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First published online August 26, 2004
doi: 10.1242/10.1242/jcs.01323

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The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor

¹ Division of Psychiatry Research, University of Zurich, August Forel-Str. 1, 8008 Zurich, Switzerland
² Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Kita 12, Nishi-6, Sapporo, 060-0812, Japan

View larger version (46K): [in a new window]   Fig. 1. Confocal sections through nuclei show that AFT-complexes of AICD, Fe65 and Tip60 are localized to spherical nuclear spots. (A,B) Both AICD (yellow) and Fe65 (red) localized homogenously throughout nuclear (blue) and cytosolic compartments in transfected HEK293 cells. Co-expression of Fe65 and AICD resulted in the same localization. (C) In the absence of AICD and Fe65 co-expression, the histone acetylase Tip60 (cyan) localized to speckle-like nuclear structures. (D) In the absence of Fe65 co-expression, Tip60 had no effect on AICD distribution. (E) In the absence of AICD, Fe65 colocalized with Tip60 in nuclei and reorganized the speckle-like morphology into spherical nuclear spots. (F,G) In cells co-expressing AICD, Fe65 and Tip60, AICD was relocalized from the cytosol into AFT-complexes predominantly found in spherical nuclear spots. AFT-complexes were found throughout nuclei in all cells co-expressing the three proteins. Confocal xz sections of the same nuclei confirmed nuclear localization. (H) The NASA mutant of the putative Fe65-binding motif NKSY in Tip60 ineffectively localized some Fe65 and AICD to speckle-like nuclear structures, but completely abolished the formation of spherical nuclear spots. Bar, 10 µm.

View larger version (39K): [in a new window]   Fig. 3. Fe65 transports AICD to nuclei where it can regulate transcription. (A) Blocking nuclear export with leptomycin B for 9 hours leads to an accumulation of Fe65 and AICD in nuclei. In cells co-expressing Fe65 and AICD, leptomycin B caused the formation of nuclear AICD-containing spots that were never observed in cells without the cotransfection of Fe65, or in the absence of leptomycin B (compare Fig. 1A,B). Bar, 10 µm. (B) Leptomycin B (24 hours) increased fluorescence signal intensity ratios of nuclear versus extranuclear staining for AICD and Fe65 in co-expressing cells. Without the expression of Fe65, leptomycin B treatment did not change the nuclear/extranuclear signal ratio for AICD. (C) Western blots of nuclear (n) and post-nuclear supernatant (PNS) fractions from AICD- and Fe65-expressing cells treated with lepomycin B (0 to 24 hours) showed increased nuclear retention of Fe65. The nuclear/PNS signal ratio for Fe65 increased to 247% after 9 hours and to 527% after 24 hours, as compared with a 100% baseline. Marker proteins are 19, 28, 39, 51, 64, 97 and 191 kDa. (D,E) Real-time PCR of clonal cell lines with and without the induced expression of citrine-AICD revealed that AICD increased the expression of APP, BACE and Tip60 (Mann-Whitney U, P<0.05). AICD did not change the expression of Fe65, Tip60, ADAM10, PSEN1, or the Notch-effector gene Hes1. The AICD-induced expression of KAI1 and Gsk3ß was confirmed. All values were normalized against GAPDH expression levels and expressed as fold baseline without induction of AICD expression. Data represent means ± s.e.m. of five experiments, each done in triplicate. (F,G) Clonal HEK293 grown for 48 hours without (F) or with (G) the induction of AICD-citrine expression. 6E10-staining for endogenous APP reveals increased protein levels induced by AICD. Bar, 20 µm. (H) Whole cell extracts of naive HEK293 and AICD-expressing clonal cell lines. Western blots were analyzed with antibodies against the N- or C-terminus of APP, against GFP to detect the citrine-AICD fusion protein and GAPDH as a loading control. Naive HEK293 (C, lane 1) were also treated with the -secretase inhibitor DAPT, which leads to accumulation of - and ß-stubs (DAPT, lane 2). Clonal HEK293 were induced for 3, 6 or 48 hours (lane 4 to 6) to express citrine-AICD. Bar diagram shows densitometric analysis of full-length APP and -together with ß-stubs from three independent experiments. Relative protein levels are determined with respect to uninduced cells (0 hrs).

View larger version (39K): [in a new window]   Fig. 2. Both FRET and immunoprecipitation reveal a close molecular association of Tip60 and AICD in AFT-complexes. (A) Two cells with nuclear AFT-complexes. Sensitized emission of citrine after excitation of cyan fluorescent protein (CFP) was calculated from raw FRET values with correction for CFP bleach-through and citrine cross-excitation. To prevent quenching of citrine emission, Fe65 was labeled with a Cy5-conjugated antibody (data not shown). (B) Control cells co-expressing CFP and citrine targeted to the nucleus with nuclear localization signals showed very little sensitized emission of citrine. (C) FRET measurements of AFT-complex-containing spherical nuclear spots showed sensitized emission from citrine-AICD in the absence of the Cy5-conjugated antibodies against Fe65. (D) Citrine-AICD was bleached in one nucleus and the intensity of CFP measured in single spots before (pre) and after bleaching (post). The graphs show the mean pixel intensity of the measured CFP-fluorescence. The pre/post values of single spots are connected by a line. After bleaching, CFP fluorescence intensities increased in spots in the bleached nucleus, but not in surrounding spots in unbleached nuclei. Bar, 10 µm. (E) Immunoprecipitation (IP) of AFT-complex components from nuclear fractions. Antibodies against AICD or Tip60 co-precipitate Fe65 as detected by western blot (WB) of the HA tag. IP with antibodies against AICD precipitated Tip60 and, vice versa, antibodies against Tip60 precipitated AICD, as shown by staining with an antibody against fluorescent proteins that detects both CFP and citrine. The lower band in panel 4 is unspecific, and results from the detection of the antibody used for immunoprecipitation by the secondary antibody used in western blots. Marker proteins are 19, 28, 39, 51, 64, 97 and 191 kDa.

View larger version (79K): [in a new window]   Fig. 4. APP-interacting proteins Jip1b and X11 have different effects on nuclear AICD localization. (A) Extranuclear localization of Jip1b. Binding of AICD to Jip1b was associated with decreased nuclear levels of AICD. (B) Co-expression of Tip60 relocalized Jip1b into nuclear speckles. (C) Additional co-expression of AICD resulted in the formation of nuclear AICD-Jip1-Tip60 (AJT) complexes with a distinct speckle-like morphology, as compared with the smaller spherical spots generated by AFT-complexes. (D) Exclusive extranuclear localization of X11. Binding of AICD to X11 was associated with decreased nuclear levels of AICD. (E) Co-expression of Tip60 had no effect on the extranuclear localization of either X11 or AICD. Bar, 10 µm.

View larger version (33K): [in a new window]   Fig. 5. AICD derived from full-length APP translocates to the nucleus and generates AFT-complexes. (A) Cells expressing APP fused to a C-terminal HA tag (red) followed in tandem by citrine (yellow). As expected, full-length APP was localized in the ER/Golgi and in vesicles in the processes and somata, but not in nuclei. Staining of APP with antibodies directed against the C-terminal HA tag colocalized with citrine fluorescence in extranuclear localizations. Confocal xz-scans through nuclei confirmed the extranuclear localization of full-length APP. (B) Cells co-expressing full-length APP together with Fe65 and Tip60 formed nuclear AFT-complexes with spherical spot morphology. (C) In contrast to the extranuclear colocalization of HA antibody staining with citrine fluorescence, HA antibodies failed to detect the nuclear AFT-complexes that emitted strong citrine fluorescence signals. (D) FRET measurements showed close molecular association of APP-derived AICD with Tip60 in nuclear AFT-complexes. Antibodies against the C-terminal HA tag of APP, stained with Cy5 (blue), consistently failed to detect AICD in AFT-complexes. Bar, 10 µm in A to C, 20 µm in D upper row and 5 µm in D lower row.

View larger version (51K): [in a new window]   Fig. 6. Pharmacological inhibition of -secretase processing of APP blocks the formation of nuclear AFT-complexes. (A,B) The -secretase inhibitors DAPT and L-685,458 prevented the localization of APP-derived AICD into nuclear AFT-complexes. In cells with lower APP expression Fe65 was targeted to nuclear spots, whereas in cells with high levels of APP, Fe65 was retained at extranuclear localizations of APP including the ER/Golgi and vesicular structures. (C) Control experiment in the absence of -secretase inhibitors confirmed the formation of APP-derived nuclear AFT-complexes. Bar, 10 µm.

View larger version (33K): [in a new window]   Fig. 7. Formation of APP-derived nuclear AFT- and AJT-complexes in differentiated human neuroblastoma cells. (A) Nuclear and cytoplasmic localization of Fe65 expressed in SH-SY5Y cells differentiated with retinoic acid and BDNF. (B) Co-expression of Tip60, Fe65 and AICD led to the formation of nuclear AFT-complexes with spherical spot morphology. (C) Co-expression of Tip60, Jip1b and AICD led to the formation of nuclear AJT-complexes with speckle-like morphology. (D) AICD derived from full-length APP is targeted to nuclear AFT-complexes with spherical spot morphology. The inset shows a higher magnification of the soma, with a lower exposure to visualize nuclear spots, which are over-exposed in the overview. Bar in A: 20 µm for A,D; 10 µm for B,C,D inset.