Circ-Vps41 positively modulates Syp and its overexpression improves memory ability in aging mice

Abstract

Introduction: Age is an established risk factor for neurodegenerative disorders. Aging-related cognitive decline is a common cause of memory impairment in aging individuals, in which hippocampal synaptic plasticity and hippocampus-dependent memory formation are damaged. Circular RNAs (circRNAs) have been reported in many cognitive disorders, but their role in aging-related memory impairment is unclear.Methods: In this study, we aimed to investigate the effects of circ-Vps41 on aging-related hippocampus-dependent memory impairment and explore the potential mechanisms. Here, D-galactose was used to produce a conventional aging model resulting in memory dysfunction.

Results: Circ-Vps41 was significantly downregulated in D-galactose-induced aging in vitro and in vivo. The overexpression of circ-Vps41 could upregulate synaptophysin (Syp), thereby promoting the synaptic plasticity and alleviating cognitive impairment in aging mice. Mechanistically, we found that circ-Vps41 upregulated Syp expression by physically binding to miR-24-3p. Moreover, the miR-24-3p mimics reversed the circ-Vps41 overexpression-induced increase in Syp expression.

Discussion: Overexpression of circ-Vps41 alleviated the synaptic plasticity and memory dysfunction via the miR-24-3p/Syp axis. These findings revealed circ-Vps41 regulatory network and provided new insights into its potential mechanisms for improving aging-related learning and memory impairment.

Keywords: aging; circ-Vps41; learning and memory; miR-24a-3p; synaptophysin.

Figure 1

circ-Vps41 expression was downregulated in the D-galactose-induced aging model. (A) Cell growth assays (CCK8) demonstrate that treatment with D-galactose leads to decreased viability in HT22 cells, which (B,C) is confirmed by the high percentage of senescent cells in β-galactosidase staining (scale bars = 200 μm). ANOVA was used to test for differences among all groups. (D) The expression level of circ-Vps41 was tested by qRT–PCR in HT22 cells in the control (con) and 75 mM D-galactose (D-gal) groups. The con group was normalized to 1. (E) SA-β-gal-positive cells could be observed in the hippocampus at CA1, CA3 and DG areas of D-galactose-induced aging mice. (Scale bars = 100 μm). (F–H) Digitized for analysis by ImageJ software, stained levels of SA-β-gal-positive cells for D-gal group at CA1, CA3 and DG areas of the hippocampus in the ipsilateral side compared to the control groups (n = 4 for control group, n = 4 for the D-gal group). (I,J) ORT test of mice at 2 and 24 h. (K) Time taken by mice to reach the platform (average latency) during the training trials. (L) Percentage of time in the target quadrants of mice during the training trials. (M,N) Numbers of platform crossings of mice and representative swimming paths in the 7-day MWM. (O,P) Average swim speed and total path length by mice to reach the platform during the training trials (n = 8 for the control group, n = 8 for the D-gal group). (Q) The expression level of circ-Vps41 was tested by qRT–PCR in the hippocampus of mice in the control and D-gal groups (n = 3 for the control group, n = 3 for the D-gal group). The control group was normalized to 1. Data are shown as the mean ± SEM. p values, two-tailed t-test, one-way ANOVA. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 2

Overexpression of circ-Vps41 improves dendritic spine density and Syp expression in D-galactose-induced aging mice (A) Representative image of stereotactic injections in the hippocampal CA1 area. (Scale bars = 500 μm). (B) The expression level of circ-Vps41 was tested by qRT–PCR in the hippocampus of mice (n = 4 for the control+AAV-GFP group, n = 4 for the D-gal+AAV-GFP group, n = 4 for the D-gal+AAV-circ-Vps41 group). The control+AAV-GFP group was normalized to 1. (C,D) Representative images and quantification of Golgi-Cox staining in the hippocampus of the three groups of mice. (n = 3 for the control+AAV-GFP group, n = 3 for the D-gal + AAV-GFP group, n = 3 for the D-gal+AAV-circ-Vps41 group, scale bars = 10 μm). (E,F) Representative images and quantification of Syp in the hippocampus of the three groups of mice were tested by western blot. (G) The expression level of Syp mRNA was tested by qRT–PCR in the hippocampus of mice in the three groups. The control + AAV-GFP group was normalized to 1. (H–K) Representative images and quantification of Syp in the hippocampus of the three groups of mice were tested by immunohistochemical staining (n = 3 for the control + AAV-GFP group, n = 3 for the D-gal + AAV-GFP group, n = 3 for the D-gal + AAV-circ-Vps41 group, scale bars = 100 μm). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. control + AAV-circRNA NC group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. D-gal+AAV-GFP group; Data shown as the mean ± SEM. p values, one-way ANOVA.

Figure 3

circ-Vps41 regulates the expression of Syp in vitro (A) The knockdown and overexpression efficiency of circ-Vps41 were measured by qRT–PCR in HT22 cells. (B,C) Representative images and quantification of Syp in HT22 cells after knockdown and overexpression of circ-Vps41 were tested by western blot. (D) The expression level of Syp mRNA was tested by qRT-PCR in HT22 cells after knockdown and overexpression of circ-Vps41. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. Vec-NC group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. si-NC group. (E) The expression level of circ-Vps41 was tested by qRT–PCR after the overexpression of circ-Vps41 in D-galactose-induced aging HT22 cells. (F,G) Representative images and quantification of Syp after the overexpression of circ-Vps41 in D-galactose-induced aging HT22 cells were tested by western blot. (H) The expression level of Syp mRNA was tested by qRT-PCR after the overexpression of circ-Vps41 in D-galactose-induced aging HT22 cells. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. con + Vec-NC group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. D-gal + Vec-NC group. Data are shown as the mean ± SEM. p values, two-tailed t-test, one-way ANOVA. The controls group were normalized to 1.

Figure 4

circ-Vps41 acts as a sponge of miR-24-3p. (A) Predicted binding sites of miR-24-3p in circ-Vps41; the mutation sequence is shown in red. (B) The regulatory effect of circ-Vps41 on the expression of miR-24-3p was verified by dual-luciferase reporter gene detection. (C–E) Representative FISH images of circ-Vps41, miR-24-3p, and 18S in the CA1 region of the hippocampus; Scale bars = 50 μm. The Control group was normalized to 1. (F) The expression level of miR-24-3p was tested by qRT–PCR in HT22 cells after knockdown and overexpression of circ-Vps41. ^*p < 0.05, ^**p < 0.01 vs. Vec-NC group; ^#p < 0.05 vs. si-NC group. The Vec-NC group and si-NC were normalized to 1. (G) The expression level of miR-24-3p was tested by qRT–PCR in the hippocampus of D-galactose-treated aging mice after overexpression of circ-Vps41. (n = 3 for the D-gal + AAV-GFP group, n = 3 for the D-gal + AAV-circ-Vps41 group). The D-gal + AAV-GFP group was normalized to 1. Data are shown as the mean ± SEM. p values two-tailed t-test, one-way ANOVA. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 5

Circ-Vps41 improves Syp expression depending on miR-24-3p. (A) Predicted binding sites of miR-24-3p in the Syp mRNA 3′UTR, and the mutation sequence is shown in red. (B) The regulatory effect of miR-24-3p on the expression of miR-24-3p was verified by dual-luciferase reporter gene detection. (C,D) Representative images and quantification of Syp after the overexpression of miR-24-3p in HT22 cells were tested by western blot. (E) The expression level of Syp mRNA was tested by qRT–PCR after the overexpression of miR-24-3p in HT22 cells. The NC mimics group was normalized to 1. (F,G) Representative images and quantification of Syp were tested by western blot after cotransfecting Vec-circ-Vps41 and miR-24-3p mimics into HT22 cells treated with D-galactose. (H) The expression level of Syp mRNA was tested by qRT-PCR after cotransfecting Vec-circ-Vps41 and miR-24-3p mimics into HT22 cells treated with D-galactose. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. control + Vector + mimics NC group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. D-gal+circ-Vps41 + mimics NC group; The control + Vector + mimics NC group was normalized to 1. Data are shown as the mean ± SEM. p values, two-tailed t-test, one-way ANOVA. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 6

Overexpression of circ-Vps41 reversed D-galactose-induced learning and memory impairment in mice (A) Experimental schematic diagram. After 6 weeks of continuous intraperitoneal injection with D-galactose, the adeno-associated virus vector with fluorescence-specific overexpression of circ-Vps41 (AAV-circ-Vps41) and the control GFP were injected by brain stereotactic technology into the hippocampal CA1 area of D-galactose-treated mice. (B,C) ORT test of mice at 2 h and 24 h. (D,E) Total path length and average swim speed by mice to reach the platform during the training trials. (F,G) Time taken by mice to reach the platform and percentage of time in the target quadrants during the training trials. (H,I) Representative swimming paths of mice and numbers of platform crossings on Day 7 of the MWM (n = 8 for the control+AAV-GFP group, n = 8 for the D-gal+AAV-GFP group, n = 8 for the D-gal+AAV-circ-Vps41 group). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. control+AAV-circRNA NC group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. D-gal+AAV-GFP group; Data are shown as the mean ± SEM. p values, two-tailed t-test, one-way ANOVA, repeated measures and multivariate analysis of variance.

Figure 7

Schematic representation of the molecular mechanisms. circ-Vps41 acts as a competitive endogenous RNA and regulates the expression of Syp by sponging miR-24-3p, ultimately improving the learning and memory of aging mice.