A ternary complex consisting of AICD, FE65, and TIP60 down-regulates Stathmin1

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Abstract

The ternary complex consisting of AICD/FE65/TIP60 is thought to play a role in gene expression and was suggested to have a crucial impact in Alzheimer's disease. AICD is the intracellular subdomain of the amyloid precursor protein (APP) and able to bind the adapter protein FE65 and the histone acetyltransferase TIP60 setting up a nuclear dot-like phenotype. Within this work we readdressed the generation of the complex as a function of its compartments. Subsequently, we studied the proteome of AFT expressing cells vs. controls and identified Stathmin1 significantly down-regulated in AFT cells. Stathmin1 functions as an important regulatory protein of microtubule dynamics and was found associated with neurofibrillary tangles in brains of Alzheimer's disease patients. We validated our results using an independent label-free mass spectrometry based method using the same cell culture model. In a reversal model with diminished APP expression, caused by simultaneous knock-down of all three members of the APP family, we further confirmed our results, as Stathmin1 was regulated in an opposite fashion. We hypothesize that AICD-dependent deregulation of Stathmin1 causes microtubule disorganization, which might play an important role for the pathophysiology of Alzheimer's disease.

Highlights

► We studied the proteome of AICD–FE65–TIP60 (AFT) expressing cells vs. controls. ► We identified a new candidate protein significantly down-regulated in AFT cells. ► We confirmed our findings in an independent knock-down model. ► We confirm that AFT establishes a nuclear dot-like phenotype. ► We demonstrate that wtAPP and swAPP are able to generate the AFT complex as well.

Introduction

APP is the precursor of the β-amyloid peptide, which is found in the amyloidogenic plaques of Alzheimer brains. The dissociation of this crucial domain from its precursor is well known, caused by the successive cleavage of APP by the β- and γ-secretase complex [1]. Another important cleavage product is the intracellular domain of APP, the AICD. AICD exists in several isoforms, which is the result of γ-secretase activity and of other enzymes like caspases [2], [3]. More than 20 proteins are known to bind to AICD, among them are adapter proteins like FE65, NUMB, DAB1 and MINT that enable binding of other proteins [4], [5], [6], [7]. Much interest aroused the AICD–FE65 interaction as this dual complex was described to enter the nucleus where it associates to a third protein, the histone acetyltransferase HTATIP (TIP60) [8]. The function of the complex is controversially discussed: many publications suggest a role as transcription factor regulating mRNA levels of KAI1 [8], GSK3β [9], or cytoskeletal associated genes [10], respectively. Those results were obtained from cell culture experiments or mouse models and could often not be validated by independent labs [11], [12]. However, pharmacological inhibition of transcription resulted in a phenotype shift of the complex [13], supporting the idea of a role in gene expression. Our understanding of AICD-related mechanisms is further complicated as AICD was found to be unstable in its unassociated form [14], [15], [16], which might also be the reason why the isolated domain could not be identified in human brain samples so far. Supporter of the AICD hypothesis counter that FE65, which is one of the most important AICD stabilizing proteins, is highly expressed in regions of the hippocampus in which the earliest abnormalities of Alzheimer's disease are evident [17]. In this context, a recent finding is of special interest demonstrating that nuclear signaling of AICD predominantly occurs through the amyloidogenic pathway, which is causative for the generation of β-amyloid [18]. The group of S. Pimplikar generated a transgenic AICD mouse model demonstrating impaired adult neurogenesis accompanied with neuroinflammation [19]. Of note, consistent with findings of deregulated genes involved in cytoskeletal dynamics, a loss of organized filamentous actin structures was found as well as a disrupted mitochondrial function [10], [20]. Those results are in good agreement to the finding that AICD plays a pivotal role in cell death [21].

However, the role of the APP intracellular domain and its interacting proteins is still poorly understood. Here, we readdressed and extended AICD related characteristics and applied for the first time a comprehensive proteome approach to study relevant protein abundance changes caused by AICD and its associated proteins FE65 and TIP60. Finally, we present a promising candidate undergoing a significant AICD-dependent abundance shift.

Section snippets

Vector constructs, transfections, and confocal microscopy

AICD50 was amplified from full length APP vector (wtAPP) and cloned into pEGFP-N1. 2N3T-TIP60 expression vector was kindly provided by Dr. Didier Trouche, Université Paul Sabatier, Toulouse, France. FE65-EGFP expression vector was kindly provided by Dr. Tomasso Russo, Università di Napoli Federico, Italy. pIRESbleo3_hFE65 was generated by subcloning full length human FE65 cDNA from FE65-EGFP in the pIRESbleo3 vector. wtAPP and swAPP vectors were kindly provided by Dr. Christoph Kaether,

Results

The amyloid precursor protein intracellular domain (AICD) is part of a nuclear complex with potential role in the modification of gene expression. Our first aim was to prove and extend results from other groups studying the localization of the AICD in concert with its interacting proteins. Readdressing those (in part) known aspects was done due to the controversies in the field. When AICD is expressed as AICD50 isoform (c-terminal fused to EGFP, not containing parts of the transmembrane domain

Discussion

Over the last decade the APP intracellular domain (AICD) aroused increasing interest in basic research of AD. AICD was suggested to play a role in gene expression, cell death, and cytoskeletal dynamics [30]. AICD enters the nucleus when bound to its adapter protein FE65 and generates a nuclear complex together with the histone acetyltransferase TIP60. This complex can be identified as nuclear dot-like structure when all three components of the complex are over-expressed [31] and was suggested

Acknowledgment

This work was funded by the EU (cNEUPRO, 6th EU FP, project LSHM-CT-2007-037950) and by FoRUM (Forschungsförderung Ruhr-Universität Bochum Medizinische Fakultät) AZ-F616-08 and AZ-F680-09.

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