Fe65 is the sole member of its family that mediates transcription regulated by the amyloid precursor protein.

The amyloid precursor protein (APP), a central molecule in Alzheimer's disease (AD), has physiological roles in cell adhesion and signaling, migration, neurite outgrowth and synaptogenesis. Intracellular adapter proteins mediate the function of transmembrane proteins. Fe65 (also known as APBB1) is a major APP-binding protein. Regulated intramembrane proteolysis (RIP) by γ-secretase releases the APP intracellular domain (AICD), together with the interacting proteins, from the membrane. We studied the impact of the Fe65 family (Fe65, and its homologs Fe65L1 and Fe65L2, also known as APBB2 and APBB3, respectively) on the nuclear signaling function of the AICD. All Fe65 family members increased amyloidogenic processing of APP, generating higher levels of ß-cleaved APP stubs and AICD. However, Fe65 was the only family member supporting AICD translocation to nuclear spots and its transcriptional activity. Using a recently established transcription assay, we dissected the transcriptional activity of Fe65 and provide strong evidence that Fe65 represents a transcription factor. We show that Fe65 relies on the lysine acetyltransferase Tip60 (also known as KAT5) for nuclear translocation. Furthermore, inhibition of APP cleavage reduces nuclear Tip60 levels, but this does not occur in Fe65-knockout cells. The rate of APP cleavage therefore regulates the nuclear translocation of AICD-Fe65-Tip60 (AFT) complexes, to promote transcription by Fe65.

Non-invasive imaging of tau-targeted probe uptake by whole brain multi-spectral optoacoustic tomography.

PURPOSE: Abnormal tau accumulation within the brain plays an important role in tauopathies such as Alzheimer's disease and frontotemporal dementia. High-resolution imaging of tau deposits at the whole-brain scale in animal disease models is highly desired. METHODS: We approached this challenge by non-invasively imaging the brains of P301L mice of 4-repeat tau with concurrent volumetric multi-spectral optoacoustic tomography (vMSOT) at ~ 115 µm spatial resolution using the tau-targeted pyridinyl-butadienyl-benzothiazole derivative PBB5 (i.v.). In vitro probe characterization, concurrent vMSOT and epi-fluorescence imaging of in vivo PBB5 targeting (i.v.) was performed in P301L and wild-type mice, followed by ex vivo validation using AT-8 antibody for phosphorylated tau. RESULTS: PBB5 showed specific binding to recombinant K18 tau fibrils by fluorescence assay, to post-mortem Alzheimer's disease brain tissue homogenate by competitive binding against [11C]PBB3 and to tau deposits (AT-8 positive) in post-mortem corticobasal degeneration and progressive supranuclear palsy brains. Dose-dependent optoacoustic and fluorescence signal intensities were observed in the mouse brains following i.v. administration of different concentrations of PBB5. In vivo vMSOT brain imaging of P301L mice showed higher retention of PBB5 in the tau-laden cortex and hippocampus compared to wild-type mice, as confirmed by ex vivo vMSOT, epi-fluorescence, multiphoton microscopy, and immunofluorescence staining. CONCLUSIONS: We demonstrated non-invasive whole-brain imaging of tau in P301L mice with vMSOT system using PBB5 at a previously unachieved ~ 115 µm spatial resolution. This platform provides a new tool to study tau spreading and clearance in a tauopathy mouse model, foreseeable in monitoring tau targeting putative therapeutics.

Lysine acetyltransferase Tip60 acetylates the APP adaptor Fe65 to increase its transcriptional activity.

Proteolytic processing of the amyloid precursor protein (APP) releases the APP intracellular domain (AICD) from the membrane. Bound to the APP adaptor protein Fe65 and the lysine acetyltransferase (KAT) Tip60, AICD translocates to the nucleus. Here, the complex forms spherical condensates at sites of endogenous target genes, termed AFT spots (AICD-Fe65-Tip60). We show that loss of Tip60 KAT activity prevents autoacetylation, reduces binding of Fe65 and abolishes Fe65-mediated stabilization of Tip60. Autoacetylation is a prerequisite for AFT spot formation, with KAT-deficient Tip60 retained together with Fe65 in speckles. We identify lysine residues 204 and 701 of Fe65 as acetylation targets of Tip60. We do not detect acetylation of AICD. Mutation of Fe65 K204 and K701 to glutamine, mimicking acetylation-induced charge neutralization, increases the transcriptional activity of Fe65 whereas Tip60 inhibition reduces it. The lysine deacetylase (KDAC) class III Sirt1 deacetylates Fe65 and pharmacological modulation of Sirt1 activity regulates Fe65 transcriptional activity. A second acetylation/deacetylation cycle, conducted by CBP and class I/II KDACs at different lysine residues, regulates stability of Fe65. This is the first report describing a role for acetylation in the regulation of Fe65 transcriptional activity, with Tip60 being the only KAT tested that supports AFT spot formation.

A fluorescent protein-readout for transcriptional activity reveals regulation of APP nuclear signaling by phosphorylation sites.

Signaling pathways that originate at the plasma membrane, including regulated intramembrane proteolysis (RIP), enable extracellular cues to control transcription. We modified the yeast Gal4 transcription system to study the nuclear translocation of transcriptionally active complexes using the fluorescent protein citrine (Cit) as a reporter. This enabled highly sensitive quantitative analysis of transcription in situ at the single cell level. The Gal4/UAS-Cit transcription assay displayed a sigmoidal response limited by the number of integrated reporter cassettes. We validated the assay by analyzing nuclear translocation of the amyloid precursor protein (APP) intracellular domain (AICD) and confirmed the requirement of Fe65 for nuclear translocation of AICD. In addition to the strong on-off effects on transcriptional activity, the results of this assay establish that phosphorylation modifies nuclear signaling. The Y682F mutation in APP showed the strongest increase in Cit expression, underscoring its role in regulating Fe65 binding. Together, we established a highly sensitive fluorescent protein-based assay that can monitor transcriptional activity at the single cell level and demonstrate that AICD phosphorylation affects Fe65 nuclear activity. This assay also introduces a platform for future single cell-based drug screening methods for nuclear translocation.

Early alterations in functional connectivity and white matter structure in a transgenic mouse model of cerebral amyloidosis.

Impairment of brain functional connectivity (FC) is thought to be an early event occurring in diseases with cerebral amyloidosis, such as Alzheimer's disease. Regions sustaining altered functional networks have been shown to colocalize with regions marked with amyloid plaques burden suggesting a strong link between FC and amyloidosis. Whether the decline in FC precedes amyloid plaque deposition or is a consequence thereof is currently unknown. The sequence of events during early stages of the disease is difficult to capture in humans due to the difficulties in providing an early diagnosis and also in view of the heterogeneity among patients. Transgenic mouse lines overexpressing amyloid precursor proteins develop cerebral amyloidosis and constitute an attractive model system for studying the relationship between plaque and functional changes. In this study, ArcAß transgenic and wild-type mice were imaged using resting-state fMRI methods across their life-span in a cross-sectional design to analyze changes in FC in relation to the pathology. Transgenic mice show compromised development of FC during the first months of postnatal life compared with wild-type animals, resulting in functional impairments that affect in particular the sensory-motor cortex already in preplaque stage. These functional alterations were accompanied by structural changes as reflected by reduced fractional anisotropy values, as derived from diffusion tensor imaging. Our results suggest cerebral amyloidosis in mice is preceded by impairment of neuronal networks and white matter structures. FC analysis in mice is an attractive tool for studying the implications of impaired neuronal networks in models of cerebral amyloid pathology.

Dysregulation of hypoxia-inducible factor by presenilin/γ-secretase loss-of-function mutations.

Presenilin (PSEN) 1 and 2 are the catalytic components of the γ-secretase complex, which cleaves a variety of proteins, including the amyloid precursor protein (APP). Proteolysis of APP leads to the formation of the APP intracellular domain (AICD) and amyloid ß that is crucially involved in the pathogenesis of Alzheimer's disease. Prolyl-4-hydroxylase-domain (PHD) proteins regulate the hypoxia-inducible factors (HIFs), the master regulators of the hypoxic response. We previously identified the FK506 binding protein 38 (FKBP38) as a negative regulator of PHD2. Genetic ablation of PSEN1/2 has been shown to increase FKBP38 protein levels. Therefore, we investigated the role of PSEN1/2 in the oxygen sensing pathway using a variety of genetically modified cell and mouse lines. Increased FKBP38 protein levels and decreased PHD2 protein levels were found in PSEN1/2-deficient mouse embryonic fibroblasts and in the cortex of forebrain-specific PSEN1/2 conditional double knock-out mice. Hypoxic HIF-1α protein accumulation and transcriptional activity were decreased, despite reduced PHD2 protein levels. Proteolytic γ-secretase function of PSEN1/2 was needed for proper HIF activation. Intriguingly, PSEN1/2 mutations identified in Alzheimer patients differentially affected the hypoxic response, involving the generation of AICD. Together, our results suggest a direct role for PSEN in the regulation of the oxygen sensing pathway via the APP/AICD cleavage cascade.

Relationship Between Reactive Astrocytes, by [18F]SMBT-1 Imaging, with Amyloid-Beta, Tau, Glucose Metabolism, and TSPO in Mouse Models of Alzheimer's Disease.

Reactive astrocytes play an important role in the development of Alzheimer's disease (AD). Here, we aimed to investigate the temporospatial relationships among monoamine oxidase-B, tau and amyloid-ß (Aß), translocator protein, and glucose metabolism by using multitracer imaging in AD transgenic mouse models. Positron emission tomography (PET) imaging with [18F]SMBT-1 (monoamine oxidase-B), [18F]florbetapir (Aß), [18F]PM-PBB3 (tau), [18F]fluorodeoxyglucose (FDG), and [18F]DPA-714 (translocator protein) was carried out in 5- and 10-month-old APP/PS1, 11-month-old 3×Tg mice, and aged-matched wild-type mice. The brain regional referenced standard uptake value (SUVR) was computed with the cerebellum as the reference region. Immunofluorescence staining was performed on mouse brain tissue slices. [18F]SMBT-1 and [18F]florbetapir SUVRs were greater in the cortex and hippocampus of 10-month-old APP/PS1 mice than in those of 5-month-old APP/PS1 mice and wild-type mice. No significant difference in the regional [18F]FDG or [18F]DPA-714 SUVRs was observed in the brains of 5- or 10-month-old APP/PS1 mice or wild-type mice. No significant difference in the SUVRs of any tracer was observed between 11-month-old 3×Tg mice and age-matched wild-type mice. A positive correlation between the SUVRs of [18F]florbetapir and [18F]DPA-714 in the cortex and hippocampus was observed among the transgenic mice. Immunostaining validated the distribution of MAO-B and limited Aß and tau pathology in 11-month-old 3×Tg mice; and Aß deposits in brain tissue from 10-month-old APP/PS1 mice. In summary, these findings provide in vivo evidence that an increase in astrocyte [18F]SMBT-1 accompanies Aß accumulation in APP/PS1 models of AD amyloidosis.

Aquaporin 4 is differentially increased and dislocated in association with tau and amyloid-beta.

AIMS: Neurovascular-glymphatic dysfunction plays an important role in Alzheimer's disease and has been analysed mainly in relation to amyloid-beta (Aß) pathology. Here, we aim to investigate the neurovascular alterations and mapping of aquaporin 4 (AQP4) distribution and dislocation associated with tau and Aß. MATERIALS AND METHODS: Perfusion, susceptibility weighted imaging and structural magnetic resonance imaging (MRI) were performed in the pR5 mouse model of 4-repeat tau and the arcAß mouse model of amyloidosis. Immunofluorescence staining was performed using antibodies against AQP4, vessel, astroglia, microglia, phospho-tau and Aß in brain tissue slices from pR5, arcAß and non-transgenic mice. KEY FINDINGS: pR5 mice showed regional atrophy, preserved cerebral blood flow, and reduced cerebral vessel density compared to non-transgenic mice, while arcAß mice showed cerebral microbleeds and reduced cerebral vessel density. AQP4 dislocation and peri-tau enrichment in the hippocampus and increased AQP4 levels in the cortex and hippocampus were detected in pR5 mice compared to non-transgenic mice. In comparison, cortical AQP4 dislocation and cortical/hippocampal peri-plaque increases were observed in arcAß mice. Increased expression of reactive astrocytes were detected around the tau inclusions in pR5 mice and Aß plaques in arcAß mice. SIGNIFICANCE: We demonstrated the neurovascular alterations, microgliosis, astrogliosis and increased AQP4 regional expression in pR5 tau and arcAß mice. We observed a divergent region-specific AQP4 dislocation and association with phospho-tau and Aß pathologies.

Interactome of the amyloid precursor protein APP in brain reveals a protein network involved in synaptic vesicle turnover and a close association with Synaptotagmin-1.

Knowledge of the protein networks interacting with the amyloid precursor protein (APP) in vivo can shed light on the physiological function of APP. To date, most proteins interacting with the APP intracellular domain (AICD) have been identified by Yeast Two Hybrid screens which only detect direct interaction partners. We used a proteomics-based approach by biochemically isolating tagged APP from the brains of transgenic mice and subjecting the affinity-purified complex to mass spectrometric (MS) analysis. Using two different quantitative MS approaches, we compared the protein composition of affinity-purified samples isolated from wild-type mice versus transgenic mice expressing tagged APP. This enabled us to assess truly enriched proteins in the transgenic sample and yielded an overlapping set of proteins containing the major proteins involved in synaptic vesicle endo- and exocytosis. Confocal microscopy analyses of cotransfected primary neurons showed colocalization of APP with synaptic vesicle proteins in vesicular structures throughout the neurites. We analyzed the interaction of APP with these proteins using pulldown experiments from transgenic mice or cotransfected cells followed by Western blotting. Synaptotagmin-1 (Stg1), a resident synaptic vesicle protein, was found to directly bind to APP. We fused Citrine and Cerulean to APP and the candidate proteins and measured fluorescence resonance energy transfer (FRET) in differentiated SH-SY5Y cells. Differentially tagged APPs showed clear sensitized FRET emission, in line with the described dimerization of APP. Among the candidate APP-interacting proteins, again only Stg1 was in close proximity to APP. Our results strongly argue for a function of APP in synaptic vesicle turnover in vivo. Thus, in addition to the APP cleavage product Aß, which influences synaptic transmission at the postsynapse, APP interacts with the calcium sensor of synaptic vesicles and might thus play a role in the regulation of synaptic vesicle exocytosis.

Nuclear signaling by the APP intracellular domain occurs predominantly through the amyloidogenic processing pathway.

Proteolytic processing of the amyloid precursor protein (APP) occurs via two alternative pathways, localized to different subcellular compartments, which result in functionally distinct outcomes. Cleavage by a beta-gamma sequence generates the Abeta peptide that plays a central role in Alzheimer's disease. In the case of alpha-gamma cleavage, a secreted neurotrophic molecule is generated and the Abeta peptide cleaved and destroyed. In both cases, a cytosolic APP intracellular domain (AICD) is generated. We have previously shown that coexpression of APP with the APP-binding protein Fe65 and the histone acetyltransferase Tip60 results in the formation of nuclear complexes (termed AFT complexes), which localize to transcription sites. We now show that blocking endocytosis or the pharmacological or genetic inhibition of the endosomal beta-cleavage pathway reduces translocation of AICD to these nuclear AFT complexes. AICD signaling further depends on active transport along microtubules and can be modulated by interference with both anterograde and retrograde transport systems. Nuclear signaling by endogenous AICD in primary neurons could similarly be blocked by inhibiting beta-cleavage but not by alpha-cleavage inhibition. This suggests that amyloidogenic cleavage, despite representing the minor cleavage pathway of APP, is predominantly responsible for AICD-mediated nuclear signaling.

Generation of antibodies specific for beta-amyloid by vaccination of patients with Alzheimer disease.

To characterize antibodies produced in humans in response to Abeta42 vaccination, we carried out immunohistochemical examinations of the brains of both transgenic mice and human patients with beta-amyloid pathology. We collected sera from patients with Alzheimer disease who received a primary injection of pre-aggregated Abeta42 followed by one booster injection in a placebo-controlled study. Antibodies in immune sera recognized beta-amyloid plaques, diffuse Abeta deposits and vascular beta-amyloid in brain blood vessels. The antibodies did not cross-react with native full-length beta-amyloid precursor protein or its physiological derivatives, including soluble Abeta42. These findings indicate that vaccination of AD patients with Abeta42 induces antibodies that have a high degree of selectivity for the pathogenic target structures. Whether vaccination to produce antibodies against beta-amyloid will halt the cognitive decline in AD will depend upon clinical assessments over time.

APP Binds to the EGFR Ligands HB-EGF and EGF, Acting Synergistically with EGF to Promote ERK Signaling and Neuritogenesis.

The amyloid precursor protein (APP) is a transmembrane glycoprotein central to Alzheimer's disease (AD) with functions in brain development and plasticity, including in neurogenesis and neurite outgrowth. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) are well-described neurotrophic and neuromodulator EGFR ligands, both implicated in neurological disorders, including AD. Pro-HB-EGF arose as a putative novel APP interactor in a human brain cDNA library yeast two-hybrid screen. Based on their structural and functional similarities, we first aimed to verify if APP could bind to (HB-)EGF proforms. Here, we show that APP interacts with these two EGFR ligands, and further characterized the effects of APP-EGF interaction in ERK activation and neuritogenesis. Yeast co-transformation and co-immunoprecipitation assays confirmed APP interaction with HB-EGF. Co-immunoprecipitation also revealed that APP binds to cellular pro-EGF. Overexpression of HB-EGF in HeLa cells, or exposure of SH-SY5Y cells to EGF, both resulted in increased APP protein levels. EGF and APP were observed to synergistically activate the ERK pathway, crucial for neuronal differentiation. Immunofluorescence analysis of cellular neuritogenesis in APP overexpression and EGF exposure conditions confirmed a synergistic effect in promoting the number and the mean length of neurite-like processes. Synergistic ERK activation and neuritogenic effects were completely blocked by the EGFR inhibitor PD 168393, implying APP/EGF-induced activation of EGFR as part of the mechanism. This work shows novel APP protein interactors and provides a major insight into the APP/EGF-driven mechanisms underlying neurite outgrowth and neuronal differentiation, with potential relevance for AD and for adult neuroregeneration.

Antibodies against beta-amyloid slow cognitive decline in Alzheimer's disease.

To test whether antibodies against beta-amyloid are effective in slowing progression of Alzheimer's disease, we assessed cognitive functions in 30 patients who received a prime and a booster immunization of aggregated Abeta(42) over a 1 year period in a placebo-controlled, randomized trial. Twenty patients generated antibodies against beta-amyloid, as determined by tissue amyloid plaque immunoreactivity assay. Patients who generated such antibodies showed significantly slower rates of decline of cognitive functions and activities of daily living, as indicated by the Mini Mental State Examination, the Disability Assessment for Dementia, and the Visual Paired Associates Test of delayed recall from the Wechsler Memory Scale, as compared to patients without such antibodies. These beneficial clinical effects were also present in two of three patients who had experienced transient episodes of immunization-related aseptic meningoencephalitis. Our results establish that antibodies against beta-amyloid plaques can slow cognitive decline in patients with Alzheimer's disease.

Abeta oligomer-mediated long-term potentiation impairment involves protein phosphatase 1-dependent mechanisms.

Amyloid beta (Abeta) oligomers are derived from proteolytic cleavage of amyloid precursor protein (APP) and can impair memory and hippocampal long-term potentiation (LTP) in vivo and in vitro. They are recognized as the primary neurotoxic agents in Alzheimer's disease. The mechanisms underlying such toxicity on synaptic functions are complex and not fully understood. Here, we provide the first evidence that these mechanisms involve protein phosphatase 1 (PP1). Using a novel transgenic mouse model expressing human APP with the Swedish and Arctic mutations that render Abeta more prone to form oligomers (arcAbeta mice), we show that the LTP impairment induced by Abeta oligomers can be fully reversed by PP1 inhibition in vitro. We further demonstrate that the genetic inhibition of endogenous PP1 in vivo confers resistance to Abeta oligomer-mediated toxicity and preserves LTP. Overall, these results reveal that PP1 is a key player in the mechanisms of AD pathology.

Amyloid-beta immunisation for Alzheimer's disease.

Alzheimer's disease is the main cause of dementia in elderly people and is becoming an ever greater problem as societies worldwide age. Treatments that stop or at least effectively modify disease course do not yet exist. In Alzheimer's disease, the conversion of the amyloid-beta peptide (Abeta) from a physiological water-soluble monomeric form into neurotoxic oligomeric and fibrillar forms rich in stable beta-sheet conformations is an important event. The most toxic forms of Abeta are thought to be oligomers, and dimers might be the smallest neurotoxic species. Numerous immunological approaches that prevent the conversion of the normal precursor protein into pathological forms or that accelerate clearance are in development. More than ten new approaches to active and passive immunotherapy are under investigation in clinical trials with the aim of producing safe methods for immunological therapy and prevention. A delicate balance between immunological clearance of an endogenous protein with acquired toxic properties and the induction of an autoimmune reaction must be found.

Fe65-PTB2 Dimerization Mimics Fe65-APP Interaction.

Physiological function and pathology of the Alzheimer's disease causing amyloid precursor protein (APP) are correlated with its cytosolic adaptor Fe65 encompassing a WW and two phosphotyrosine-binding domains (PTBs). The C-terminal Fe65-PTB2 binds a large portion of the APP intracellular domain (AICD) including the GYENPTY internalization sequence fingerprint. AICD binding to Fe65-PTB2 opens an intra-molecular interaction causing a structural change and altering Fe65 activity. Here we show that in the absence of the AICD, Fe65-PTB2 forms a homodimer in solution and determine its crystal structure at 2.6 Å resolution. Dimerization involves the unwinding of a C-terminal α-helix that mimics binding of the AICD internalization sequence, thus shielding the hydrophobic binding pocket. Specific dimer formation is validated by nuclear magnetic resonance (NMR) techniques and cell-based analyses reveal that Fe65-PTB2 together with the WW domain are necessary and sufficient for dimerization. Together, our data demonstrate that Fe65 dimerizes via its APP interaction site, suggesting that besides intra- also intermolecular interactions between Fe65 molecules contribute to homeostatic regulation of APP mediated signaling.

Gains and losses on the road to understanding Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder and the most common cause for dementia, which affects approximately 120 thousand people in Switzerland and 35 million worldwide. Aging is a major risk factor for developing AD and thus, as our societies are growing older, we face great challenges to find treatment strategies. The disease is characterised by loss of memory, deposition of extracellular amyloid plaques containing Aß peptides and intraneuronal tangles of the tau protein. To date, there is no effective treatment and the cause of the disease is still debated. The Schweizerische Alzheimervereinigung states that we need "continuous manifold research" into all possible causes of AD to find a cure for this disease. Fitting this proposition, a recent publication by Xia et al. (2015) described a novel mouse model that for the first time reproduces cortical neuron death as observed in human AD cases. At the same time, this publication questions the major theory of AD pathogenesis and points towards different treatment avenues that should be followed to find a cure for AD.

RanBP9 modulates AICD localization and transcriptional activity via direct interaction with Tip60.

Proteolytic processing of the amyloid-ß protein precursor (AßPP) occurs via alternative pathways, culminating with the production of the AßPP intracellular domain (AICD). AICD can translocate to the nucleus and regulate transcription, but its activity is modulated by interactions with other proteins. In the nucleus, AICD, FE65, and Tip60 associate into AFT complexes, which are targeted to nuclear spots which correspond to transcription factories. Here we report that RanBP9 interacts with the cytoplasmic domain of AßPP, through the NPXY internalization motif. Moreover, RanBP9 interaction with Tip60 is also described. The RanBP9-Tip60 interaction dramatically relocated RanBP9 from a widespread cellular distribution to nuclear speckles. AßPP processing is a central aspect in determining the protein's function and that of its resulting proteolytic fragments, among them AICD. The latter results from the amyloidogenic pathway and is the peptidic species predominantly involved in nuclear signaling. Of note RanBP9 transfection was previously demonstrated to increase amyloid-ß generation. Here we show that RanBP9 relocates AICD to the Tip60-enriched nuclear speckles, and prevented the formation of nuclear spots formation, having therefore a negative effect on AICD mediated nuclear signaling and consequently AFT complex formation. Furthermore, by transfecting cells with increasing amounts of RanBP9, the expression of AICD-regulated genes, including AßPP itself, was reduced. Given the data presented, one can deduce that RanBP9 has an inhibitory regulatory effect on AICD-mediated transcription and the effect is mediated by relocating AICD away from transcription factories.

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-, ß-, 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.

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 ß-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 ß-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.