Association of CaMK2A and MeCP2 signaling pathways with cognitive ability in adolescents

Abstract

The glutamatergic signaling pathway is involved in molecular learning and human cognitive ability. Specific single variants (SNVs, formerly single-nucleotide polymorphisms) in the genes encoding N-methyl-D-aspartate receptor subunits have been associated with neuropsychiatric disorders by altering glutamate transmission. However, these variants associated with cognition and mental activity have rarely been explored in healthy adolescents. In this study, we screened for SNVs in the glutamatergic signaling pathway to identify genetic variants associated with cognitive ability. We found that SNVs in the subunits of ionotropic glutamate receptors, including GRIA1, GRIN1, GRIN2B, GRIN2C, GRIN3A, GRIN3B, and calcium/calmodulin-dependent protein kinase IIα (CaMK2A) are associated with cognitive function. Plasma CaMK2A level was correlated positively with the cognitive ability of Taiwanese senior high school students. We demonstrated that elevating CaMK2A increased its autophosphorylation at T286 and increased the expression of its downstream targets, including GluA1 and phosphor- GluA1 in vivo. Additionally, methyl-CpG binding protein 2 (MeCP2), a downstream target of CaMK2A, was found to activate the expression of CaMK2A, suggesting that MeCP2 and CaMK2A can form a positive feedback loop. In summary, two members of the glutamatergic signaling pathway, CaMK2A and MeCP2, are implicated in the cognitive ability of adolescents; thus, altering the expression of CaMK2A may affect cognitive ability in youth.

Keywords: Calcium/calmodulin-dependent protein kinase IIα (CaMK2A); Cognitive function; Glutamatergic signaling pathway; Methyl-CpG binding protein 2 (MeCP2); Single-nucleotide variant (SNV).

Fig. 1

Schematic outline (not to scale) of the Flp-In™ stable cell lines with inducible expression of CaMK2A–DsR fused gene. The CaMK2A–DsR expression cassette is integrated into a distinct Flp recombination target site and is under the control of the Tet repressor. CaMK2A expression is induced by tetracycline. P: promoter; SV40: simian virus 40; CMV: cytomegalovirus; lacZ: β-galactosidase ORF, Amp: ampicillin ORF, DsR: DsRed ORF, pUC ori: Replication origin of plasmid pUC, TetO2: Tetracycline operator 2, and BGH pA: Bovine growth hormone polyadenylation signal

Fig. 2

Increased CaMK2A activated autophosphorylation. Total soluble protein from HEK293-derived cells was harvested at 2, 4, and 6 days after induction of CaMK2A expression with doxycycline. A Representative immunoblots displaying CaMK2A, phospho-CaMK2A (T286), GluA1, and phospho-GluA1 (S831) expression. H3.3B was considered the loading control. B, C Autophosphorylation of CaMK2A and phosphorylation of GluA1 increased after CaMK2A induction. Quantification of relative protein levels. Data are presented as mean ± standard deviation. *p < 0.05, n = 5, Student’s t test

Fig. 3

MeCP2 is involved in CaMK2A-mediated phosphorylation. Total soluble protein from stably transfected SH-SY5Y cells was harvested at 2, 4, and 6 days after induction of MeCP2 expression with doxycycline. A Representative immunoblots displaying phospho-MeCP2 (S80), MeCP2, phospho-CaMK2A (T286), CaMK2A, and pro-BDNF expression. H3.3B was considered the loading control. B, C Quantification of relative protein expression levels. Data are presented as mean ± standard deviation. *p < 0.05, n = 5, Student’s t test

Fig. 4

MeCP2 binds to GRIA1 and GRID1 promoters. ChIP was performed using anti-MeCP2 antibodies on sheared chromatin from SH-SY5Y neuroblastoma cells expressing MeCP2 on days 0, 2, 4, and 6. Purified DNA from immunoprecipitated chromatin was amplified using optimized primers for the promoters (GRIA1, GRID1, and GRIN1). Molecular weight markers are on the left in bases. Normal rabbit IgG was used as a negative immunoprecipitation control. +DNA: Purified chromosomal DNA, used as a positive control

Fig. 5

MeCP2 interaction with CaMK2A and CaMK2A phosphorylation signaling pathway plays a critical role in synaptogenesis and molecular mechanism of learning