevented neuronal cell death and promoted neuronal cell differentiation from neuronal progenitor cells in Aβ1-42-infused mice. These data indicate that PD-MSC mediates neuroprotection by regulating neuronal death, neurogenesis, glia cell activation in hippocampus, and altering cytokine expression, suggesting a close link between the therapeutic effects of MSCs and the damaged CNS in Alzheimer's disease.

Figure 1

The scheme of experimental study on an Aβ_1–42-infused mouse model. After Aβ infusion for 14 days, the Morris water maze test, probe test, and passive avoidance test were conducted as shown

Figure 2

Inhibitory effects of PD-MSCs on memory impairment in Aβ_1–42-infused mice model. (a–c) Training trial was performed three times a day for 7 days. Swimming time (a) and swimming distance (b) to arrive at platform were automatically recorded. At 24 h after training trials, a probe test was performed. The time spent in the target quadrant and target site crossing within 60 s was represented (c). Each value is presented as mean±S.E.M. from 10 mice. (d) To perform passive avoidance test, the mice were given electric shock when entered into the dark compartment for training on learning day. After 1 day, the retention time in illuminated compartment was recorded. Each value is presented as mean±S.E.M. from 10 mice. Significant difference from control mice (*P<0.05, **P<0.01, and ***P<0.001). Significant difference from Aβ_1–42-infused mice (^#P<0.05 and ^##P<0.01)

Figure 3

Inhibitory effects of PD-MSCs on Aβ_1–42, BACE1 and APP in the brain of Aβ_1–42-infused mice. (a–c) Aβ_1–42, BACE1, and APP were observed by immunohistochemical analysis as described in the Materials and Methods. The sections of brains were incubated with anti-Aβ_1–42 (a), anti-BACE1 (b), and anti-APP (c) antibodies, followed by biotinylated secondary antibody. Immunoperoxidase staining was expressed as brown color. (d) The expression of APP and BACE1 were detected by western blotting using specific antibodies in brain lysates. β-Actin was used as an loading control. (e) Aβ_1–42 level was measured in mouse brains by ELISA as described in the Materials and Methods. Value is mean±S.E.M. of 8 mice. (f and g) The activities of β-secretase (f) and γ-secretase (g) were carried out using each assay kit as described in the Materials and Methods. Values measured from each group of mice were calibrated by amount of protein and expressed as mean±S.E.M. from 8 mice. Significant difference from control mice (*P<0.05 and **P<0.01). Significant difference from Aβ_1–42-infused mice (^#P<0.05 and ^##P<0.01). The experiments shown in Figure 2 were repeated in triplicate with similar results

Figure 4

PD-MSC inhibits the activation of astrocytes and microglia, and reduces the expression of COX-2 and iNOS. (a and b) The effect of PD-MSC on reactive astrocytes and activated microglia cells was measured by immunohistochemical analysis. The sections were incubated with antiglial fibrillary acidic protein (GFAP) (a) and anti-ionized calcium binding adaptor molecule 1 (Iba1) (b) antibodies, followed by the biotinylated secondary antibody (n=8). Immunoperoxidase staining was expressed as brown color. (c and d) The sections were incubated with anti-iNOS (c) and anti-COX-2 (d) antibodies, and then followed by the biotinylated secondary antibody (n=8). Immunoperoxidase staining was expressed as brown color. (e) The expression of GFAP, Iba1, iNOS, and COX-2 was detected by western blotting using specific antibodies. The signals of GFAP, Iba1, iNOS, and COX2 were normalized using β-actin and the numerical values were expressed as relative fold of control. Significant difference from control mice (*P<0.05 and **P<0.01). Significant difference from Aβ_1–42-infused mice (^#P<0.05). The experiments shown in Figure 3 were repeated in triplicate with similar results

Figure 5

PD-MSC prevents Aβ_1–42-induced apoptotic cell death and promotes neuronal differentiation. (a–c) Representative photographs (original magnification × 100) of each region of control mice, Aβ_1–42-infused mice, and PD-MSC (1 × 10⁶ cells) mice. Apoptotic cell death in hippocampus CA1 (a), CA3 (b), and DG (c) of mouse brain was determined by DAPI and TUNEL staining as described in the Materials and Methods. (d) DAPI- and TUNEL-positive cells were counted in three separate locations and expressed as % of control. Significant difference from control mice (*P<0.05, **P<0.01, and ***P<0.001). Significant difference from Aβ_1–42-infused mice (^#P<0.05). (e and f) The effect of PD-MSC on newly generated neurons and proliferation of neural stem cells was measured by immunohistochemical analysis. The sections were incubated with anti-doublecortin (DCX) (e) and anti-antigen Ki-67 (Ki-67) (f) antibodies, followed by the biotinylated secondary antibody (n=8). Significant difference from control mice (**P<0.01). Significant difference from Aβ_1–42-infused mice (^#P<0.05). The experiments shown in Figure 4 were repeated in triplicate with similar results

Figure 6

The localization of PD-MSCs with NeuN, DCX, and GFAP in Aβ_1–42-infused mice. (a–c) Immunofluorescence analysis using confocal microscope in Aβ_1–42-infused mice. The sections were fixed and permeabilized. Mature neuron (a), newborn neuron (b), and astrocytes (c) (green, left) were immunostained with anti-NeuN, anti-DCX, and anti-GFAP antibodies, and then followed by Alexa488-conjugated secondary antibodies. PD-MSC (red, middle) was immunostained with anti-CD90 antibody, and then followed by Alexa555-conjugated secondary antibodies. The right panels of (a–c) show the merged images of the left and middle panels. The experiments shown in Figure 5 were repeated in triplicate with similar results