Micro-RNA-137 Inhibits Tau Hyperphosphorylation in Alzheimer's Disease and Targets the CACNA1C Gene in Transgenic Mice and Human Neuroblastoma SH-SY5Y Cells

Abstract

BACKGROUND Alzheimer's disease (AD) results in cognitive impairment. The calcium voltage-gated channel subunit alpha-1 C CACNA1C gene encodes an alpha-1 C subunit of L-type calcium channel (LTCC). The aim of this study was to investigate the role of micro-RNA-137 (miR-137) and the CACNA1C gene in APPswe/PS1ΔE9 (APP/PS1) double-transgenic AD mice and in human neuroblastoma SH-SY5Y cells. MATERIAL AND METHODS Six-month-old APP/PS1 double-transgenic AD mice (N=6) and age-matched normal C57BL/6 mice (N=6) underwent a Morris water maze (MWM) test, expression levels of amyloid-β (Aβ), LTCC, the CACNA1C gene, and miR-137 were measured in the rat hippocampus and cerebral cortex in both groups of mice. A luciferase assay was used to evaluate the effect of miR-137 on the expression of CACNA1C in SH-SY5Y human neuroblastoma SH-SY5Y cells. Western blotting was used to detect the CACNA1C, phosphorylated-tau (p-tau), and Aβ proteins. RESULTS In APP/PS1 transgenic AD mice, spatial learning and memory was significantly reduced, levels of Aβ1-40 and Aβ1-42 were increased in the serum, hippocampus, and cerebral cortex, expression levels of miR-137 were reduced, expression of CACNA1C protein was increased in the hippocampus and cerebral cortex, compared with normal control mice. miR-137 regulated the expression of the CACNA1C gene. Increased expression levels of p-tau (Ser202, Ser396, and Ser404) induced by Aβ1-42 were inhibited by miR-137 mimics in SH-SY5Y human neuroblastoma cells in vitro. CONCLUSIONS In a transgenic mouse model of AD, miR-137 and expression of the CACNA1C gene inhibited the hyperphosphorylation of tau protein.

Figure 1

Behavioral evaluation of the APPswe/PS1ΔE9 (APP/PS1) double-transgenic mouse Alzheimer’s disease (AD) model by the Morris water maze (MWM) test. (A) The mean daily latencies of escape in the normal C57BL/6 control mice (N=6) and the APPswe/PS1ΔE9 (APP/PS1) double-transgenic Alzheimer’s disease (AD) mouse model (N=6). (B) Representative tracks for the AD mouse model and the normal C57BL/6 control mice in the Morris water maze (MWM) test at day 6. (C, D) The times across the platform and percentage of time in each platform quadrant, at day 6 of the MWM. The comparison is between the two groups, the AD mouse model and the normal C57BL/6 control mice. Statistically significant differences between groups are denoted by * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

Figure 2

Levels of amyloid-β (Aβ) peptides, Aβ_1–40 and Aβ_1–42 in the mouse serum, hippocampus, and cerebral cortex in the APPswe/PS1ΔE9 (APP/PS1) double-transgenic Alzheimer’s disease (AD) mice and the normal C57BL/6 control mice, measured by enzyme-linked immunosorbent assay (ELISA). (A, B) Levels of the Aβ_1–42 amyloid-β (Aβ) peptides Aβ_1–40 (A) and Aβ_1–42 (B) in the normal C57BL/6 control mice (N=6) and the APPswe/PS1ΔE9 (APP/PS1) double-transgenic Alzheimer’s disease (AD) mice (N=6) are measured by an enzyme-linked immunosorbent assay (ELISA). Differences between groups are denoted by * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

Figure 3

Expression levels of micro-RNA-137 (miR-137), the CACNA1C protein, and amyloid-β (Aβ) in the normal C57BL/6 control mice (N=6) and the APPswe/PS1ΔE9 (APP/PS1) double-transgenic Alzheimer’s disease (AD) mice (N=6). (A) The expression levels of micro-RNA-137 (miR-137) in the hippocampus and cerebral cortex of healthy control mice (N=6) and the APP/PS1 Alzheimer’s disease (AD) mice are measured by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. (B) Western blot shows that the protein level of calcium voltage-gated channel subunit alpha-1 (CACNA1C) is increased in the APP/PS1 AD mice. (C) The immunofluorescence (IF) assay shows increased expression levels of CACNA1C protein and amyloid-β (Aβ)-containing plaques in both the hippocampus and cerebral cortex in APP/PS1 AD mice compared with healthy controls. Scale bar, 100 μm.

Figure 4

micro-RNA-137 (miR-137) targets the CACNA1C gene in human neuroblastoma SH-SY5Y cells. (A) (left panel). The predicted micro-RNA-137 (miR-137) binding sites on the 3′-UTR of CACNA1C transcript (from TargetScanHuman, Version 7.1, June 2016). (A) (right panel). Luciferase reporter assay on the interaction between miR-137 and CACNA1C 3′-UTR. A mutant CACNA1C (mut-CACNA1C) is tested to validate the specific binding. (B) The regulatory effects of miR-137 on the expression of CACNA1C in human neuroblastoma SH-SY5Y cells. The expression levels of CACNA1C protein measured by Western blot analysis. Statistically significant differences between groups are denoted by * P<0.05.

Figure 5

micro-RNA-137 (miR-137) suppresses the phosphorylation of tau protein (p-tau) in human neuroblastoma SH-SY5Y cells. (A, B) The levels of p-tau (ser202), p-tau (ser396), p-tau (ser404) and tau protein in amyloid-β (Aβ) peptide Aβ_1–42-induced human neuroblastoma SH-SY5Y cells transfected with micro-RNA-137 (miR-137) mimics or miR-137 inhibitors are analyzed by Western blot.