DL0410 ameliorates cognitive disorder in SAMP8 mice by promoting mitochondrial dynamics and the NMDAR-CREB-BDNF pathway

Abstract

Alzheimer's disease (AD) is a worldwide problem and there are no effective drugs for AD treatment. Previous studies show that DL0410 is a multi-target, anti-AD agent. In this study, we investigated the therapeutic effect of DL0410 and its action mechanism in SAMP8 mice. DL0410 (1-10 mg·kg^-1·d^-1) was orally administered to 8-month-old SAMP mice (SAMP8) for 8 weeks. We showed that DL0410 administration effectively ameliorated the cognitive deficits in the Morris water maze test, novel object recognition test, and nest building test. We revealed that DL0410 dose-dependently increased the expression levels of the mitochondrial proteins (PGC-1α, Mitofusin 2, OPA1, and Drp1), and subsequently ameliorated the processes of mitochondrial biosynthesis, fusion, and fission in the cortex and hippocampus of SAMP8 mice. Furthermore, DL0410 administration promoted the expression of synaptic proteins (synaptophysin and PSD95) in the brain of SAMP8 mice, and upregulated the protein phosphorylation in NMDAR-CAMKII/CAMKIV-CREB pathway responsible for the synaptic plasticity. DL0410 administration dose-dependently increased the expression of BDNF and TrkB, and the neurotrophic effect was mediated via the ERK1/2 and PI3K-AKT-GSK-3β pathways. DL0410 administration upregulated Bcl-2, increased the Bcl-2/Bax ratio and the level of caspase 3 and PARP-1, alleviating neuronal apoptosis. We proposed that the NMDAR-CREB-BDNF pathway might establish a positive feedback loop between synaptic plasticity and neurotrophy, with CREB at the center. In summary, DL0410 promotes synaptic function and neuronal survival, thus ameliorating cognitive deficits in SAMP8 mice via improved mitochondrial dynamics and increased activity of the NMDAR-CREB-BDNF pathway. DL0410 is a promising candidate to treat aging-related AD, and deserves more research and development in future.

Keywords: CREB; DL0410; mitochondrial dynamics; neurotrophy; synapse plasticity.

Fig. 1. The chemical structure of DL0410.

DL0410 is the active compound with multiple drug targets against AD.

Fig. 2. There was cognitive impairment in the 8-month-old SAMP8 mice compared with the SAMR1 mice in the step-down test.

Data are expressed as the mean±SEM (n = 14–15). a The latency of the SAMP8 mice was shorter than that of the SAMR1 mice, and b the error times of SAMP8 mice tended to increase. ^#P < 0.05 and ^##P < 0.01 vs the SAMR1 mice.

Fig. 3. The effect of DL0410 on the memory of the SAMP8 mice in the behavioral tests.

Data are expressed as the mean±SEM (n = 12–18). The SAMP8 mice were randomly divided into 5 groups: the SAMP8 group (SAMP8+DL0410-0 mg/kg), DL-1 mg/kg group (SAMP8+DL0410-1 mg/kg), DL-3 mg/kg group (SAMP8+DL0410-3 mg/kg), DL-10 mg/kg group (SAMP8+DL0410-10 mg/kg), and donepezil group (SAMP8+donepezil). SAMR1 mice were used as the control group. a There was no difference among the groups in locomotor activity. b DL0410 increased the DI of the SAMP8 mice in the novel object recognition test. c In the nest building test, DL0410 improved the nesting score of the SAMP8 mice at 12 and 24 h. In the navigation trials of the Morris water maze, the latency decreased as the training progressed (d). On the fourth day, there was no difference among the groups in the swimming speed (e); in addition, DL0410 shortened the latency (f) and swimming distance (g); in terms of searching strategy, DL0410 increased the ratio of searching time (h) and searching distance (i) in the target quarter. In the probe trial, DL0410 decreased the latency onto the platform (j) and increased the entry times onto the platform (k). In terms of searching strategy, DL0410 could increase the searching time (l) and distance (m) in the target quarter. ^##P < 0.01, ^###P < 0.001 vs the SAMR1 group; *P < 0.05, **P < 0.01, and ***P < 0.001 vs the SAMP8 group.

Fig. 4. The effect of DL0410 on the mitochondrial protein in the brains of the SAMP8 mice.

Data are expressed as the mean±SEM (n = 6–8). DL0410 could improve the levels of PGC-1α (a), Drp1 (b), Mitofusin 2 (d) and OPA1 (e) in the cortex and hippocampus of the SAMP8 mice. c The representative bands of Mitofusin 2 and OPA1 in Western blots. SAMR1 mice were set as the control group, and SAMP8 mice were randomly divided into 5 groups: the SAMP8 group (SAMP8+DL0410-0 mg/kg), DL-1 mg/kg group (SAMP8+DL0410-1 mg/kg), DL-3 mg/kg group (SAMP8+DL0410-3 mg/kg), DL-10 mg/kg group (SAMP8+DL0410-10 mg/kg), and donepezil group (SAMP8+donepezil). In the histogram, the SAMR1 group, SAMP8 group, DL-1 mg/kg group, DL-3 mg/kg group, DL-10 mg/kg group, and donepezil group are listed from left to right. ^##P < 0.01 and ^###P < 0.001 vs the SAMR1 group; *P < 0.05, **P < 0.01, and ***P < 0.001 vs the SAMP8 group.

Fig. 5. The effect of DL0410 on synaptic proteins in the brains of the SAMP8 mice.

Data are expressed as the mean±SEM (n = 6–8). The IHC results of synaptophysin in the cortex (a, 200×) and CA1 area (b, 200×) and the Western blot results (c) are shown; DL0410 showed a tendency to increase synaptophysin expression in the cortex and hippocampus of the SAMP8 mice. DL0410 increased PSD95 expression in the SAMP8 mice, as shown by Western blots (f), and IHC of the cortex (d, 200×) and CA1 area (e, 200×) showed similar results. SAMR1 mice were set as the control group, and SAMP8 mice were randomly divided into 5 groups: the SAMP8 group (SAMP8+DL0410-0 mg/kg), DL-1 mg/kg group (SAMP8+DL0410-1 mg/kg), DL-3 mg/kg group (SAMP8+DL0410-3 mg/kg), DL-10 mg/kg group (SAMP8+DL0410-10 mg/kg), and donepezil group (SAMP8+donepezil). In the histogram, the SAMR1 group, SAMP8 group, DL-1 mg/kg group, DL-3 mg/kg group, DL-10 mg/kg group, and donepezil group are listed from left to right. ^##P < 0.01 and ^###P < 0.001 vs the SAMR1 group; *P < 0.05 and ***P < 0.001 vs the SAMP8 group.

Fig. 6. The effect of DL0410 on the expression of phosphorylated NMDAR2B, CAMKII-α, CAMKIV, and CREB in the brains of the SAMP8 mice.

Data are expressed as the mean ± SEM (n = 6–8). a Representative bands of Western blotting. DL0410 promoted the phosphorylation of NMDAR2B (b), CAMKII-α (c), CAMKIV (d) and CREB (e) in the cortex and hippocampus of the SAMP8 mice. SAMR1 mice were set as the control group, and SAMP8 mice were randomly divided into 5 groups: the SAMP8 group (SAMP8+DL0410-0 mg/kg), DL-1 mg/kg group (SAMP8+DL0410-1 mg/kg), DL-3 mg/kg group (SAMP8+DL0410-3 mg/kg), DL-10 mg/kg group (SAMP8+DL0410-10 mg/kg), and donepezil group (SAMP8+donepezil). In the histogram, the SAMR1 group, SAMP8 group, DL-1 mg/kg group, DL-3 mg/kg group, DL-10 mg/kg group and donepezil group are listed from left to right. ^###P < 0.001 vs the SAMR1 group; *P < 0.05, **P < 0.01, and ***P < 0.001 vs the SAMP8 group.

Fig. 7. The effect of DL0410 on the expression of BDNF, TrkB, and phosphorylated ERK1/2 in the brains of the SAMP8 mice.

Data are expressed as the mean±SEM (n = 6–8). a Representative bands of Western blotting. DL0410 increased the levels of BDNF (b) and TrkB (c) and promoted the phosphorylation of ERK1/2 (d) in the cortex and hippocampus of the SAMP8 mice. SAMR1 mice were set as the control group, and SAMP8 mice were randomly divided into 5 groups: the SAMP8 group (SAMP8+DL0410-0 mg/kg), DL-1 mg/kg group (SAMP8+DL0410-1 mg/kg), DL-3 mg/kg group (SAMP8+DL0410-3 mg/kg), DL-10 mg/kg group (SAMP8+DL0410-10 mg/kg), and donepezil group (SAMP8+donepezil). In the histogram, the SAMR1 group, SAMP8 group, DL-1 mg/kg group, DL-3 mg/kg group, DL-10 mg/kg group and donepezil group are listed from left to right. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs the SAMR1 group; *P < 0.05, **P < 0.01, and ***P < 0.001 vs the SAMP8 group.

Fig. 8. The effect of DL0410 on the PI3K-AKT-GSK-3β pathway in the brains of the SAMP8 mice.

Data are expressed as the mean±SEM (n = 6–8). a Representative Western blot bands. DL0410 increased the expression of PI3K p110α (b) and the phosphorylation of PI3K p85 (c), AKT (d) and GSK-3β (Ser9) (e) in the cortex and hippocampus of the SAMP8 mice. SAMR1 mice were set as the control group, and SAMP8 mice were randomly divided into 5 groups: the SAMP8 group (SAMP8+DL0410-0 mg/kg), DL-1 mg/kg group (SAMP8+DL0410-1 mg/kg), DL-3 mg/kg group (SAMP8+DL0410-3 mg/kg), DL-10 mg/kg group (SAMP8+DL0410-10 mg/kg), and donepezil group (SAMP8+donepezil). In the histogram, the SAMR1 group, SAMP8 group, DL-1 mg/kg group, DL-3 mg/kg group, DL-10 mg/kg group and donepezil group are listed from left to right. ^###P < 0.001 vs the SAMR1 group; *P < 0.05, **P < 0.01, and ***P < 0.001 vs the SAMP8 group.

Fig. 9. The effect of DL0410 on the apoptotic proteins in the brains of the SAMP8 mice.

Data are expressed as the mean ± SEM (n = 6–8). a Representative Western blot bands. DL0410 increased Bcl-2 expression (b) and raised the ratio of Bcl-2/Bax (c) in the cortex and hippocampus of the SAMP8 mice. The levels of Caspase 3 (d) and PARP-1 (e) induced by DL0410 were high in the cortex and hippocampus of the SAMP8 mice. SAMR1 mice were set as the control group, and SAMP8 mice were randomly divided into 5 groups: the SAMP8 group (SAMP8+DL0410-0 mg/kg), DL-1 mg/kg group (SAMP8+DL0410-1 mg/kg), DL-3 mg/kg group (SAMP8+DL0410-3 mg/kg), DL-10 mg/kg group (SAMP8+DL0410-10 mg/kg), and donepezil group (SAMP8+donepezil). In the histogram, the SAMR1 group, SAMP8 group, DL-1 mg/kg group, DL-3 mg/kg group, DL-10 mg/kg group and donepezil group are listed from left to right. ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 vs the SAMR1 group; *P < 0.05, **P < 0.01 and ***P < 0.001 vs the SAMP8 group.

Fig. 10. Illustration of the mechanism of DL0410.

The mechanism is related to mitochondrial dynamics, synaptic plasticity, neurotrophic effects and neuronal apoptosis. The improvement in mitochondrial dynamics and the NMDAR-CREB-BDNF pathway are vital mechanisms of DL0410 in promoting synaptic function and inhibiting neuronal apoptosis, and ultimately ameliorating cognitive impairments in SAMP8 mice.