The role of PTB domain containing adaptor proteins on PICALM-mediated APP endocytosis and localization

One hallmark of Alzheimer's disease (AD) is the presence of amyloid plaques, which mainly consist of the amyloid precursor protein (APP) cleavage product amyloid β (Aβ). For cleavage to occur, the APP must be endocytosed from the cell surface. The phosphatidylinositol binding clathrin assembly protein (PICALM) is involved in clathrin-mediated endocytosis and polymorphisms in and near the gene locus were identified as genetic risk factors for AD. PICALM overexpression enhances APP internalization and Aβ production. Furthermore, PICALM shuttles into the nucleus, but its function within the nucleus is still unknown. Using co-immunoprecipitation, we demonstrated an interaction between PICALM and APP, which is abrogated by mutation of the APP NPXY-motif. Since the NPXY-motif is an internalization signal that binds to phosphotryrosine-binding domain-containing adaptor proteins (PTB-APs), we hypothesized that PTB-APs can modulate the APP-PICALM interaction. We found that interaction between PICALM and the PTB-APs (Numb, JIP1b and GULP1) enhances the APP-PICALM interaction. Fluorescence activated cell sorting analysis and internalization assays revealed differentially altered APP cell surface levels and endocytosis rates that depended upon the presence of PICALM and co-expression of distinct PTB-APs. Additionally, we were able to show an impact of PICALM nuclear shuttling upon co-expression of PTB-APs and PICALM, with the magnitude of the effect depending on which PTB-AP was co-expressed. Taken together, our results indicate a modulating effect of PTB-APs on PICALM-mediated APP endocytosis and localization.status: publishe

Data from: Metabolic characterization of intact cells reveals intracellular amyloid beta but not its precursor protein to reduce mitochondrial respiration

One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases

raw data, Schaefer et al.

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Ubiquitination and the proteasome rather than caspase‐3‐mediated C‐terminal cleavage are involved in the EAAT2 degradation by staurosporine‐induced cellular stress

Glutamate (Glu) released synaptically by neurons and extrasynaptically by the cystine (Cys)/Glu antiporter system xc‐ in astrocytes and microglia (µGlia) is rapidly taken up by the astrocytic glutamate transporter EAAT2. EAAT2 is prominently down‐regulated in neurodegenerative diseases, probably leading to overstimulation of glutamate receptors (GluR), neuronal calcium overload (Ca++) and finally excitotoxicity. When recombinantly over‐expressed in neuroectodermal cells, EAAT2 is rapidly down‐regulated by staurosporine (STR)‐induced cellular stress associated with caspase 3 activation. We found that C‐terminal (COOH) caspase 3 cleavage does not play a role in this down‐regulation. Instead, C‐terminal lysines (Lys) are required for proteasomal degradation of EAAT2 following ubiquitination. Diminished glutamate (Glu) uptake via the excitatory amino acid transporter EAAT2, which normally accounts for ~90% of total forebrain EAAT activity, may contribute to neurodegeneration via Glu‐mediated excitotoxicity. C‐terminal cleavage by caspase‐3 (C3) was reported to mediate EAAT2 inactivation and down‐regulation in the context of neurodegeneration. For a detailed analysis of C3‐dependent EAAT2 degradation, we employed A172 glioblastoma as well as hippocampal HT22 cells and murine astrocytes over‐expressing VSV‐G‐tagged EAAT2 constructs. C3 activation was induced by staurosporine (STR). In HT22 cells, STR‐induced C3 activation‐induced rapid EAAT2 protein degradation. The mutation of asparagine 504 to aspartate (D504N), which should inactivate the putative C3 cleavage site, increased EAAT2 activity in A172 cells. In contrast, the D504N mutation did not protect EAAT2 protein against STR‐induced degradation in HT22 cells, whereas inhibition of caspases, ubiquitination and the proteasome did. Similar results were obtained in astrocytes. Phylogenetic analysis showed that C‐terminal ubiquitin acceptor sites—but not the putative C3 cleavage site—exhibit a high degree of conservation. Moreover, C‐terminal truncation mimicking C3 cleavage increased rather than decreased EAAT2 activity and stability as well as protected EAAT2 against STR‐induced ubiquitination‐dependent degradation. We conclude that cellular stress associated with endogenous C3 activation degrades EAAT2 via a pathway involving ubiquitination and the proteasome but not direct C3‐mediated cleavage. In addition, C3 cleavage of EAAT2, described to occur in other models, is unlikely to inactivate EAAT2. However, mutation of the highly conserved D504 within the putative C3 cleavage site increases EAAT2 activity via an unknown mechanism

Bioenergetic alterations of metabolic redox coenzymes as NADH, FAD and FMN by means of fluorescence lifetime imaging techniques

Metabolic FLIM (fluorescence lifetime imaging) is used to image bioenergetic status in cells and tissue. Whereas an attribution of the fluorescence lifetime of coenzymes as an indicator for cell metabolism is mainly accepted, it is debated whether this is valid for the redox state of cells. In this regard, an innovative algorithm using the lifetime characteristics of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) to calculate the fluorescence lifetime induced redox ratio (FLIRR) has been reported so far. We extended the FLIRR approach and present new results, which includes FLIM data of the various enzymes, such as NAD(P)H, FAD, as well as flavin mononucleotide (FMN). Our algorithm uses a two-exponential fitting procedure for the NAD(P)H autofluorescence and a three-exponential fit of the flavin signal. By extending the FLIRR approach, we introduced FLIRR1 as protein-bound NAD(P)H related to protein-bound FAD, FLIRR2 as protein-bound NAD(P)H related to free (unbound) FAD and FLIRR3 as protein-bound NAD(P)H related to protein-bound FMN. We compared the significance of extended FLIRR to the metabolic index, defined as the ratio of protein-bound NAD(P)H to free NAD(P)H. The statistically significant difference for tumor and normal cells was found to be highest for FLIRR1

APP overexpression reduces mitochondrial respiration in Neuro-2a cells.

<p><b>a)</b> APP and BACE1 overexpression in N2a cells stably transfected with HA-phCMV3, APP695-CMV(myc) or BACE-phCMV3(HA) detected by western blotting using α-HA antibody (Sigma) and 9E10 α-myc antibody (Sigma). β-actin as a loading control was detected using AC-15 α-β-actin antibody (Sigma). <b>b)</b> Calculated doubling time in the 48h period between seeding of the cells and high-resolution respirometry. The mean of 5 independent experiments is shown. Error bars indicate confidence intervals (95%). <b>c)</b> High-resolution respirometry of stably transfected N2a cells in an Oxygraph-2k. Titration protocol: addition of 2mio cells in their conditioned medium (Routine respiration), 1.25μM oligomycin (Leak respiration), titration of FCCP to a final concentration of ~2.5μM (ETS capacity), 0.5μM rotenone and 5μM antimycin A (ROX). Oxygen consumption of the cells was corrected for ROX and normalized to the Routine respiration of N2a HA, which was set to 1. The means of 10 independent experiments, each measured in duplicate, are shown. Error bars indicate standard error. Significance was calculated using Kruskal-Wallis test and Dunn´s multiple comparison test. <b>d)</b> Mitochondrial mass measured as Mitotracker Green FM fluorescence using FACS analysis. The mean fluorescence in the FL1 channel was normalized to control cells, which was set to 1. The mean values of 5 independent experiments measured in triplicate are shown. Error bars indicate confidence intervals (95%).</p

APP as against BACE1 overexpression results in higher intracellular Aβ and a reallocation towards mitochondria.

<p><b>a)</b> Reduction of transduction strength quantified by measuring mCherry fluorescence of living cells by FACS analysis. Fluorescence was normalized to the first day of measurement (day 0) which was set to 1. Means of 3 independent experiments each with at least 2 time points of analysis per time interval are displayed. Error bars indicate standard error. Significance versus control cells was calculated using One-way ANOVA with Bonferroni´s Multiple comparison. <b>b)</b> Intracellular Aβ40 levels measured by ELISA and displayed as absolute raw counts normalized to control cells, which were set to 1. Error bars indicate standard error. Significance was calculated using unpaired two-tailed t-test. <b>c)</b> High-resolution respirometry of naïve HEK293 cells treated with conditioned medium from HEK pUltra, HEK APP pUltra or HEK BACE pUltra for 18h, Oxygen consumption of the cells was corrected for ROX and normalized to the Routine respiration of HEK sn pUltra, which was set to 1. Means of 6 independent experiments measured in duplicates are shown. Error bars indicate standard error. Significance was evaluated versus control using Kruskal-Wallis test with Dunn´s multiple comparison for routine respiration and One-way ANOVA with Bonferroni´s multiple comparison for Leak respiration and ETS capacity. <b>d)</b> Quantification of mitochondrially localized portion of APP/Aβ determined by immunoelectron microscopy. Per experiment, at least 20 images of mitochondria-rich regions were taken and all immunogold particles were counted manually. Means of 3 independent experiments are displayed. Error bars indicate standard error. Significance was calculated using unpaired two-tailed t-test. <b>e)</b> Exemplary image for the immunogold-staining of Aβ/APP in HEK293 cells.</p

Intracellular but not extracellular Aß correlates with the rescue of mitochondrial respiration following GSI treatment.

<p><b>a)</b> Quantification of Aβ38, Aβ40 and Aβ42 secretion rate using ELISA. Secretion rates were corrected for the collection time and the mean cell number during collection time. Results were normalized to the mean values of untreated control cells, which were set to 1. The mean values of at least 3 independent experiments measured in technical duplicates are shown. Error bars indicate standard error. <b>b)</b> Intracellular Aβ40 levels measured by ELISA and displayed as absolute raw counts normalized to untreated control cells, which were set to 1. Error bars indicate standard error. <b>c)</b> Accumulation of APP-CTF detected by western blotting using α APP C-terminus-antibody (Sigma). <b>d)</b> Fold change of APP-CTF accumulation in HEK pUltra and HEK APP pUltra normalized to untreated cells (set to 1) by quantification of western blots. Western blots of three independent protein samples per condition were analyzed with 3 images each. Equal loading was ensured by BCA protein assay. Error bars indicate standard error.</p