Fig 1: HistopathologyHistopathology of patient II-1 TA biopsy (A–E, G–K, M–N, P) shows myopathic changes: fiber size variation and numerous internal nuclei, some with central pallor in H&E (A, arrowheads) and rimmed vacuoles in Herovici (B, arrowheads). Subsarcolemmal TNPO3 accumulation is observed in TNPO3 IHC staining (C, arrowheads). A fiber with small cytoplasmic bodies is seen in Gomori trichrome staining (D, black arrowhead) and a fiber with myofibrillar pathology (D, white arrowhead). In a serial section, mitochondrial NADH staining reveals an uneven staining pattern in central parts of the muscle fibers (E). Gomori trichrome of patient I-3 TA biopsy (F) shows a ragged red fiber (black arrowhead) and myofibrillar pathology (white arrowhead). Large (arrowhead) and small myotilin accumulations are observed in IHC staining (G). In IF double staining, p62 (green) and TDP-43 (red) colocalize in inclusion bodies (H, arrowheads) in the rimmed vacuolar fiber. The mitochondrial CHCHD10 shows subsarcolemmal accumulation in several muscle fibers (I). IF double staining of desmin (green) and alpha-B-crystallin (red) shows both cytoplasmic and subsarcolemmal overexpression and colocalization (J, arrowheads), as does desmin (green) and tropomyosin (red) in K. Notably, desmin (green) and tropomyosin (red) show overexpression in patient III-1 muscle at only age 16 months (L). In confocal microscopy, RBM4 (green) is often excluded from the nuclei (M, arrowheads) in the patient biopsies, whereas SRRM2 (green) in N shows strictly nuclear localization in the patient, a pattern similar to control muscles. Confocal analysis of TNPO3 shows normal nuclear localization in patient III-1 (O), but often perinuclear accumulation of TNPO3 in adult patient biopsy (P, arrowheads), which shows as a nuclear rim. (A–L), scale bar = 100 μm; (M–P), scale bar = 50 μm. IF = immunofluorescent; IHC = immunohistochemistry; TA = tibialis anterior; TNPO3 = transportin-3.
Fig 2: Erk1/2 phosphorylates SRRM2 at Ser1068. a Kinase prediction by NetworKIN (http://networkin.info/). Erk1/MAPK3 and Clk1 showed scores of >1.0 for Ser1068 of mouse SRRM2 and the counterpart Ser1103 of human SRRM2. b Western blot detection of Erk1/2 (MAPK3/1) in the cerebral cortex of 5xFAD mice from 1 to 12 months of age. The lower graph shows the results of quantitative analyses. c Downstream target proteins of Erk1/2 but not of Clk1 are activated in 5xFAD mice. Proteins shown in red indicate increased phosphorylation at 1 month of age in whole cerebral cortex tissues of 5xFAD mice. White: detected by mass analysis but unchanged, red: detected and increased significantly in Welch’s test with post-hoc BH procedure (p < 0.05), gray: undetected by mass analysis. Diamond: kinase protein, circle: non-kinase protein. In lower panel, the ratio of phosphorylated proteins / total proteins was compared between inside and outside of kinase downstream by Fisher’s exact test (p < 0.05). The high quality figure is posted at http://suppl.atgc.info/021/. d To confirm that these activated kinases are responsible for SRRM2 phosphorylation at Ser1068, in vitro phosphorylation was performed with SRRM2 peptide substrate (GSLSRSSSPVTELTARSPVK) and Erk1/MAPK3, Erk2/MAPK1, or Clk1 was purified followed by detection of phosphorylated substrates by mass spectrometry. The peaks in LC that were identified by MS/MS as phosphorylated or non-phosphorylated SRRM2 peptides are indicated. e SiRNA-mediated knockdown of Erk1 or Erk2 suppresses SRRM2 phosphorylation at Ser1068. Lower graphs show quantitative analyses of band signal intensities from three mice (N = 3). Double asterisks indicate p < 0.01 in Tukey’s HSD test
Fig 3: pSer1068 prevents interaction with TCP1a and decreases SRRM2. a Experimental procedure to detect binding proteins specific to phosphorylated and/or non-phosphorylated SRRM2. b A specific band was detected in the precipitate of the non-phosphorylated SRRM2 mimicry mutant (1068 A) and more faintly in the precipitate of the wild-type SRRM2. #1 and #2 were cut out from the gel and subjected to mass spectrometry. c Results of mass spectrometry of the bands cut out from the silver stained gel. d Phosphorylated/non-phosphorylated SRRM2 mimicry mutants were transiently expressed in HeLa cells, and interaction with endogenous TCP1a was tested by immunoprecipitation. e Primary cortical neurons prepared from mouse E15 embryos were transiently transfected with TCP1a or scrambled siRNA, and multiple protein levels in the nucleus and cytoplasm fractions were evaluated by western blot analysis. f Immunocytochemistry of primary neurons confirmed that TCP1a-KD decreased SRRM2. g The TCP1a-KD-induced reduction of the SRRM2 protein was inhibited by addition of MG132 to the culture medium
Fig 4: 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-Aß antibody (82E1). No extracellular Aß aggregates were detected, while intracellular Aß accumulation was detected at 1 month of age. Extracellular Aß 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-Aß 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-Aß antibody revealed that intracellular Aß 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
Fig 5: SRRM2 deficiency destabilizes PQBP1. a Interactome database (String ver.10.5) predicted the interaction of PQBP1 and SC35 with SRRM2. b Immunoprecipitation revealed the interaction between endogenous SRRM2 and PQBP1 or between SRRM2 and SC35. c SiRNA-mediated SRRM2 knockdown reduced PQBP1 in primary cortical neurons prepared from E15 mouse embryos (white arrow). d Expression of EGFP-wild-type or 1068A-mutant SRRM2 recovered PQBP1, whereas EGFP-1068D-mutant or 1068E-mutant SRRM2 did not recover PQBP1 sufficiently. e Western blot analysis confirmed SRRM2 knockdown by siRNA and the accompanying decrease of PQBP1 in HeLa cells. f Western blot analysis of whole cerebral cortex lysates from 5xFAD mice and sibling mice (B6/SJL) at each age revealed that PQBP1 decreased during aging and the decrease occurred more rapidly in 5xFAD mice. g Immunohistochemistry was performed with parietal lobe tissues of human AD patients with PS1 Met146Leu mutation (N = 3) and non-neurological disease controls (N = 3). Reduction of SRRM2 and PQBP1 was obvious in AD patients, and representative images of cortical neurons are shown. h Western blot analysis was performed with the same AD and non-neurological disease patients. The right graphs show the quantitative analysis of ß-actin-corrected PQBP1 and SRRM2 signals (N = 3, *p < 0.05, Student’s t-test). i Human iPS cells carrying the APP KM670/671NL heterozygous or homozygous mutation generated by Crispr-cas9 based genome editing were used to show that SRRM2 and PQBP1 are lower in the nuclei and that pSer1068-SRRM2 is increased in the cytoplasm of differentiated neurons. Cytoplasmic levels of TCP1a in human iPS cells carrying the APP KM670/671NL mutation and in human normal iPS cell-derived neurons were similar. j Western blot analysis of neurons differentiated from human iPS cells carrying the APP KM670/671NL heterozygous or homozygous mutation. Right panels show quantification of the band intensities
Supplier Page from Abcam for Anti-SRRM2 antibody