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

APP overexpression results in an increased mitochondrial membrane potential.

<p><b>a)</b> Mitochondrial membrane potential was measured by staining the cells with the fluorescent dyes DiOC6, TMRM or JC-1 and subsequent analysis using FACS. Dead cells were excluded by gating in the FSC/SSC plot. Mean fluorescent intensities or the ratio of FL1/FL2 for JC-1 were corrected for the uncoupled control cells and normalized to control cells, which were set to 1. Means of 7 (DiOC6), 6 (TMRM) and 3 (JC-1) independent experiments are shown, each measured in triplicates. Error bars indicate standard error. Significance versus control was calculated using Kruskal-Wallis test with Dunn´s multiple comparison for DiOC6 and TMRM and One-way ANOVA with Bonferroni´s multiple comparison for JC-1. <b>b)</b> Calculated doubling time in the 48h period between seeding of the cells and high-resolution respirometry. The mean of 10 independent experiments is shown. Error bars indicate confidence intervals (95%). <b>c)</b> Measurement of lactic acid concentration in the conditioned medium in which also respirometry is performed in. Results of 5 independent experiments are shown, each measured in technical duplicates. Outcomes were corrected for collection time and mean cell number during collection time, and were finally normalized to the release rate of control cells, which was set to 1. Error bars indicate confidence intervals (95%).</p

High-resolution respirometry reveals a reduced mitochondrial respiration following APP but not BACE overexpression.

<p><b>a)</b> High-resolution respirometry performed in an Oroboros Oxygraph-2k. The blue curve shows the oxygen concentration in the sealed chamber and the red curve shows the oxygen consumption of the cells. 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 ~3μM (ETS capacity), 0.5μM rotenone and 5μM antimycin A (residual oxygen consumption; ROX). <b>b)</b> Measurement of complex IV capacity after blockage of complex III. Addition of 2mM ascorbate and 0.5mM TMPD (Asc+TMPD) results in maximum complex IV respiration plus auto-oxidation (intervals 1–3). Addition of 50μM sodium sulfide (Sulf) results in oxygen consumption just by auto-oxidation, which is dependent on oxygen concentration (intervals 4–9). <b>c)</b> Calculation of complex IV capacity. Pairs of values of oxygen flux and the corresponding oxygen concentration are displayed. The black rhombs show the dependence of the auto-oxidation on the oxygen concentration, whereas the blue squares show the sum of auto-oxidation and complex IV respiration in relation to the oxygen concentration. Thus, complex IV capacity can be seen as the distance between the blue and black regression curves. <b>d)</b> Oxygen consumption of the cells was corrected for ROX and normalized to the Routine respiration of HEK pUltra, which was set to 1. The mean of 7 (HEK pUltra), 8 (HEK APP pUltra) and 8 (HEK BACE) independent experiments, each measured in duplicate, are shown. Error bars indicate standard error. Significance versus control was calculated using Kruskal-Wallis test with Dunn´s method for correction of multiple comparison. <b>e)</b> Mitochondrial mass measured as Mitotracker Green FM fluorescence (MT Green) using FACS analysis or citrate synthase activity (CS activity). The mean values of 5 independent experiments measured in triplicate (MT Green) and 6 independent experiments measured in technical duplicates (CS activity) are shown, all normalized to control cells. Error bars indicate confidence intervals (95%). Significance versus control was calculated using Kruskal-Wallis test with Dunn´s method for correction of multiple comparison.</p

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

APP processing in HEK293 cells overexpressing APP or BACE1.

<p><b>a)</b> APP and BACE1 expression in HEK293 cells transduced with APP pUltra-hot, BACE pUltra-hot or the empty control vector pUltra-hot detected by western blotting using 6E10 α-Aβ antibody (Covance) and D10E5 α-BACE1 antibody. β-actin as a loading control was detected using AC-15 α-β-actin antibody (Sigma). <b>b)</b> Fold change of protein expression in HEK APP pUltra and HEK BACE pUltra versus control cells (HEK pUltra) by quantification of western blots. Western blots of three independent protein samples were analyzed with a total of 9 images. APP and BACE1 levels were corrected for β-actin loading control and normalized to the levels of control cells, which were set to 1. Error bars indicate standard error. <b>c)</b> Quantification of sAPP and Aβ secretion rate using ELISA and corrected for the collection time and the mean cell number during collection time. Results were normalized to the mean values of control cells, which were set to 1. The mean values of 3 independent experiments measured in duplicates are shown. Error bars indicate standard error.</p

Gamma secretase inhibitor treatment rescues mitochondrial respiration in HEK APP pUltra.

<p><b>a)</b> High-resolution respirometry of HEK pUltra and HEK APP treated with 0/0.1/1/5nM GSI, performed in an Oroboros 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 ~3μM (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 HEK pUltra, which was set to 1. Means of at least 8 independent experiments measured in duplicates are shown. Error bars indicate standard error. Significance was evaluated for different GSI concentrations versus untreated control using Kruskal-Wallis test and Dunn´s multiple comparison test. <b>b)</b> Differences in respiration (normalized to Routine of control, which was set to 1) of HEK APP pUltra to the respective control cells treated with the same concentration of GSI. Lower asterisks indicate significances of HEK APP pUltra versus respective control, upper asterisks indicate significances between the differences (HEK APP pUltra to HEK pUltra) of GSI treated to untreated cells. Significance was calculated using Kruskal-Wallis test and Dunn´s multiple comparison test.</p

Levels of antigen-bound and free Aβ-autoantibodies (OD at 450 nm) in serum of healthy adults.

<p>The mean levels of Aβ-IgG immune complexes are significantly higher than those of free Aβ-autoantibodies; <i>t</i>₍₃₅₎  = 10.12, <i>p</i><0.0001.</p

Antigen-Bound and Free β-Amyloid Autoantibodies in Serum of Healthy Adults

<div><p>Physiological β-amyloid autoantibodies (Aβ-autoantibodies) are currently investigated as potential diagnostic and therapeutic tools for Alzheimer’s disease (AD). In previous studies, their determination in serum and cerebrospinal fluid (CSF) using indirect ELISA has provided controversial results, which may be due to the presence of preformed Aβ antigen-antibody immune complexes. Based on the epitope specificity of the Aβ-autoantibodies, recently elucidated in our laboratory, we developed (a) a sandwich ELISA for the determination of circulating Aβ-IgG immune complexes and (b) an indirect ELISA for the determination of free Aβ-autoantibodies. This methodology was applied to the analysis of serum samples from healthy individuals within the age range of 18 to 89 years. Neuropsychological examination of the participants in this study indicated non-pathological, age-related cognitive decline, revealed especially by tests of visual memory and executive function, as well as speed-related tasks. The ELISA serum determinations showed significantly higher levels of Aβ-IgG immune complexes compared to free Aβ-autoantibodies, while no correlation with age or cognitive performance of the participants was found.</p> </div

Pearson’s <i>r</i> correlations between the levels of Aβ-IgG immune complexes (<i>n</i> = 39) versus free Aβ-autoantibodies (<i>n</i> = 39) and cognitive performance.

<p>Benton test (correct answers; range 0–20); Benton Test (errors; range 0–30); Digit span test (HAWIE-R; range 0–28); Digit-symbol substitution test (HAWIE-R; range 0–93); Figure recall (CERAD-NP-plus; range 0–14); Mosaic test (HAWIE-R; range 0–51); Phonetic/Semantic fluency (CERAD-NP-plus); TMT-A/B – Trail making test part A/B (CERAD-NP-plus; A: range 0–180 sec.; B: range 0–300 sec.); Word list learning (CERAD-NP-plus; range 0–30); Word recall (CERAD-NP-plus; range 0–10).</p>*<p>Significant correlation between cognitive test performance and age.</p>**<p>Significant correlation between cognitive test performance and age after correction for multiple correlation coefficients according to Holm.</p