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C-terminal fragments of amyloid precursor protein exert neurotoxicity by inducing glycogen synthase kinase-3ß expression¹

HYE-SUN KIM^*,2, EUN-MEE KIM^*,2, JEAN-PYO LEE, CHEOL HYOUNG PARK^*, SEONGHAN KIM^*, JI-HEUI SEO^*, KEUN-A CHANG^*, EUNAH YU^*, SUNG-JIN JEONG^*, YOUNG HAE CHONG and YOO-HUN SUH^*,3

^* Department of Pharmacology, College of Medicine, National Creative Research Initiative Center for Alzheimer’s Dementia and Neuroscience Research Institute, MRC, Seoul National University, Seoul 110-799, South Korea;
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA; and
Departments of Microbiology, College of Medicine, Division of Neuroscience, Medical Research Center, Ewha Womans University, Seoul, 158-056, South Korea

³Correspondence: Department of Pharmacology, College of Medicine, National Creative Research Initiative Center for Alzheimer’s Dementia, Seoul National University, Seoul, 110-799, South Korea. E-mail; yhsuh{at}plaza.snu.ac.kr

SPECIFIC AIMS

To elucidate the neurotoxic mechanisms of AICD (APP intracellular domain: C57, C59), the -secretase cleaved C-terminal fragments of APP and C31, the caspase-cleaved C-terminal fragment of APP, based on the hypothesis that C-terminal fragments of APP (APP-CTs) could exert neurotoxicity by transcriptional regulation of some genes, we investigated the interacting proteins with APP-CTs in the nucleus and their gene-inducing effects. We examined whether AICD or C31 interacted with CP2/LSF/LBP1 transcription factor, which has been reported to bind to Fe65 in the nucleus. We examined the effects of AICD and C31 on GSK-3ß gene expression, a gene known to be regulated by CP2/LSF/LBP1 transcription factor. Its downstream events were investigated, including the phosphorylation of tau, a substrate for GSK-3ß whose hyperphosphorylated form is the main component of the neurofibrillary tangles found in Alzheimer’s disease (AD) brain.

PRINCIPAL FINDINGS

1. AICD or C31 interacted with Fe65 and CP2/LSF/LBP1 transcription factor in the nucleus of differentiated PC12 cells
Using C9 antibody, we found that APP-CTs were also seen in the nuclear fractions of the mock and deletion mutants of C59 and C31 without YENPTY, an Fe65-interacting domain (dC59, dC31) transfected cell (Fig. 1 A). In Fig. 1B , APP-CTs (using C9 antibody) were observed diffusely in the cytoplasm and clearly in the nucleus (Fig. 1Bk-o) . Endogenous Fe65 was also found in the cytoplasm and nucleus (Fig. 1Bf-j) . We found that C57, C59, or C31 were colocalized with Fe65 in the nucleus (Fig. 1Bq, s, t ) while dC59 and dC31 were not. As shown in Fig. 1A , the Cy3 signal showing APP-CTs were observed in the nucleus of mock and dC31 transfected cells (Fig. 1Bk, m ), indicating the presence of endogenous APP-CTs in the nucleus of the cells. We also examined whether Fe65 or CP2/LSF/LBP1 transcription factor interacted with C31, C59, or C99 by immunoprecipitation. PC12 cells were transfected with APP-CTs C31, dC31, C59, C99, and dC99 (deletion mutant of C99 without YENPTY domain) cloned in pEGFP vector. Immunoprecipitation with anti-Fe65 antiserum followed by anti-GFP antibody showed an APP-CT-GFP band of 34 kDa in the nuclear fraction of C31, C59, and C99-transfected cells, suggesting an interaction between Fe65 and APP-CTs (Fig. 1C ). The immunoprecipitation of APP-CT by anti-GFP antibody and immunoblotting for CP2/LSF/LBP1 revealed that transfected APP-CTs and endogenously existing CP2/LSF/LBP1 interact in the nuclear fraction (Fig. 1D ). Immunoprecipitation was performed with anti-CP2/LSF/LBP1 antiserum and analyzed with anti-Fe65 antiserum to examine the interaction between Fe65 and CP2/LSF/LBP1 (Fig. 1E ). In dC31- and dC99-transfected cells of Fig. 1E , Fe65 bands of 90 kDa representing the interaction with CP2/LSF/LBP1 were weaker than in C31, C59, or C99 transfected cells. Although the mechanisms of molecular interactions between these three proteins need further elucidation, our data suggest the interaction between Fe65 and CP2/LSF/LBP1 might be affected by APP-CTs via its YENPTY domain (Fig. 1E ). Our findings revealed the presence of the APP-CTs-Fe65-CP2/LSF/LBP1 ternary complex in the nucleus of C59-, C31-, or C99-transfected PC12 cells and that the interactions between the three proteins are mediated by the YENPTY domain of APP-CTs.

View larger version (28K): [in this window] [in a new window]   Figure 1. Interaction between APP-CTs and Fe65 and CP2/LSF/LBP1 in the nuclear fraction of APP-CTs transfected PC12 cells. A) At 48 h post-transfection, APP-CTs were detected in the nuclear fractions of C31-, C59-, dC31, and dC59 (cloned in pEGFP or pcDNA3-flag) -transfected PC12 cells. B) Immunocytochemical experiment analyzed by confocal laser scanning microscopy shows that C31, C57, and C59 colocalized with Fe65 in the nucleus. a–e) DAPI (1 µM) staining showing the location of the nucleus(blue); f–j) FITC-conjugated secondary antibody staining showing the location of Fe65 (green) by anti-Fe65 antibody; k–o) Cy3-conjugated secondary antibody staining representing the location of APP-CTs (red) by APP-CT antibody; p–t) merged images of each color. a, f, k, p) pcDNA3-flag transfected PC12 cells; b, g, l, q) C31-transfected cells; c, h, m, r) dC31-transfected cells; d, i, n, s) C57-transfected cells; e, j, o, t) C59-transfected cells. To explore the interactions between APP-CTs, Fe65, and CP2/LSF/LBP1, we performed immunoprecipitation (IP) and immunoblotting (IB). C) Samples obtained from the nuclear fraction of APP-CTs-transfected cells in pEGFP-N1 or pCDNA-3 flag vector, respectively, were immunoprecipitated with anti-Fe65 antiserum, then immunoblotted with anti-GFP antibody. D) Immunoprecipitation with anti-GFP antibody, immunoblotting with anti-CP2/LSF/LBP1 antiserum. E) Immunoprecipitation with anti-CP2/LSF/LBP1 antiserum, immunoblotting with anti-Fe65 antiserum.

2.Overexpression of AICD or C31 increased GSK-3ß levels and its promoter activity significantly in neuronal cells whereas that of their YENPTY domain mutants did not
Based on the result that C31, C59, or C99 form a ternary complex with Fe65 and CP2/LSF/LBP1 in the nucleus, we tested the involvement of GSK-3ß, a gene known to be regulated by the CP2/LSF/LBP1 transcription factor. We found an increase of GSK-3ß (Fig. 2A-C ) and of its active form (phosphorylated at Tyr²¹⁶ residue) (Fig. 2D ) in C57-, C59-, or C31-expressing neuronal cells. Neuronal cells expressing dC59, dC31, C59, or C31 with Y682G point mutation showed no significant increase in GSK-3ß induction (Fig. 2B, C ). To confirm the effects of APP-CTs on GSK-3ß induction, we assayed luciferase activity using the human GSK-3ß promoter in pGL2 luciferase vector. A marked increase in gene reporter activity was shown in C57-, C59-, or C31-transfected PC 12 cells. Consistent with the changes in protein and mRNA levels of GSK-3ß, its promoter activity began to increase 12 h after transfection (Fig. 2E ). C59 with a N684A point mutation showed >fourfold increases in GSK-3ß promoter activity. Meanwhile, dC57, dC59, dC31, or C59 with a Y682G point mutation showed no significant increase in GSK-3ß promoter activity in the transfected PC12 cells (Fig. 2E ). Putative CP2/LSF/LBP1 binding sites were identified at nt -1292 -1282 and nt +0 +10 by Lau et al. In this study, we made deletion mutant without nt -1292 -1282 (GCGCACACCAA) or nt +0 +10 (GCCCGGGCCAA), respectively, and performed luciferase activity assay after cotransfection of C59 or dC59 with one of these deletion mutants of human GSK-3ß promoter in pGL2 luciferase vector. As shown in Fig. 2I , deletion of CP2/LSF/LBP1 binding site +0 +10 inhibits the increase of the promoter activity by C59, whereas that of CP2/LSF/LBP1 binding site -1292 -1282 did not significantly affect the increase of the human GSK-3ß promoter activity by C59. Our findings suggest that the CP2/LSF/LBP1 binding site located at +0 +10 sequence in human GSK-3ß promoter region is essential for gene transcription by APP-CTs.

View larger version (55K): [in this window] [in a new window]   Figure 2. APP-CTs increased GSK-3ß expression and promoter activity and decreased nuclear ß-catenin in PC12 cells and rat primary cortical neurons. A) At 6, 12, 24, and 48 h post-transfection, the GSK-3ß level was checked by Western blot with anti-GSK-3ß antibody in PC12 cells. B, C) At 48 h post-transfection, GSK-3ß levels were checked in the mock, C57, dC57, C59, dC59, Y682G C59, N684A C59, C31, dC31, Y682G C31, and N684A C31-transfected PC12 cells and in rat primary cortical neurons. G) At 48 h post-transfection, p-GSK-3ß(Tyr216) and ß-catenin levels in the nuclear and cytosolic fractions of PC12 cells transfected with C31 were detected by immunoblotting using anti-p-GSK-3ß(Tyr216) antibody and anti-ß-catenin antibody, respectively. H) Promoter activity was measured by luciferase activity assay in APP-CTs and human GSK-3ß promoter cotransfected PC12 cells. I) Promoter activity was measured by luciferase activity assay in C59, dC59, and deletion mutants of human GSK-3ß promoter without nt -1292 -1282 or +0 +10. Data represent the means ± SE of 4 separate experiments. An asterisk indicates a significant difference from mock transfected group (*P<0.05, **P<0.01 by ANOVA test).

CONCLUSIONS AND SIGNIFICANCE

AICD, composed of the last 57-59 amino acids, the 6 kDa C terminus of APP, has been demonstrated to cause transcriptional activation after trans-localization into the nucleus in combination with Fe65 and Tip60, a histone acetyltransferase, suggesting a role of AICD in gene regulation. In addition to AICD, it has been reported that C31, generated from caspase-8 and -9 cleavage, was detected in the brains of AD patients and had a proapoptotic and a toxic effect on neuronal cells. However, the neurotoxic mechanisms of AICD and C31 remain to be elucidated.

In this study, we tested the hypothesis that the neurotoxicity induced by AICD and C31 might be mediated by transcriptional events in the nucleus. We demonstrated for the first time that APP-CTs existed as a ternary complex with Fe65 and CP2/LSF/LBP1 in the nuclear extracts of AICD- and C31-transfected PC12 cells and that this complex affects the transcription of GSK-3ß, followed by an increase in tau phosphorylation and reduced ß-catenin level whereas deletion mutants and point mutant (Y682G) of them did not. Fe65 has also been reported to bind the CP2/LSF/LBP1 family transcription factor through the PTB1 domain. Numerous results demonstrated that CP2/LSF/LBP1 was a housekeeping factor regulating a variety array of genes, including the human globulin gene, the serum amyloid A3 gene, GSK-3ß, and 2-macroglobulin.

The following reports on human AD brain are significantly correlated with our findings that APP-CTs may exert toxicity by trans-localizing into the nucleus and up-regulating the expression of GSK-3ß, binding to the CP2/LSF/LBP1 binding site in the promoter region and this may contribute to the pathogenesis of AD. The in vivo evidence is 1) clear nuclear staining of APP-CT, but not the N-terminal fragment of APP, has been detected, colocalizing with Fe65 in post mortem AD brain; the amount of nuclear staining related well to disease status; 2) in AD brains, increased levels of GSK-3ß and active form of GSK-3ß have been found to be accumulated in pretangled neurons.

View larger version (38K): [in this window] [in a new window]   Figure 3. Proposed neurotoxic mechanism of AICD and C31 via transcriptional regulation. AICD and C31 generated from APP by -secretase and caspases, respectively, trans-localize into the nucleus and form a ternary complex with Fe65 and CP2/LSF/LBP1 transcription factor and induce the transcription of GSK-3ß by binding to its promoter region (CP2/LSF/LBP1 binding site +0 +10). Up-regulation of GSK-3ß is followed by the phosphorylation of tau, a substrate for GSK-3ß, leading to apoptosis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0106fje; doi: 10.1096/fj.03-0106fje

² H.-S.K. and E.-M.K. contributed equally to this study.

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