Alzheimer’s disease associated AKAP 9 I2558M mutation alters posttranslational modification and interactome of tau and cellular functions in CRISPR‐edited human neuronal cells

Alzheimer's disease (AD) is a pervasive neurodegeneration disease with high heritability. Whole exome sequencing study identified two rare African American (AA)-specific variants in AKAP9, a kinase anchor protein 9, (rs144662445 and rs149979685), that increased the odds of AD more than 2.75-fold. However, the functional impact of the AKAP9 rs144662445 (I2558M) mutation in the central nervous system and the underlying mechanism in AD remain unclear.AKAP9 I2558M mutation was introduced by CRISPR-mediated knock-in and validated by Sanger sequence in SH-SY5Y cells stably expressing P301L Tau (SH-SY5Y P301L cells), which was further differentiated into neuronal cells. Tau phosphorylated level between AKAP9 WT and I2558M cells was evaluated by immunocytochemistry and ELISA assay. Moreover, we analyzed the tau interactome in AKAP9 I2558M cells by affinity purification-mass spectrometry. In silico analysis was performed to compare the differences between AKAP9 WT and mutation in tau-interacting proteins. The protein synthesis and oxidative activity in SH-SY5Y P301L neurons with and without I2558M were measured by using the Click-iT protein synthesis kit and CellRox dye, respectively.No difference in the neuronal cell morphology was observed between differentiated WT and mutant SH-SY5Y P301L cells. The AD-associated AKAP9 I2558M mutation resulted in a significant increase in phosphorylated tau protein at residues S396 and S404 site in a differentiated SH-SY5Y P301L cell line. The presence of this mutation altered the composition of tau-interacting proteins, by increasing interaction of proteins associated with translation, RNA localization and oxidative activity. Importantly, functional studies showed a significant reduction in protein synthesis activity and excessive oxidative stress in AKAP9 mutant compared to wild type cells.Our results demonstrated specific effects of rs14462445 on mis-processing of tau and suggest a potential role of AKAP9 in AD pathogenesis.