Fig. 1.

SRRM2 phosphorylation at pSer1068 was increased in 5xFAD mice. a Immunohistochemistry of 5xFAD mouse brains at 1 and 6 months of age with the most sensitive anti-Aβ antibody (82E1). No extracellular Aβ aggregates were detected, while intracellular Aβ accumulation was detected at 1 month of age. Extracellular Aβ aggregates were detected throughout the brain except cerebellum at 6 months of age. b Mass analyses were performed with whole cortex tissues from 5xFAD mice at 1, 3, 6, and 12 months of age. Phosphorylation was higher at two sites (Ser1068 and Ser2535) of SRRM2 at 1 month in 5xFAD mice than in the background B6/SJL mice (N = 3, *p < 0.05). c Immunohistochemistry of 5xFAD and B6/SJL mice at 1, 3, and 6 months of age revealed an increase of pSer1068-SRRM2 in neurons. Cytoplasmic staining of neurons was ubiquitously detected in the retrosplenial dysgranular cortex (RSD) and frontal association cortex (FrA) in 5xFAD mice at 1 month, whereas the signals were lower at later ages. The other brain regions are shown in Supplementary Fig. 2. Quantitative analyses of intensities are shown in graphs at each time point and in each area. Signal intensities were determined in six cytoplasmic areas of a single cell, and the mean was adjusted to the background intensity. The corrected mean values from 30 cells in each brain area were used to calculate the representative value of a mouse. Statistical analysis was performed with the values of three mice in each area at each time point by Student’s t-test. Right panels show co-staining of pSer1068-SRRM2 with MAP2 or GFAP. d Western blot analyses of mouse cortex tissues at 1, 3, 6, and 12 months confirmed the increase at 1 month and subsequent decline of pSer1068-SRRM2 in 5xFAD mice. e FACS-sorted cells from cerebral cortex of 5xFAD mice at 1 month were blotted with anti-pSer1068-SRRM2 antibody (left panels) or anti-Aβ antibody (82E1) (right panels). The ratios of neurons (MAP2-positive), astrocytes (GFAP-positive) and other cells (MAP2-negative and GFAP-negative) are shown in lower graph. Western blot with anti-Aβ antibody revealed that intracellular Aβ already formed ADDLs or protofibrils and a small part of reached to the fibril state. f Intracellular localization of EGFP-tagged phosphorylation mimicry mutants (S1068A, S1068D, and S1068E) of SRRM2 in Hela cells and primary cortical neurons prepared from E15 mouse embryos 48 h after transient transfection