To explore whether N⁶-methyl adenosine (m⁶A) is involved in arsenic-induced tau protein phosphorylation.

Neuroblastoma (SH-SY5Y) cells were treated with 0, 1, 5, 10 μmol/L sodium arsenate for 24 hours. Then, the intracellular m⁶A level was detected, the mRNA expression levels of m⁶A-related enzymes in the cells were detected by qPCR,and the total tau protein expression level, phosphorylated tau protein level and m⁶A-related enzyme expression levels in the cells were detected by Western Blot. After inhibiting the intracellular m⁶A level with 3-deoxyadenosines, the changes in the intracellular m⁶A level and tau protein phosphorylation level were verified. SPSS was used for analysis of variance of the experimental results, with α=0.05.

After SH-SY5Y cells were treated with various concentrations of arsenic for 24 hours, there was no significant difference in the total tau protein level in the cells (F=3.047, P > 0.05). After the cells were treated with 5 μmol/L arsenic for 24 hours, the intracellular m⁶A level increased by 31.4% (F=4.511, P < 0.05), and the phosphorylated tau protein (at site T231) level increased by 42.6% (95%CI: 0.165-0.689, P < 0.01). The level of phosphorylated tau protein (at sites S202 + T205) increased with the increase in arsenic concentration, and the highest increase was 55.2%(95%CI: 0.050-0.409, P < 0.05) after treatment with 10 μmol/L arsenic for 24 hours. As the arsenic treatment concentration increased, the METTL3 mRNA expression in the cells increased, with the highest increase of 73.2% (95%CI: 0.201-1.423, P < 0.05) at a concentration of 10 μmol/L. The mRNA expression levels of METTL14, WTAP and FTO decreased, and they decreased to 65.4% (95%CI:-1.055 to-0.337, P < 0.01), 64.8% (95%CI:-0.389 to -0.111, P < 0.05) and 85.4% (95%CI: -0.030 to -0.010, P < 0.01) of the control group respectively after treatment with 10 μmol/L arsenic. The ALKBH5 mRNA expression first increased and then decreased, with an increase of 27.5% (95%CI: 0.033-0.147, P < 0.05) after treatment with 1 μmol/L arsenic for 24 hours; while it decreased by 30.7% (95%CI:-1.62 to -0.038, P < 0.01) after treatment with 10 μmol/L arsenic. Arsenic treatment led to an increase in METTL3 protein expression, with the highest increase of 107.1% (95%CI: 0.331-1.009, P < 0.01) after treatment with 5 μmol/L arsenic for 24 hours, while the protein expression levels of METTL14, WTAP and ALKBH5 decreased to 20.4% (95%CI: -0.788 to -0.509, P < 0.001), 23.5% (95%CI:-1.371 to -0.685, P <0.001) and 49.2% (95%CI:-0.423 to -0.183, P < 0.001) of the control group respectively after treatment of SH-SY5Y cells with 10 μmol/L arsenic for 24 hours. The FTO protein expression level showed a decreasing trend with the increase in arsenic concentration, with the lowest decrease of 45.3% (95%CI:-0.709 to -0.413, P < 0.001) after treatment with 10 μmol/L arsenic for 24 hours. After DAA inhibited the intracellular m⁶A level, the phosphorylated tau protein levels were significantly decreased (P < 0.05).

Arsenic can increase the m⁶A level in SH-SY5Y cells by increasing the expression level of the m⁶A methylase METTL3 and decreasing the expression levels of the m⁶A demethylases FTO and ALKBH5, thereby inducing the phosphorylation of tau protein in the cells.