Visualization and quantification of APP intracellular domain-mediated nuclear signaling by bimolecular fluorescence complementation

BACKGROUND: The amyloid precursor protein (APP) intracellular domain (AICD) is released from full-length APP upon sequential cleavage by either α- or β-secretase followed by γ-secretase. Together with the adaptor protein Fe65 and the histone acetyltransferase Tip60, AICD forms nuclear multiprotein complexes (AFT complexes) that function in transcriptional regulation. OBJECTIVE: To develop a medium-throughput machine-based assay for visualization and quantification of AFT complex formation in cultured cells. METHODS: We used cotransfection of bimolecular fluorescence complementation (BiFC) fusion constructs of APP and Tip60 for analysis of subcellular localization by confocal microscopy and quantification by flow cytometry (FC). RESULTS: Our novel BiFC-constructs show a nuclear localization of AFT complexes that is identical to conventional fluorescence-tagged constructs. Production of the BiFC signal is dependent on the adaptor protein Fe65 resulting in fluorescence complementation only after Fe65-mediated nuclear translocation of AICD and interaction with Tip60. We applied the AFT-BiFC system to show that the Swedish APP familial Alzheimer's disease mutation increases AFT complex formation, consistent with the notion that AICD mediated nuclear signaling mainly occurs following APP processing through the amyloidogenic β-secretase pathway. Next, we studied the impact of posttranslational modifications of AICD on AFT complex formation. Mutation of tyrosine 682 in the YENPTY motif of AICD to phenylalanine prevents phosphorylation resulting in increased nuclear AFT-BiFC signals. This is consistent with the negative impact of tyrosine phosphorylation on Fe65 binding to AICD. Finally, we studied the effect of oxidative stress. Our data shows that oxidative stress, at a level that also causes cell death, leads to a reduction in AFT-BiFC signals. CONCLUSION: We established a new method for visualization and FC quantification of the interaction between AICD, Fe65 and Tip60 in the nucleus based on BiFC. It enables flow cytometric analysis of AICD nuclear signaling and is characterized by scalability and low background fluorescence

Turnover of amyloid precursor protein family members determines their nuclear signaling capability

The amyloid precursor protein (APP) as well as its homologues, APP-like protein 1 and 2 (APLP1 and APLP2), are cleaved by α-, β-, and γ-secretases, resulting in the release of their intracellular domains (ICDs). We have shown that the APP intracellular domain (AICD) is transported to the nucleus by Fe65 where they jointly bind the histone acetyltransferase Tip60 and localize to spherical nuclear complexes (AFT complexes), which are thought to be sites of transcription. We have now analyzed the subcellular localization and turnover of the APP family members. Similarly to AICD, the ICD of APLP2 localizes to spherical nuclear complexes together with Fe65 and Tip60. In contrast, the ICD of APLP1, despite binding to Fe65, does not translocate to the nucleus. In addition, APLP1 predominantly localizes to the plasma membrane, whereas APP and APLP2 are detected in vesicular structures. APLP1 also demonstrates a much slower turnover of the full-length protein compared to APP and APLP2. We further show that the ICDs of all APP family members are degraded by the proteasome and that the N-terminal amino acids of ICDs determine ICD degradation rate. Together, our results suggest that different nuclear signaling capabilities of APP family members are due to different rates of full-length protein processing and ICD proteasomal degradation. Our results provide evidence in support of a common nuclear signaling function for APP and APLP2 that is absent in APLP1, but suggest that APLP1 has a regulatory role in the nuclear translocation of APP family ICDs due to the sequestration of Fe65