MicroRNA-210-5p Contributes to Cognitive Impairment in Early Vascular Dementia Rat Model Through Targeting Snap25

Abstract

Vascular dementia (VD) is the most common form of dementia in elderly people. However, little is understood about the role of microRNAs (miRNAs) involved in cognitive impairment in early VD. Here, a VD model induced by chronic cerebral ischemia and fetal bovine serum (FBS)-free cell model that detects synapse formation was established to investigate the function of miRNAs in early VD. The microarray analysis and real-time reverse transcription polymerase chain reaction (RT-PCR) showed that miR-210-5p increased significantly in the hippocampus of rats with 4 weeks of ischemia. The VD model rats also displayed significant cognitive deficits and synaptic loss. The overexpression of miR-210-5p decreased the synaptic number in primary hippocampal neurons, whereas specific suppression of miR-210-5p resulted in the formation of more synapses. Additionally, intracerebroventricular (ICV) injection of miR-210-5p agomir to VD rats aggravated phenotypes of cognitive impairment and synaptic loss. These VD-induced phenotypes were effectively attenuated by miR-210-5p antagomir. Moreover, bioinformatic prediction revealed that synaptosomal-associated protein of 25 KDa (Snap25) mRNA is targeted by miR-210-5p. The miR-210-5p decreased the luciferase activities of 3' untranslated region (3'UTR) of Snap25 mRNA. Mutation of predicted miR-210-5p binding sites in the 3' UTR of Snap25 mRNA abolished the miR-210-5p-induced decrease in luciferase activity. Western blot and immunofluorescence staining confirmed that miR-210-5p targets Snap25. Finally, RT-quantitative PCR (qPCR) and immunofluorescence staining detected that miR-210-5p agomir downregulated Snap25 expression in the cornu ammonis1 (CA1) region of hippocampi in VD rats, whereas miR-210-5p antagomir upregulated Snap25 expression. Altogether, miR-210-5p contributes to cognitive impairment in chronic ischemia-induced VD model through the regulation of Snap25 expression, which potentially provides an opportunity to develop a new therapeutic strategy for VD.

Keywords: Snap25; cognitive impairment; miR-210-5p; synaptic loss; vascular dementia.

Figure 1

Chronic ischemic rat model exhibits the decline of spatial learning capability (n = 13/group) and synapse loss in the hippocampus relative to control group (n = 6/group). (A) The timeline of animal experiment. (B) The escape latency of individual rats was processed by a Morris water maze that lasts for 5 days (four times per day). (C) The number of platform crossings measured within 120 s. (D) The motor function was assessed by the swimming speed. (E) Synapses were indicated with black triangles; bar chart showed the number of synapses. Data are shown as mean ± standard deviation (SD) values, and statistical significance between both groups is defined as *p < 0.05; **p < 0.01.

Figure 2

The expression of microRNA (miRNA)-210-5p is significantly increased in hippocampal tissues of vascular dementia (VD) model rats when compared with control rats. (A) miRNA profile was measured through microarrary (n = 4/group). In cluster diagram, red color represents relatively high quantity of the miRNAs expression, and the green means relatively low. (B) mir210 expression was tested by quantitative PCR (qPCR; n = 6/group). Data are shown as mean ± SD values, and statistical significance between both groups is defined as **p < 0.01.

Figure 3

miR-210-5p downregulates postsynaptic density. (n = 3/group, 2 images per sample). (A) Green puncta were stained using Synapsin1 (Syn1) antibody, which represents pre-synapses. Red puncta stained with PSD-95 means post-synapses. The puncta colocalized with Syn1 and PSD-95 indicates functional synapses. (B) Quantification results of puncta in each channel; the colocalized synaptic density is normalized by pre-synapses respectively. Data are shown as mean ± SD values, and statistical significance between both groups is defined as *p < 0.05; **p < 0.01.

Figure 4

Intracerebroventricular (ICV) injection of miRNA-210-5p agomir aggregates cognitive impairment level and antagomir attenuates cognitive impairment in VD model (n = 13/group). (A) Timeline. (B–D) The latency, the moving trails of rats in platform crossing test and number of rats crossing over the platform. (E) Synapses were indicated with black triangles; a: Control group, b: Model group, c: Agomir group, d: Antagomir group; bar chart showed the number of synapses (n = 6/group). Data are shown as mean ± SD values, and statistical significance between both groups is defined as *p < 0.05; **p < 0.01.

Figure 5

miR-210 can specifically target on synaptosomal-associated protein of 25KDa (Snap25; n = 3/group, 2 images per sample). (A) The target sites of miR-210-5p in Snap25 mRNA 3’ untranslated region (3’UTR) were analyzed by Targetscan 7.0. (B) The construction profile of psiCHECK2-Snap25 wild-type (WT) and Mut vectors are shown above. Snap25 luciferase reporter (Snap25 3’ UTR WT) and the corresponding mutant construct (Snap25 3’ UTR Mut) were co-transfected into PC12 cells together with miR-210 mimics and inhibitor respectively. Luciferase activity was measured 24 h post transfection. Values are expressed relatively to the internal firefly luciferase activity. (C) Immunofluorescence and its intensity analysis showed miR-210-5p mimics decreased Snap25 protein expression in Primary neuron cells, whereas miR-210-5p inhibitor increased snap 25 protein expression. (D) Western blot results showed a decrease of Snap25 protein expression in primary neuron cells treated with miR-210-5p mimics, whereas they showed an increase of snap 25 expression in cells with miR-210-5p inhibitor. Data are shown as mean ± SD values, and statistical significance between both groups is defined as *P < 0.05; **p < 0.01.

Figure 6

ICV injection of miRNA-210 antagomir effectively restore hippocampal snap25 expression in VD model (n = 6/group). (A) miR-210-5p and Snap25 mRNA expression detected by qPCR (n = 6/group). (B) Snap25 protein expression visualized through immunofluorescence. (C) Snap 25 protein expression in hippocampus tested by western blot (n = 6/group). Data are shown as mean ± SD values, and statistical significance between both groups is defined as *p < 0.05; **p < 0.01.