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Fig. 3 | Acta Neuropathologica Communications

Fig. 3

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

Fig. 3

PML forms container-like structures within the complex and precipitates with APP-CT. (A) A direct co-localization of APP-CT (blue, BFP) with PML (green, EGFP) was only observed to some extent (cells with nuclear aggregates), but most cells revealed a uniform staining pattern of APP-CT50 and PML in the cytosol and nucleus. (B) Additional co-expression of FE65 (red, mcherry) enriched the aggregation of PML in the nucleus, but a strong co-localization to APP-CT50/FE65 was not observable in the imaging study. (C) Additional transfections showed that p53 (red, mCherry) is part of the PML aggregates in the nucleus that also contained APP-CT50, as indicated by the visible co-localization (for example see white arrows). (D) Notably, another suspected binding partners for PML like the DNA helicase BLM (blue, BFP), which was identified as binding protein in the APP-CT50 complex, is not co-localized with the PML aggregates. (E) Confocal 3D imaging validated the co-localization of PML (green, EGFP) and APP-CT50 (red, mCherry) in the nucleus (FE65/TIP60 were co-transfected w/o fluorophore). (F) Interaction of APP-CT with PML was shown using co-immunoprecipitation assay. Precipitation using anti-GFP antibody revealed detection of APP-CT-EGFP (as expected) as well as PML (PML1 isoform was used, white arrow). This was true for two different APP-CT isoforms (APP-CT50 and CT57), whereas control conditions revealed no unspecific co-precipitation. The before mentioned isoforms of APP’s c-terminal domain differ in their respective amino acid length generated through ε-cleavage. While APP-CT50 represents the most common form, APP-CT57 is less common.Results were the same for two different PML1 tags: in the left panel of blots PML1-HA was used, whereas PML1-myc was used in the right panel. (G) High-resolution STED imaging specified the localization of APP-CT50 (green, EGFP) within the PML bodies (red, mCherry). Different phenotypes were evident, either with a uniform localization within the bodies or with APP-CT50 signal at the inside wall of the PML body

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Acta Neuropathologica Communications

ISSN: 2051-5960

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