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. 2010 Jan;31(1):58-73.
doi: 10.1016/j.neurobiolaging.2008.03.001. Epub 2008 Apr 10.

Co-localization of the amyloid precursor protein and Notch intracellular domains in nuclear transcription factories

Uwe Konietzko  1 , Zoë V Goodger, Michelle Meyer, Bernhard M Kohli, Jérôme Bosset, Debomoy K Lahiri, Roger M Nitsch
Affiliations

Co-localization of the amyloid precursor protein and Notch intracellular domains in nuclear transcription factories

Uwe Konietzko et al. Neurobiol Aging. 2010 Jan.

Abstract

The beta-amyloid precursor protein (APP) plays a major role in Alzheimer's disease. The APP intracellular domain (AICD), together with Fe65 and Tip60, localizes to spherical nuclear AFT complexes, which may represent sites of transcription. Despite a lack of co-localization with several described nuclear compartments, we have identified a close apposition between AFT complexes and splicing speckles, Cajal bodies and PML bodies. Live imaging revealed that AFT complexes were highly mobile within nuclei and following pharmacological inhibition of transcription fused into larger assemblies. We have previously shown that AICD regulates the expression of its own precursor APP. In support of our earlier findings, transfection of APP promoter plasmids as substrates resulted in cytosolic AFT complex formation at labeled APP promoter plasmids. In addition, identification of chromosomal APP or KAI1 gene loci by fluorescence in situ hybridization showed their close association with nuclear AFT complexes. The transcriptional activator Notch intracellular domain (NICD) localized to the same nuclear spots as occupied by AFT complexes suggesting that these nuclear compartments correspond to transcription factories. Fe65 and Tip60 also co-localized with APP in the neurites of primary neurons. Pre-assembled AFT complexes may serve to assist fast nuclear signaling upon endoproteolytic APP cleavage.

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Figures

Figure 1
Figure 1
AICD is _targeted to nuclear spots with Fe65 dominating this localization in competition with other APP-binding proteins. (A) HEK 293 cells were treated with Leptomycin B (LMB, 10 or 20 ng/ml) for 24 hours to block nuclear export. Staining of endogenous APP with a C-terminal antibody reveals the accumulation of AICD in nuclear spots. (B-D) A clonal HEK 293 cell line with inducible expression of Citrine-AICD was co-transfected with CFP-Tip60 and a combination of HA-Fe65 and FLAG-Jip1b (B) or Myc-Fe65 and HA-X11α/MINT1 (C) or FLAG-Jip1b and HA-X11α (D). Confocal microscopy can clearly discriminate CFP, Citrine, Cy3 (red), Cy5 (Blue) and DAPI (grey) emissions. _targeting of AICD to nuclear spots was seen in all cells with Fe65 expression regardless of the co-expressed proteins. Bar, 10 μm.
Figure 2
Figure 2
AFT complexes associate with Cajal and PML bodies but are distinct from splicing speckles. Clonal AICD-expressing cells were transfected with CFP-Tip60 and HA-Fe65 to generate AFT complexes. Antibodies against marker proteins of nuclear compartments were used to identify the nature of AFT complexes. No co-localization was seen with Nucleoli (A) or splicing speckles (B). The few cases where AFT complexes are seen inside the nucleolar territorium are probably due to spots lying over the nucleolus. Staining of Cajal bodies (C) and PML bodies (D), both of which have a spherical structure shows that the majority of these compartments are closely associated with an AFT spot. Bar, 5 μm.
Figure 3
Figure 3
Live cell imaging shows that AFT complexes reside in highly mobile nuclear compartments that fuse upon inhibition of transcription. Live cell imaging was performed with the confocal microsope heated to 37°C. (A) Tip60 speckles imaged via the fused CFP were relatively stable, whereas AFT complexes visualized by the fused Citrine are highly mobile. The first lane shows a single confocal stack. In the second lane three consecutive stacks imaged 45 seconds apart are color-coded in RGB. In the third lane the consecutive stacks are imaged 90 seconds apart. HEK cells are mobile in culture, resulting in concomitant movement of the nucleus, which can be seen by red coloring of Tip60 speckles at the bottom and the blue at the top. On top of this movement AFT complexes are highly mobile with different velocities (see also supplementary movies). (B) Inhibition of transcription by ActD had no effect on Tip60 speckles but caused a rapidly initiated fusion of AFT complexes. (C) The different behavior of Tip60 speckle and AFT spot compartments is also mirrored in the fact that these two compartments do not co-localize in cells where not all of the Tip60 has been relocated to AFT complexes. Bar, 7.5 μm in A upper, 5 μm in A lower, 20 μm in B upper, 10 μm in B lower, 5 μm in C.
Figure 4
Figure 4
APP promoter sequences can induce the formation of extranuclear AFT complexes. (A) Co-transfection of Dig-labeled (red) promoter plasmids into cells transfected to generate AFT complexes induced the formation of extranuclear AFT complexes close to sites of labeled promoter plasmid (arrow). (B) Fe65 and Tip60 were transfected to generate AFT complexes and APP promoter plasmids were subsequently transfected 3 hours prior to fixation. Extranuclear AFT complexes can now be observed at greater distances from the nucleus (arrow) with nearby labeling of promoter DNA. (C) Extensive washing removes much of the extracellular promoter plasmid DNA, while extranuclear AFT complexes are still found to be associated with APP promoters. Bar, 5 μm.
Figure 5
Figure 5
AFT complexes are closely associated with the chromosomal loci of AICD-regulated genes. Fluorescence in situ hybridization was performed on clonal cells transfected to generate AFT complexes. (A) Hybridization with a BAC containing sequences of the 3′ end of the APP gene. Close association of the FISH signal with an AFT spot is marked by an arrow. (B) FISH signals from a BAC containing sequences from the APP promoter are similarly associated with AFT complexes (arrows). (C) Hybridization with a BAC containing sequences of the KAI1 gene. Two of the labeled genomic loci are associated with AFT complexes. Bar, 7.5 μm.
Figure 6
Figure 6
AICD expression increases the turnover of APP. (A) Autoradiograph of SDS PAGE from clonal AICD-expressing cell lines. Cells without or with induction of AICD expression were pulsed with Easy Tag 35S Label Mix and chased for various times. Immunoprecipitation with an antibody against the C-terminus of APP retrieves endogenous APP and the induced Citrine-AICD (arrows). Full-length APP quickly matures and is thereafter degraded. (B) Quantification at 45 minutes post pulse revealed a reduced half-life when Citrine AICD was expressed, which was significant for immature APP (n = 5). No changes in the levels of Citrine-AICD, either due to leakage expression or induction, where seen in 45 minutes.
Figure 7
Figure 7
NICD localizes to the same nuclear structures as AICD. (A) Transfection of Cerulean-NICD into HEK 293 showed that it localizes to nuclear complexes, whereas HA-Fe65 is distributed throughout the cells with diffuse accumulation in the nucleus. (B) Co-transfection of NICD and Fe65 _targeted Fe65 to the same nuclear complexes. (C) Clonal Citrine-AICD expressing cells were transfected with Myc-Tip60 and HA-Fe65 to generate AFT complexes. Co-transfected NICD localizes to all nuclear AFT complexes. (D) APP knockout fibroblasts were transfected with Fe65 and Tip60. Together they localize in nuclear complexes, whereas Tip60 alone is seen in speckles. (E) NICD transfected together with Tip60 and Fe65 in APP knockout fibroblasts localizes to identical nuclear complexes. Bar, 15 μm.
Figure 8
Figure 8
AICD localizes to nuclear AFT complexes in neurons and astrocytes. (A) Primary astrocytes (upper row) and neurons (lower row) were co-transfected with APP-Citrine, HA-Fe65 and CFP-Tip60. Spherical nuclear complexes are formed in both cell types. (B) Staining for endogenous APP shows that AICD is localized to nuclear spots. Inhibition of nuclear export with LMB leads to accumulation of AICD in spots. The lower row shows zoomed neuronal somata with the outline of the DAPI stained nucleus. Bar, 15 μm in A upper, 30 μm and 7.5 μm in A lower, 10 μm in B.
Figure 9
Figure 9
Tip60 undergoes nucleo-cytoplasmic cycling and co-localizes with APP and Fe65 throughout the neurites. (A) Myc-tagged Tip60 is localized to nuclear speckles in HEK 293. Increased photomultiplier gain revealed that Tip60 is also present throughout the cytosol. Cells were counterstained with two different DNA-binding dyes (DAPI and DRAQ5) that both stain nuclei. (B) Endogenous Tip60 localizes to nuclear speckles in HEK 293 cells. Inhibition of nuclear export with LMB for 24 hours leads to a strong accumulation of Tip60 in nuclear speckles as can be seen in confocal images acquired with identical photomultiplier settings. (C) Co-transfection of Myc-Tip60, RFP-Fe65 and APP-3HA into primary neurons cultured on astrocytes. Cells were fixed 20 hours after transfection and viewed in the confocal microscope. AFT complexes were formed throughout the neurites in control cells. Inhibition of γ-secretase with DAPT leads to a strong accumulation of AFT complexes in the neurites. Boxed insert is zoomed threefold. Bar, 15 μm in A, 10 μm in B 30 μm in C and 10 μm for the enlarged insert.
Figure 10
Figure 10
APP and Notch interact at different levels of their nuclear signaling pathways and their intracellular domains co-localize in transcription factories. Notch and APP can directly interact (1) at the plasma membrane (PM) (15, 40). Both proteins undergo ectodomain shedding by the same α-secretases and regulated intramembrane proteolysis by γ-secretase complexes (13, 29) leading to competition for these proteolytic cleavages (2) that are necessary for the translocation of the intracellular domains to the nucleus (N). Several adaptor proteins such as Fe65, Jip1 or numb can physically associate with AICD or NICD (3), thereby influencing their capacity of nuclear translocation and transcriptional activity (16, 19, 49). AICD is transported to the nucleus by Fe65 and together with Tip60 forms spherical nuclear AFT complexes (57) that represent transcription factories (TF). AICD and NICD co-localize in nuclear transcription factories, where they both can interact with Fe65 (4) and mutually influence transcriptional activity (17, 21). Transcription factories containing AFT complexes associate with the genomic loci of regulated genes, i.e. APP and KAI1. Increased generation of AICD enhances the expression of APP and the β-secretase BACE1 (57), leading to enhanced APP turnover due to this positive feedback loop. NICD can associate with Fe65 and AICD in transcription factories and negatively (21) or positively (17) modulate the expression of AICD-regulated genes.
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