ller ‡ §ʈʈ FE65 is a cytosolic adapter protein and an important binding partner of amyloid precursor protein. Dependent on Thr668 phosphorylation in amyloid precursor protein, which influences amyloidogenic amyloid precursor protein processing, FE65 undergoes nuclear translocation, thereby transmitting a signal from the cell membrane to the nucleus. As this translocation may be relevant in Alzheimer disease, and as FE65 consists of three proteinprotein interaction domains able to bind and affect a variety of other proteins and downstream signaling pathways, the identification of the FE65 interactome is of central interest in Alzheimer disease research. In this study, we identified 121 proteins as new potential FE65 interacting proteins in a pulldown/mass spectrometry approach using human post-mortem brain samples as protein pools for recombinantly expressed FE65. Co-immunoprecipitation assays further validated the interaction of FE65 with the candidates SV2A and SERCA2. In parallel, we investigated the whole cell proteome of primary hippocampal neurons from FE65/FE65L1 double knockout mice. Notably, the validated FE65 binding proteins were also found to be differentially abundant in neurons derived from the FE65 knockout mice relative to wild-type control neurons. SERCA2 is an important player in cellular calcium homeostasis, which was found to be up-regulated in double knockout neurons. Indeed, knock-down of FE65 in HEK293T cells also evoked an elevated sensitivity to thapsigargin, a stressor specifically targeting the activity of SERCA2. Thus, our results suggest that FE65 is involved in the regulation of intracellular calcium homeostasis. Whereas transfection of FE65 alone caused a typical dotlike phenotype in the nucleus, co-transfection of SV2A significantly reduced the percentage of FE65 dot-positive cells, pointing to a possible role for SV2A in the modulation of FE65 intracellular targeting. Given that SV2A has a signaling function at the presynapse, its effect on FE65 intracellular localization suggests that the SV2A/FE65 interaction might play a role in synaptic signal transduction. Molecular

SummaryThe intracellular domain of the amyloid precursor protein (AICD) is generated following cleavage of the precursor by the c-secretase complex and is involved in membrane to nucleus signaling, for which the binding of AICD to the adapter protein FE65 is essential. Here we show that FE65 knockdown causes a downregulation of the protein Bloom syndrome protein (BLM) and the minichromosome maintenance (MCM) protein family and that elevated nuclear levels of FE65 result in stabilization of the BLM protein in nuclear mobile spheres. These spheres are able to grow and fuse, and potentially correspond to the nuclear domain 10. BLM plays a role in DNA replication and repair mechanisms and FE65 was also shown to play a role in DNA damage response in the cell. A set of proliferation assays in our work revealed that FE65 knockdown in HEK293T cells reduced cell replication. On the basis of these results, we hypothesize that nuclear FE65 levels (nuclear FE65/BLM containing spheres) may regulate cell cycle reentry in neurons as a result of increased interaction of FE65 with BLM and/or an increase in MCM protein levels. Thus, FE65 interactions with BLM and MCM proteins may contribute to the neuronal cell cycle re-entry observed in brains affected by Alzheimer's disease.