Comparable dimerization found in wildtype and familial Alzheimer's disease amyloid precursor protein mutants

Department of Medicine Graduate Program in Molecular Medicine, Boston University School of Medicine 72 East Concord Street, K-304, Boston, MA, 02118, USA.
American Journal of Neurodegenerative Diseases 03/2013; 2(1):15-28.
Source: PubMed


Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disorder marked by memory impairment and cognitive deficits. A major component of AD pathology is the accumulation of amyloid plaques in the brain, which are comprised of amyloid beta (Aβ) peptides derived from the amyloidogenic processing of the amyloid precursor protein (AβPP) by β- and γ-secretases. In a subset of patients, inheritance of mutations in the AβPP gene is responsible for altering Aβ production, leading to early onset disease. Interestingly, many of these familial mutations lie within the transmembrane domain of the protein near the GxxxG and GxxxA dimerization motifs that are important for transmembrane interactions. As AβPP dimerization has been linked to changes in Aβ production, it is of interest to know whether familial AβPP mutations affect full-length APP dimerization. Using bimolecular fluorescence complementation (BiFC), blue native gel electrophoresis, and live cell chemical cross-linking, we found that familial Alzheimer's disease (FAD) mutations do not affect full-length AβPP dimerization in transfected HEK293 and COS7 cells. It follows that changes in AβPP dimerization are not necessary for altered Aβ production, and in FAD mutations, changes in Aβ levels are more likely a result of alternative proteolytic processing.

Download full-text


Available from: Christina E Khodr, Nov 06, 2015
  • Source
    • "The two structurally well-defined regions E1 and E2 are predestined to represent sites of such high-affinity dimerization . An initial dimerization at the single transmembrane helix of APP is unlikely to be the physiologically primary driving force, as also evidenced by a competing binding of this region to cholesterol (Song et al., 2013) and the fact that mutations in this segment do not affect the oligomerization of APP (So et al., 2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The amyloid precursor protein (APP) and its cellular processing are believed to be centrally involved in the etiology of Alzheimer’s disease (AD). In addition, many physiological functions have been described for APP, including a role in cell-cell- and cell-ECM-adhesion as well as in axonal outgrowth. We show here the molecular determinants of the oligomerization/dimerization of APP, which is central for its cellular (mis)function. Using size exclusion chromatography (SEC), dynamic light scattering and SEC-coupled static light scattering we demonstrate that the dimerization of APP is energetically induced by a heparin mediated dimerization of the E1 domain, which results in a dimeric interaction of E2. We also show that the acidic domain (AcD) interferes with the dimerization of E1 and propose a model where both, cis- and trans-dimerization occur dependent on cellular localization and function.
    Full-text · Article · Jul 2014 · Journal of Structural Biology
  • Source
    • "In the field of neurodegenerative diseases, BiFC has so far been used to study disease mechanisms in Alzheimer’s and Parkinson’s disease [20], such as the oligomerization of α-synuclein [21,22]. For APP, BiFC was employed to demonstrate the formation of APP homodimers in the endoplasmatic reticulum and Golgi apparatus and to study the differential dimerization properties of different isoforms and familial AD mutations of APP [23-25]. In another set of experiments, BiFC revealed the heterodimerization of APP with Notch2 [26,27]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: 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. To develop a medium-throughput machine-based assay for visualization and quantification of AFT complex formation in cultured cells. 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). 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. 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.
    Full-text · Article · Sep 2013 · PLoS ONE
  • Source

    Full-text · Dataset · Oct 2014
Show more