The molecular mechanism of miRNA-195-5p regulating ERK involvement in abnormal phosphorylation of Tau protein by aluminum maltol in PC12 cells
Authors: Gao Dan , Yin Jinzhu , Zhang Yunwei , Zhao Dan , Han Xiao , Huan Jiaping , Wang Tianshu , Xu Shimeng , Wang Linping , Song Jing , Zhang Huifang , Niu Qiao , Lu Xiaoting 
Published: December 2023

Abstract
Although aluminum is ubiquitously present on Earth, it is not necessary for life. Aluminum is a metal element that can induce neurotoxicity. The neurotoxicity of aluminum is mainly caused by the aggregation of abnormally phosphorylated tau protein to form neurofibrillary tangles (NFT). The phosphorylation of tau is regulated by both kinases and phosphatases. ERK is involved in PHF-type tau hyperphosphorylation. Recent studies have revealed that the interaction between microRNAs (miRNAs) and the ERK/MAPK cascade is related to maintaining the normal function of the nervous system. miR-195 is involved in the early development of AD with a potential impact on cognition. Therefore, we speculate that miRNA-195 may regulate ERK activity, thereby causing hyperphosphorylation of tau protein and neurotoxicity. Objective: To explore the role of miRNA-195-5p in regulating ERK in the process of Al (mal)3-induced tau hyperphosphorylation. Methods: PC12 cells were exposed to Al(mal)3. The ERK activation inhibitor U0126 and miRNA-195-5p plasmid were selected for intervention. The exposure groups included the control group, 100 µmol/L Al(mal)3 group, 200 µmol/L Al(mal)3 group, and 400 µmol/L Al(mal)3 group. The intervention groups of U0126 included the control group, 200 µmol/L Al(mal)3 group, DMSO group, 50 µmol/L U0126 group, and 50 µmol/L U0126 + 200 µmol/L Al(mal)3 group. The intervention groups of miRNA-195-5p included the control group, 200 µmol/L Al(mal)3 exposure group, blank plasmid group, and miRNA-195-5p overexpression + 200 µmol/L Al(mal)3 group. Cell viability was detected by CCK8 assay. The distribution of aluminum and the expression of tau protein in PC12 cells were observed by immunofluorescence. ERK, P-ERK, tau5, PHF and NFT were detected by Western blotting. mRNA-ERK and miRNA-195-5p were detected by RT‒PCR. Results: The fluorescence signal showed that aluminum is mainly distributed in the cytoplasm. As the concentration of Al(mal)3 increases, the fluorescence signal gradually increases. With the increase in Al(mal)3 concentration, PC12 cell viability decreased, the expression of miRNA-195-5p decreased, and the expression of P-ERK, tau5, PHF and NFT increased. After U0126 intervention, the expression levels of tau5, PHF, and NFT protein in the U0126 + 200 µmol/L Al(mal)3 group decreased compared to those in the 200 µmol/L Al(mal)3 group. There is an interaction effect between U0126 and Al(mal)3on the expression of tau5, PHF, and NFT proteins. After transfection with microRNA-195-5P, the expression levels of tau5, PHF, and NFT protein in the miRNA-195-5p overexpression + 200 µmol/L Al(mal)3 group decreased compared to those in the the 200 µmol/L Al(mal)3 group. There is an interaction effect between miRNA-195-5p and Al(mal)3on the expression of tau5, PHF, and NFT proteins. Conclusion: miRNA-195-5p regulates ERK involvement in the abnormal phosphorylation of Tau protein by aluminum maltol in PC12 cells.