Neurotropin reduces memory impairment and neuroinflammation via BDNF/NF-κB in a transgenic mouse model of Alzheimer's disease

Abstract

Alzheimer's disease (AD) is a devastating neurodegenerative disease with limited treatments and no cure. Neurotropin (NTP) is an analgesic drug widely prescribed for neuropathic pain. Increasing evidence suggests that NTP may also protect against neurodegeneration, but NTP's treatment potential against memory impairments of AD remains to be explored. APP/PS1 mice, which model AD, were given NTP for three months then cognitively tested with the Morris water maze. Their Aβ burden, microglial and astrocytic activation, and BDNF levels were compared to untreated controls using immunofluorescent staining. Expression of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and NF-κB pathway related proteins (p65 and IκB-α) were examined by ELISA or Western blots in vivo and in vitro in the microglia cell line. Lastly, BV-2 cells were pre-treated with the selective BDNF inhibitor ANA-12 and with NTP to examine mechanistic pathways. Taken together, NTP treatment reduced cognitive impairment, Aβ deposits, and glial activation in cortex and hippocampus APP/PS1 mice. IL-1β, IL-6 and TNF-α also decreased after NTP treatment in vivo and in vitro, and BDNF levels rose. Also, NTP reduced p65 and IκB-α activation and the effect of NTP on pro-inflammatory cytokines and NF-κB pathway related proteins was abolished by BDNF inhibitor. Our results indicate that NTP reduces neuroinflammation and improves the cognitive deficits in APP/PS1 mice possibly via BDNF/NF-κB pathway. NTP may be a new promising drug candidate for patients with AD.

Keywords: Alzheimer’s disease; NF-κB; brain derived neurotrophic factor; memory impairment; neuroinflammation; neurotropin.

Figure 1

Neurotropin attenuates cognitive impairment of APP/PS1 mice. A. The escape latencies of the mice in each group of mice. B. The normalized escape latencies of each group of mice. C. Representative path images of the mice finding the platform. D. The average distances of the mice swimming to find the platform. E. The times of the mice swimming across the target quadrants. The results are presented as mean ± SE from at least eight mice in each group. **P < 0.01, and NS, nonsignificant.

Figure 2

Neurotropin decreases Aβ accumulation of APP/PS1 mice. A. Aβ plaques were detected by Bielschowsky silver staining in the cortex and hippocampus. B. Aβ plaques were detected with immunofluorescent staining in the cortex and hippocampus. C. Quantification of Aβ plaque load using Bielschowsky silver staining. D. Statistical analysis of Aβ plaque burden with immunofluorescent staining. E and G. Soluble and insoluble Aβ_1-40 in the brain of TG and TG+NTP mice. F and H. Soluble and insoluble Aβ_1-42 in the brain of TG mice and TG+NTP mice. The results presented as means ± SE from six independent experiments. *P < 0.05 and **P < 0.01 versus TG mice.

Figure 3

Neurotropin alleviates astrocyte activation with attenuation of reactive gliosis and neuroinflammation. The coronal sections of the cortex and hippocampus in TG group and TG+NTP group of the mice were stained for (A). Aβ, Iba1 and DAPI, (B) Aβ, GFAP and DAPI. The percentage of the areas of microglial (C) and astrocytes (D) in the cortex and hippocampus. Analysis of the levels of IL-1β (E), IL-6 (F), and TNF (G) in the cortex and hippocampus of each group by ELISA. Data are presented as mean ± SE from six mice in each group. *P < 0.05, and **P < 0.01.

Figure 4

Neurotropin enhances levels of BDNF in APP/PS1 mice. BDNF was detected with immunofluorescent staining in the cortex (A) and the hippocampus (B) of each group. Analysis of the levels of BDNF (C), NGF (D), and NT-3 (E) in the cortex and the hippocampus with ELISA. Data are presented as mean ± SE from six mice in each group. *P < 0.05, **P < 0.01, and N.S., nonsignificant.

Figure 5

Chronic Neurotropin treatment regulates NF-κB pathways in APP/PS1 mice. A. Western blot analyses of the levels of p-65 and p-IκB. B and C. The relative levels of p-P65, p-IκB-α, and β-actin as a loading control in each group of mice, were quantified. Data are presented as mean ± SE from at least three mice in each group. *P < 0.05, and **P < 0.01.

Figure 6

The effect of NTP on BV-2 cells was abolished by BDNF inhibitor. A-C. IL-1β, IL-6 and TNF-α were found highly expressed after LPS treatment by comparing with control group. IL-1β, IL-6 and TNF-α increased after a selective, non-competitive BDNF receptor antagonist, ANA12, administration. D. Cell viability was assayed by CCK8 after treatment with ANA12. E. BDNF level was detected after NTP and ANA12 treatment. F-H. Both p-p65 and p-IκB-α were activated by LPS and inactivated by NTP. The activation of p-p65 and p-IκB-α was abolished by ANA12.