Amyloid precursor protein causes fusion of promyelocytic leukemia nuclear bodies in human hippocampal areas with high plaque load

Abstract

The amyloid precursor protein (APP) is a type I transmembrane protein with unknown physiological function but potential impact in neurodegeneration. The current study demonstrates that APP signals to the nucleus causing the generation of aggregates comprising its adapter protein FE65 and the tumour suppressor proteins p53 and PML. The PML nuclear body generation, known to be of relevance in virus defence and cell division, is induced and fusion occurs over time depending on APP signalling. We further show that the nuclear aggregates of APP C-terminal (APP-CT) fragments together with PML and FE65 are present in the aged human brain but not in cerebral organoids differentiated from iPS cells.Notably, human Alzheimer's disease brains reveal a highly significant loss of these nuclear aggregates in areas with high plaque load compared to plaque-free areas of the same individual. Based on these results we conclude that APP-CT signalling to the nucleus takes DPBS (Gibco) and fixed in 4 % paraformaldehyde in PBS. Cover glasses were mounted with the Shandon™ Immu-Mount™ solution (Thermo Scientific) on glass slides and dried overnight at RT. For immunofluorescence staining, HEK293T cells were seeded in 8-well-µslide-ibi Treat (ibidi ® , Martinsried, Germany) and transfected using calcium phosphate transfection after 24 h. Cells were fixed with Roti ® -Histofix 4 % (4 % phosphate buffered formaldehyde solution; Roth, Karlsruhe, DE) for 20 min at 37 °C, and permeabilized and blocked with 5 % normal goat serum (NGS) in 0.3 % (w/v) Triton X-100/PBS for 30 min at RT. The cells were incubated with primary antibodies diluted in 1 % BSA/0.3 % Triton X-100/DPBS (mouse anti-HA (BioLegend, 901501; 1:1000), mouse anti-myc (NEB/Cell Signaling, 2276; 1:1500) o/n at 4 °C. For the secondary antibody staining and the cell staining, goat-anti-mouse AF568 (Invitrogen, A11004, 1:1000) was used, together with HCS CellMask™ Deep Red Stain (ThermoFisher Scientific, H32721, 1:5000), Hoechst33342 (10 mg/ml in H2O, Applichem, A0741, 1:1000) in DPBS (1 % BSA, 0.3 % Triton) and incubated for 1 h at RT.Immunoprecipitation HEK 293T cells were seeded in 10 cm dishes and co-transfection was performed 24 h after seeding. Whole cell extracts were prepared 24 h after transfection by scraping the cells from the dish with a cell scraper, washing the cell pellet in ice-cold PBS, extracting with 1 ml interaction buffer (50 mM Tris pH 8, 150 mM NaCl, 5 mM EDTA, 0.5% NP40, 1 mM DTT, 1 mM PMSF, 1x complete protease inhibitor cocktail), followed by sonication (15 s at 95 % amplitude) using a Sonopuls mini20 device (Bandelin, Berlin, Germany). The lysates were centrifuged (15,000 g, 15 min, 4 °C) and the supernatant was transferred to a new reaction tube. Input samples of the lysates were stored separately. Immunoprecipitation (IP) was carried out with the µMACS isolation kits for tagged proteins from Miltenyi Biotec (Bergisch-Gladbach, Germany). The eluates, as well as input samples of the lysates were subjected to SDS-PAGE and immunoblotting.