Fig. 4

Highly mobile APP-CT50-depending complexes that are also present in the aged human brain drive PML complex generation. (A) Expression of APP-CT50/ FE65/TIP60-HA in HEK293 cells reveals a highly mobile complex moving three-dimensionally in the cellular nucleus. Ring-like structures were coloured (orange to yellow) according to z-level (confocal microscopy). The indicated structure (white arrowhead) revealed time-dependent movement. The corresponding video is given in the supplement. (B) Movement of the individual aggregates was tracked using Huygens object tracker software. Transfection in HEK293 cells included APP-CT50/FE65-mCherry/TIP60-HA with and without EGFP-PML co-expression. The FE65-mCherry signal was used for tracking, revealing lower speed in cells co-expressing PML (first diagram). In addition, the distance from the track origin (at time point 0) was analysed. Co-expression of PML revealed significantly lower distances pointing to mutual trapping of both complexes. The mean speed was 0.38 in PML versus 0.76 µm/s in non-PML co-expressing cells (last diagram). (C) This part reveals a representative image demonstrating the complex generation of APP-CT50/FE65/TIP60 (red) and PML (green aggregates). (D) Time-dependent generation of nuclear APP-CT50/PML aggregates. PML (green, EGFP) expression revealed a uniform distribution within the nucleus and cytosol (first row). Co-expression of FE65 (red, mCherry) and APP-CT50 (blue, BFP) caused initial aggregate formation after 48 h (middle row). Additional expression of TIP60-HA (w/o fluorophore) (last row) showed early generation of nuclear aggregates after 24 h. 48 h after transfection large nuclear aggregates were observed (white arrow). (E) PML (green, FITC) and APP-CT50 (red, TRITC) co-localization was studied in human brain sections. In total, 15 human hippocampal sections were analysed (different Braak stages). Confocal tile-scan imaging (5 z-stacks, then fused by maximum projection algorithm) revealed strong co-localization of PML with APP-CT50. As in cell culture experiments, nuclei containing many small aggregates (arrow) as well as nuclei with larger aggregates (arrowhead) were evident. Co-localization is further shown by intensity tracking of both fluorescent channels in the diagram for a nucleus along the dotted white arrow. (F) Similarly, co-staining of PML (green, FITC) and FE65 (red, TRITC), which confirmed the association of both proteins in the nuclei of the human brain, was performed. (G) In order to address the question whether co-localization also occurs in non-aged tissue, we differentiated human cerebral organoids from induced pluripotent stem cells. Embryonic bodies were embedded in Matrigel at day 11 followed by neuronal induction to generate organoids, which were analysed after 30 days in culture (seeding at day 0). Staining of cryosections failed to demonstrate co-localization of APP-CT50 (red, TRITC) and PML (green, FITC)