MiR-483-3p improves learning and memory abilities via XPO1 in Alzheimer's disease

Abstract

Introduction: Alzheimer's disease (AD), a common form of dementia, has been reported to influence 27 million individuals globally. Several risk factors including oxidative stress, gut microbiota imbalance, and cognitive activity are reported to be closely associated with the initiation or progression of AD. Although miR-483-3p was identified to be downregulated in AD patient serum. However, the biological role and mechanism of miR-483-3p remained unknown in AD. Here, we explored the role of miR-483-3p in AD.

Methods: Sprague-Dawley rats were injected with homocysteine (Hcy) to establish an AD animal model. The Morris water maze tests and contextual fear tests were conducted to assess the cognitive and memory abilities of rats. TUNEL staining was utilized to determine cell apoptosis. Luciferase reporter assay was used to evaluate the binding relation between miR-483-3p and exportin 1 (XPO1).

Results: Homocysteine treatment (400 μg/kg) induced the learning, cognitive and memory defects of rats. miR-483-3p was downregulated in Hcy-treated rat hippocampus. Functionally, miR-483-3p alleviated cell apoptosis and impairments of learning and memory abilities in Hcy-treated rats. In addition, miR-483-3p inhibited cell apoptosis and protein level of AD-associated factors (APP, BACE1, and Aβ1-42) in PC12 cells. In mechanism, miR-483-3p was confirmed to target XPO1 in PC12 cells. XPO1 displayed high level in rat hippocampus and was negatively correlated with miR-483-3p levels. Finally, XPO1 overexpression rescued the suppressive effect of miR-483-3p on cell apoptosis and protein levels of AD-associated factors.

Conclusions: miR-483-3p alleviates neural cell apoptosis and impairments of learning and memory abilities by targeting XPO1 in AD.

Keywords: Alzheimer's disease; PC12 cells; XPO1; homocysteine; miR-483-3p.

FIGURE 1

MiR‐483‐3p is downregulated in Hcy‐treated rats. (a) The escape latency of sham‐operated rats and Hcy‐treated rats during the training from day 41 to day 45. (b), (c) Swimming tracks of rats during the training and in Morris water maze (MWM) test. (d) The number of platform crossings in each group after removing the platform. (e) The escape latency of rats in MWM test. (f) After removing the platform, the time that rats spent in the target quadrant. (g) The percentage of freezing after the footshock in each group. (h) The assessment of fear memory of rats in contextual fear test. (i) The miR‐483‐3p level in hippocampus of sham‐operated rats and Hcy‐treated rats. n = 10/group. ^* p < .05, ^** p < .01 compared with sham group

FIGURE 2

MiR‐483‐3p alleviates impairments of learning ability, memory ability, and neural apoptosis in homocysteine‐treated rats. (a) The efficacy of AAV‐miR‐483‐3p in hippocampus of rats was detected by PCR. (b) The escape latency of rats in sham group, AAV‐miR‐483‐3p, Hcy+NC group, and Hcy+AAV‐miR‐483‐3p group during the training was recorded. (c), (d) The swimming track of rats during the training and in MWM test. (e) The number of platform crossings after removing the platform. (f), (g) The escape latency and time of rats spent in the target quadrant in MWM test. (h) The percentage of freezing after footshock. (i) Fear conditioning test for contextual fear memory assessment in rats. (j) The protein levels of APP, BACE1 and Aβ1‐42 in hippocampus of rats were analyzed by western blot. (k) TUNEL assay was conducted to assess apoptosis in hippocampus of rats in the sham, AAV‐miR‐483‐3p, Hcy+NC, and Hcy+AAV‐miR‐483‐3p groups. (l) Protein levels of apoptosis markers (Bcl‐2, Bax, and cleaved caspase‐3) in hippocampus of rats were quantified using western blot. n = 10/group. ^* p < .05, ^** p < .01 compared with sham group; ^& p < .05, ^&& p < .01 compared with Hcy group

FIGURE 3

MiR‐483‐3p overexpression inhibits the apoptosis of Aβ1‐40‐stimulated PC12 cells and downregulates levels of AD‐associated proteins. (a) The viability of PC12 cells in response to Aβ1‐40 treatment (2.5, 5, or 10 μM) was evaluated by MTT assay. (b) The miR‐483‐3p level in PC12 cells treated with different concentrations of Aβ1‐40 was analyzed by RT‐qPCR. (c) The miR‐483‐3p level in control group, Aβ1‐40 (5 μM) group, Aβ1‐40 (5 μM) + NC mimics group, and Aβ1‐40 (5 μM) + miR‐483‐3p group was analyzed by RT‐qPCR. (d) The viability of PC12 cells in above four groups was evaluated by MTT assay. (e) The apoptosis of PC12 cells in above four groups was measured by TUNEL assay. (f), (g) Protein levels of apoptotic markers (Bcl‐2, Bax, and cleaved caspase‐3) in PC12 cells with above transfection and treatment were quantified using western blot. (h), (i) APP, BACE1, and Aβ1‐42 protein levels in PC12 cells of above four groups were examined by western blot analysis. ^* p < .05, ^** p < .01, ^*** p < .001

FIGURE 4

MiR‐483‐3p targets XPO1 by binding with its 3´ UTR. (a) The overexpression efficiency of miR‐483‐3p in PC12 cells was detected by RT‐qPCR. (b) The mRNA levels of predicted mRNAs in the context of miR‐483‐3p overexpression were measured by RT‐qPCR. (c) The binding sequences between miR‐483‐3p and XPO1 3´ UTR. (d) The luciferase reporter assay was adopted to evaluate the binding relation between miR‐483‐3p and XPO1 3´ UTR. (e) The protein level of XPO1 in PC12 cells was detected by western blot analysis. (f), (g) The mRNA and protein levels of XPO1 in hippocampus of rats in Hcy, Hcy+NC, and Hcy+AAV‐miR‐483‐3p group (n = 10/group) were detected by PCR and western blot. (h) The expression correlation between miR‐483‐3p and XPO1 in hippocampus of Hcy‐treated rats (n = 20) was analyzed by Pearson's correlation analysis. ^* p < .05, ^** p < .01, ^*** p < .001

FIGURE 5

Silencing XPO1 inhibits the apoptosis of Aβ1‐40‐stimulated PC12 cells and downregulates levels of AD‐associated proteins. (a), (b) The expression levels of XPO1 in Aβ1‐40‐treated PC12 cells and its knockdown efficacy after transfection of sh‐XPO1 were detected by PCR and western blot. (c), (d) The viability and apoptosis of PC12 cells in four groups (Con, Aβ1‐40, Aβ1‐40+sh‐NC, and Aβ1‐40+sh‐XPO1) were examined by MTT and TUNEL assays. (e)–(h) Protein levels of apoptosis markers (Bcl‐2, Bax, and Cleaved caspase‐3) and AD‐associated factors (APP, BACE1, and Aβ1‐42) in PC12 cells of above four groups were quantified utilizing western blot. ^* p < .05, ^** p < .01, ^*** p < .001

FIGURE 6

XPO1 overexpression reverses the suppressive effect of miR‐483‐3p on cell apoptosis and protein levels of AD‐associated factors. (a) The overexpression efficacy of XPO1 in the context of miR‐483‐3p overexpression was validated by western blot analysis. (b), (c) The effects of overexpressing miR‐483‐3p and XPO1 on the viability and apoptosis of PC12 cells were examined by MTT assay and TUNEL assay. (d), (e) The effects of overexpressed XPO1 on apoptosis markers and APP, BACE1, and Aβ1‐42 protein levels in response to miR‐483‐3p overexpression were determined by western blot. ^* p < .05, ^** p < .01, ^*** p < .001