Repetitive Transcranial Magnetic Stimulation Improves Mild Cognitive Impairment Associated with Alzheimer's Disease in Mice by Modulating the miR-567/NEUROD2/PSD95 Axis

Abstract

Background: Mild cognitive impairment (MCI) is a typical symptom of early Alzheimer's disease (AD) and is driven by the dysfunction of microRNAs (miRs). Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive technique for handling neuropsychiatric disorders and has universally effects on the functions of miRs. In the current study, the improvement effects of rTMS on MCI associated with AD were explored by focusing on miR-567/NEUROD2/PSD95 axis.

Methods: MCI was induced in mice using scopolamine and was treated with rTMS of two frequencies (1 Hz and 10 Hz). The changes in cognitive function, brain structure, neurotrophic factor levels, and activity of miR-567/NEUROD2/PSD95 axis were assessed. The interaction between rTMS and miR-567 was further verified by inducing the level of miR-567 in AD mice.

Results: The administrations of rTMS improved the cognitive function of AD mice and attenuated brain tissue destruction, which were associated with the restored production of BDNF and NGF. Additionally, rTMS administrations suppressed the expression of miR-567 and up-regulated the expressions of NEUROD2 and PSD95, which contributed to the improved condition in central nerve system. With the induced level of miR-567, the effects of rTMS were counteracted: the learning and memorizing abilities of mice were impaired, the brain neuron viability was suppressed, and the production of neurotrophic factors was suppressed even under the administration of rTMS. The changes in brain function and tissues were associated with the inhibited expressions of NEUROD2 and PSD95.

Conclusion: The findings outlined in the current study demonstrated that rTMS treatment could protect brain against AD-induced MCI without significant side effects, and the function depended on the inhibition of miR-567.

Keywords: Alzheimer’s disease; NEUROD2; miR-567; mild cognitive impairment; repetitive transcranial magnetic stimulation.

Figure 1

Administration of rTMS improves the cognitive function of mice with Alzheimer’s disease (replicate number = 5). Mild cognitive impairment was induced in mice using scopolamine injection, and rTMS was then administered with two different frequencies (1 Hz and 10 Hz). Next, the mice were subjected to Morris water maze tests to assess the changes in cognitive functions. (A) Escaping latency in the 4-day visible platform trial. (B) Proportion of penetrating path in platform area in the 1-day probe trial. (C) Number of animals crossing the platform quadrant in the 1-day probe trial. Sham group, mice injected with normal saline. Model group, mice injected with scopolamine. L rTMS group, mice injected with scopolamine and treated with rTMS of 1 Hz. H rTMS group, mice injected with scopolamine and treated with rTMS of 10 Hz. *P<0.05 vs Sham group, ^#P<0.05 vs Model group.

Figure 2

Administrations of repetitive transcranial magnetic stimulation increases cell viability in the hippocampus regions of mice with Alzheimer’s disease (replicate number = 5). Cell apoptosis in the hippocampus regions of mice was detected with TUNEL staining, and cell viability was represented by the production of doublecortin in the hippocampus regions, as detected by IHC assay. (A) Analysis of the results and representative images of cell apoptosis by TUNEL staining. (B) Analysis of the results and representative images of IHC detection of doublecortin production. Sham group, mice injected with normal saline. Model group, mice injected with scopolamine. L rTMS group, mice injected with scopolamine and treated with rTMS of 1 Hz. H rTMS group, mice injected with scopolamine and treated with rTMS of 10 Hz. *P<0.05 vs Sham group, ^#P<0.05 vs Model group.

Figure 3

Administration of repetitive transcranial magnetic stimulation increases the production of neurotrophic BDNF and NGF in mice with AD. The production of BDNF and NGF in brain tissues of mice with AD was detected by ELISA (replicate number = 5). Analysis of the results of ELISA detection of (A) BDNF and (B) NGF. Sham group, mice injected with normal saline. Model group, mice injected with scopolamine. L rTMS group, mice injected with scopolamine and treated with rTMS of 1 Hz. H rTMS group, mice injected with scopolamine and treated with rTMS of 10 Hz. *P<0.05 vs Sham group, ^#P<0.05 vs Model group.

Figure 4

Administration of repetitive transcranial magnetic stimulation suppresses the expression of miR-567, and increases the expression of NEUROD2 and PSD95 in mice with Alzheimer’s disease (replicate number = 5). The expression of miR-567 was detected by RT-qPCR, and the expression of NEUROD2 and PSDD95 was detected by Western blotting. (A) Analysis of the results of RT-qPCR detection of miR-567. (B) Analysis of the results and representative images of the detection of NEUROD2 and PSDD95 by Western blotting. Sham group, mice injected with normal saline. Model group, mice injected with scopolamine. L rTMS group, mice injected with scopolamine and treated with rTMS of 1 Hz. H rTMS group, mice injected with scopolamine and treated with rTMS of 10 Hz. *P<0.05 vs Sham group, ^#P<0.05 vs Model group.

Figure 5

In vivo induction of miR-567 by agomir impairs the cognitive improvement function of rTMS (replicate number = 5). Mice were injected with miR-567 agomir along with rTMS treatment of 10 Hz and mild cognitive impairment model induction. (A) Escaping latency in the 4-day visible platform trial. (B) Proportion of penetrating path in platform area in the 1-day probe trial. (C) Number of animals crossing the platform quadrant in the 1-day probe trial. H rTMS group, mice injected with scopolamine and treated with rTMS of 10 Hz. NC+H rTMS group, mice injected with negative control (NC) agomir, and subjected to the injection of scopolamine and the treatment of rTMS of 10 Hz. Agomir+H rTMS group, mice injected with miR-567 agomir, and subjected to the injection of scopolamine and the treatment of rTMS of 10 Hz.

Figure 6

In vivo induction of miR-567 by agomir counteracts the brain neuron protective effects of rTMS (replicate number = 5). Mice were injected with miR-567 agomir along with rTMS treatment of 10 Hz and mild cognitive impairment model induction. (A) Analysis of the results and representative images of TUNEL detection of cell apoptosis. (B) Analysis of the results and representative images of immunohistochemical detection of doublecortin production. Analysis of the results of ELISA detection of (C) BDNF and (D) NGF. H rTMS group, mice injected with scopolamine and treated with rTMS of 10 Hz. NC+H rTMS group, mice injected with negative control (NC) agomir, and subjected to the injection of scopolamine and the treatment of rTMS of 10 Hz. Agomir+H rTMS group, mice injected with miR-567 agomir, and subjected to the injection of scopolamine and the treatment of rTMS of 10 Hz. *P<0.05 vs H rTMS group, ^#P<0.05 vs NC+H rTMS group.

Figure 7

In vivo induction of miR-567 by agomir inhibits the expression of NEUROD2 and PSD95 (replicate number = 5). Mice were injected with miR-567 agomir along with rTMS treatment of 10 Hz and mild cognitive impairment model induction. (A) Analysis of the results of reverse transcription-quantitative PCR detection of miR-567. (B) Analysis of the results and representative images of Western blot detection of NEUROD2 and PSDD95. H rTMS group, mice injected with scopolamine and treated with rTMS of 10 Hz. NC+H rTMS group, mice injected with negative control (NC) agomir, and subjected to the injection of scopolamine and the treatment of rTMS of 10 Hz. Agomir+H rTMS group, mice injected with miR-567 agomir, and subjected to the injection of scopolamine and the treatment of rTMS of 10 Hz. *P<0.05 vs H rTMS group, ^#P<0.05 vs NC+H rTMS group.