Alzheimer's disease linked Aβ42 exerts product feedback inhibition on γ-secretase impairing downstream cell signaling

Abstract

Amyloid β (Aβ) peptides accumulating in the brain are proposed to trigger Alzheimer's disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for Aβ42 toxicity that arises from its proven affinity for γ-secretases. We hypothesized that the reported increases in Aβ42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. We conducted kinetic analyses of γ-secretase activity in cell-free systems in the presence of Aβ, as well as cell-based and ex vivo assays in neuronal cell lines, neurons, and brain synaptosomes to assess the impact of Aβ on γ-secretases. We show that human Aβ42 peptides, but neither murine Aβ42 nor human Aβ17-42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75, and pan-cadherin. Moreover, Aβ42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in Aβ42 contribute to cellular toxicity via the γ-secretase inhibition, and provide a novel conceptual framework to address Aβ toxicity in the context of γ-secretase-dependent homeostatic signaling.

Keywords: Alzheimer's disease; amyloid beta; amyloid toxicity; biochemistry; chemical biology; gamma-secretase; gamma-secretase inhibition; human; mouse; neuroscience; presenilin; rat.

Figure 1.. Human Aβ42 peptide inhibits γ-secretase-mediated proteolysis of APP_C99.

(A) The scheme depicts the γ-secretase-mediated cleavage of amyloid precursor protein (APP), leading to the generation of amyloid β (Aβ) and p3 peptides. The N-terminal sequence of APP_C99 /Aβ is shown in the lower panel. The differences in the amino acid sequence of human (hu) vs murine (mu) Aβ peptides and the positions of β’- and α-cleavages (that precede the generation of Aβ11–42 and p3 17–42 peptides, respectively) are indicated. The transmembrane domain is labeled in grey and the sequence of Aβ42 is presented within a rectangle. The initial γ-secretase endopeptidase cut may occur at one of two different positions on APP, generating two different de novo substrates that are further processed by carboxypeptidase-like cleavages as follows: Aβ49→Aβ46→Aβ43→Aβ40→Aβ37 or Aβ48 →Aβ45→Aβ42→Aβ38. Aβ38, Aβ40, and Aβ42 peptides are the major products under physiological conditions. The triangles mark the sequential cleavage positions. (B) The western blot presents APP intracellular domain (AICD) products generated de novo in detergent-based γ-secretase activity assays using APP_C99-3xFLAG at 0.4 μM or 1.2 μM as substrate. To test the inhibitory properties of human Aβ1–42, the peptide was added to the activity assays at concentrations ranging from 0.5 to 10 μM. DMSO at 2.5% was used as a vehicle control. The graphs present the quantification of the western blot bands corresponding to AICDs. The pink and green lines correspond to 0.4 μM and 1.2 μM substrate concentrations, respectively. The data are normalized to the AICD levels generated in the DMSO conditions, considered as 100%. The data are presented as mean ± SEM, n=6–16. (C) Mass spectrometry-based analysis of de novo generated AICD levels in in vitro cell-free γ-secretase activity assays containing 3 μM human Aβ1–42 or vehicle (0.6% DMSO) indicate that human Aβ1–42 inhibits both product lines. (D) The western blot presents de novo generated AICDs in detergent-based γ-secretase activity assays using 0.4 μM APP_C99-3xFLAG as substrate and wild- type γ-secretases composed of different presenilin (PSEN) (1 or 2) and anterior pharynx defective 1 (APH1) (A or B) subunits in the presence of vehicle, human Aβ1–42 at 3 μM or GSI (InhX) at 10 μM concentration. (E) The graph presents ELISA quantification of Aβ1–38 peptides generated in detergent- or detergent-resistant membranes (DRM)-based γ-secretase activity assays using either human Aβ1–42 at 10 μM or APP_C99-3xFLAG at 1.5 μM as substrates. The data are presented as mean ± SEM, n=3. (F) The western blot presents de novo generated AICDs in detergent-based γ-secretase activity assays using 0.4 μM APP_C99-3xFLAG as a substrate and γ-secretase immobilized on sepharose beads. During the first round, the activity assay was supplemented with 3 μM Aβ1–42, 10 μM GSI or DMSO vehicle. After this first round, the γ-secretase-beads were washed to remove the peptide and inhibitor, respectively, fresh substrate was added, and the reaction proceeded for the second round. The analysis demonstrates the reversibility of the Aβ1–42-mediated inhibition.

Figure 1—figure supplement 1.. Cell-free γ-secretase activity assay.

(A) Analysis of AICD-3xFLAG levels that are de novo generated by purified γ-secretase from the APP_C99-3xFLAG substrate is shown. Γ-secretase inhibitor (GSI) and catalytically inactive γ-secretase mutant (DA) were used as negative controls. The left side of the blot presents the analysis of unfractionated γ-secretase activity assay samples, while the hydrophilic fraction obtained through methanol:chloroform extraction is presented on the right side. This protein extraction method allows quantification of APP intracellular domains (AICDs) without the interference of the signal coming from the excess of APP_C99-3xFLAG substrate present in the assays. * marks a hydrophobic contaminant. (B) The graph presents ELISA quantification of Aβ1–38, 1–40, and 1–42 peptides generated in detergent-based γ-secretase activity assays using either human Aβ1–42, murine Aβ1–42, or human Aβ1–45 at 10 μM as substrates. The data are presented as median±IQR, n=5.

Figure 2.. N- and C-terminus of amyloid β (Aβ) influence the inhibitory properties of the peptide.

(A, B, C) The western blots present de novo generated APP intracellular domains (AICDs) in detergent-based γ-secretase activity assays using purified protease and APP_C99-3xFLAG at 0.4 μM or 1.2 μM as a substrate. To test the inhibitory properties of (A) murine Aβ1–42, (B) human Aβ11–42, and (C) human p3 17–42, the peptides were added to the assays at concentrations ranging from 0.5 to 10 μM. DMSO at 2.5% was used as a vehicle. The graphs present the quantification of the western blot bands for AICDs. The pink and green lines correspond to 0.4 μM and 1.2 μM substrate concentrations, respectively. The grey dotted lines in the plot B present the curves recorded for human Aβ1–42 (from Figure 1, plot B) for comparison. The data are normalized to the AICD levels generated in the DMSO conditions, considered as 100%, and presented as mean ± SEM, n=3–8. (D) Detergent-based γ-secretase activity assays using purified protease and APP_C99-3xFLAG at 0.4 μM concentration were supplemented with different Aβ peptides at 1 μM concentration or DMSO. De novo generated AICDs were quantified by western blotting. The data are shown as mean ± SEM, n=6–27. The statistics were calculated using one-way ANOVA and multiple comparisons Dunnett’s test, with DMSO (black) or Aβ1–42 (blue) set as references. **p<0.01, ****p<0.0001.

Figure 3.. Human Aβ42 leads to the accumulation of APP-CTFs.

(A, B, C, H) The western blots present full-length amyloid precursor protein (APP) (APP-FL) and APP C-terminal fragments (APP-CTFs) detected in (A) SH-SY5Y, (B) PC12, (C) ReNcell VM human neural progenitor cells and (H) induced pluripotent stem cell-derived human neurons treated for 24 h with respective peptides at indicated concentrations or vehicle (DMSO). The ratio between the APP-CTF and APP-FL levels was calculated from the integrated density of the corresponding western blot bands. The data are shown as mean ± SEM, n=3–23. The statistics were calculated using one-way ANOVA and multiple comparison Dunnett’s test, with DMSO set as a reference. *p<0.05, **p<0.01, ****p<0.0001. (D) The amount of HiBiT-Aβ peptides was measured in the conditioned medium collected from human embryonic kidney (HEK) cell line stably expressing HiBiT-APP C99 treated with DMSO, Aβ1–42 (1 μM) or p3 17–42 (1 μM). The data are shown as mean ± SEM, n=8–16. The statistics were calculated using one-way ANOVA and multiple comparisons Dunnett’s test, with DMSO, set as a reference. ****p<0.0001. (E) The scheme presents the principles of the lactate dehydrogenase (LDH)-based and ATP-based cytotoxicity assays. (F) Cytotoxicity of the treatments was analyzed in SH-SY5Y cells. The cells were treated with DMSO, Aβ1–42 (1 μM), p3 17–42 (1 μM), or GSI (InhX, 2 μM) for 24 h, conditioned medium collected and subjected to the measurement of lactate dehydrogenase (LDH) activity using luminescence-based assay. TX-100 was used as a positive control expected to lead to 100% cell death. The data are shown as mean ± SEM, n=5–17. The statistics were calculated using one-way ANOVA and multiple comparisons Dunnett’s test, with DMSO set as a reference, ****p<0.0001. TX-100 led to a marked increase in the luminescent signal, while no significant toxicity of the other treatments was detected. (G) An ATP-based cell viability assay was used to determine the cytotoxicity of the treatments. We analyzed SH-SY5Y cells treated with DMSO, Aβ1–42 (1 μM), p3 17–42 (1 μM), or GSI (Inh X, 2 μM) for 24 h. The data are shown as mean ± SEM, n=9–18. The statistics were calculated using one-way ANOVA and multiple comparisons Dunnett’s test, with DMSO set as a reference. No significant toxicity of the treatments was detected.

Figure 4.. Human Aβ42 selectively accumulates in the cells and inhibits γ-secretase-mediated proteolysis.

(A) APP-FL and APP-CTF levels in SH-SY5Y cells treated for 24 h with a series of amyloid β (Aβ) peptides at 1 μM concentration are shown. The APP-CTF/FL ratio was calculated from the integrated density of the corresponding western blot bands. The data are shown as mean ± SEM, n=3–23. The statistics were calculated using one-way ANOVA and multiple comparisons Dunnett’s test, with DMSO set as a reference. ****p<0.0001. (B) PC12 cells were treated with respective Aβ or p3 peptides for 24 h and stained with anti-Aβ antibody (clone 4G8) followed by anti-mouse Alexa Fluor Plus 488 conjugated secondary antibody, Alexa Fluor Plus 555 conjugated phalloidin and nuclear stain DAPI. Scale bar: 20 μm.

Figure 5.. Human Aβ42 inhibits endogenous γ-secretase activity in neurons.

(A) The scheme presents the Fӧrster resonance energy transfer (FRET)-based probe allowing monitoring of γ-secretase activity in situ in living cells. (B, C) Spectral FRET analysis of γ-secretase activity in mouse primary neurons using a C99 Y-T probe is shown. The cells were treated with the indicated peptides/compounds at 1 μM concentration for 24 h. Γ-secretase-mediated proteolysis results in an increase in the distance between two fluorophores incorporated in the probe (YPet and Turquoise-GL). The increase in the distance translates to the reduced FRET efficiency, quantified by the YPet/Turquoise-GL fluorescence ratio. The distribution of recorded FRET efficiency, inversely correlating with γ-secretase activity, is shown in the density plots, n=4 (n=331–354). Medians are shown as dashed lines. Optimal bin number was determined using the Freedman-Diaconis rule. The statistics were calculated using the Kruskal-Wallis test and multiple comparison Dunn test. Significant differences (***p<0.001) were recorded for DMSO vs Aβ1–42, p3 17–42 vs Aβ1–42, DMSO vs GSI, and p3 17–42 vs GSI. Scale bar: 20 μm.

Figure 6.. Contribution of Aβ42-mediated inhibition of γ-secretase and general degradation mechanisms to APP-CTF accumulation.

(A) The scheme presents the experimental design of the cycloheximide (CHX)-based assay evaluating APP-FL and APP-CTF stability. (B) Western blot shows APP-FL and APP-CTF levels in SH-SY5Y cells at 0, 1, 2.5, and 5 h collection points defined in the scheme (A). (C) The integrated densities of the bands corresponding to APP-FL and APP-CTF were quantified and plotted relatively to the time point zero. The data are presented as mean ± SEM, n=6. Statistics were calculated using two-way ANOVA, followed by multiple comparisons Dunnett’s test. *p<0.05, **p<0.01. (D) Quantification of APP-CTF/ FL ratio at the zero time point is shown. The data are presented as mean ± SEM, n=6. Statistics were calculated using unpaired Student’s t-test, **p<0.01.

Figure 6—figure supplement 1.. Sheddase activation is not behind the APP-CTF accumulation in cells.

(A) The western blots present soluble amyloid precursor protein (APP) (soluble APP, sAPP) detected in conditioned medium collected from SH-SY5Y cells treated for 24 h with respective peptides at 1 μM concentration, γ-secretase (GSI) (Inh X, 2 μM) or vehicle (DMSO). The sAPP levels were calculated from the integrated density of the corresponding western blot bands. The data are shown as mean ± SEM, n=7–18. The statistics were calculated using one-way ANOVA and multiple comparisons Dunnett’s test, with DMSO set as a reference. ***p<0.001. (B) The western blots present ADAM10, BACE1, PSEN1-CTF and GAPDH detected in total lysates prepared from SH-SY5Y cells treated for 24 h with respective peptides at 1 μM concentration, GSI (InhX, 2 μM), or vehicle (DMSO). The relative expression levels of the proteases were calculated from the integrated density of the corresponding western blot bands and normalized to housekeeping protein levels (GAPDH). The data are shown as mean ± SEM, n=6, with DMSO considered as 100%. The statistics were calculated using two-way ANOVA and multiple comparisons Dunnett’s test, with DMSO set as a reference. No statistically significant differences were detected between the tested conditions.

Figure 7.. Human Aβ42 leads to the accumulation of APP-CTFs.

Scheme depicts the experimental design testing the impact of biologically-derived Aβ on the proteolysis of amyloid precursor protein (APP). Conditioned media were collected from fully differentiated wild-type (WT) human neurons (WT-CM) and human neurons expressing APP with Swedish mutation (SWE-CM). A portion of SWE-CM was subjected to amyloid β (Aβ) immunodepletion using the anti-Aβ antibody 3D6. WT, SWE, and Aβ immunodepleted (delSWE-CM) conditioned medias (CMs) were added to the PC12 cells for 24 h to analyze APP processing. As a reference, control cells treated with base media were analyzed. Representative western blots present the analysis of total cellular proteins from four cell culture sets treated with base media (control), WT-CM, SWE-CM, and delSWE-CM, respectively. The ratio between APP-CTFs and APP-FL was calculated from the integrated density of the corresponding western blot bands. The data are shown as mean ± SEM, n=3. The statistics were calculated using one-way ANOVA and multiple comparisons Dunnett’s test, with a control set as a reference, *p<0.05.

Figure 8.. Human Aβ1–42 peptides inhibit proteolysis of multiple γ-secretase substrates.

(A) The western blot presents de novo generated NICDs in detergent-based γ-secretase activity assays, using NOTCH1-3xFLAG at 0.4 μM and 1.2 μM as a substrate, supplemented with human Aβ1–42 peptides at concentrations ranging from 0.5 to 10 μM. The graphs present the quantification of the western blot bands for NICDs. The pink and green lines correspond to 0.4 μM and 1.2 μM substrate concentrations, respectively. The data are normalized to the NICD levels generated in the DMSO conditions, considered as 100%, and presented as mean ± SEM, n=3–5. (B) Analysis of the de novo intracellular domain (ICD) generation in cell-free detergent-based γ-secretase activity assays is shown. The graph presents the quantification of the western blots. The data are shown as mean ± SEM, n=3–18. The statistics were calculated using one-way ANOVA and multiple comparisons of predefined columns, with the Šidák correction test, with respective DMSO-supplemented reactions set as a reference, ****p<0.0001. (C) PanCad-FL and PanCad-CTF levels in ReNcell VM cells treated for 24 h with human Aβ1–42 peptides at 1 μM or GSI (InhX) at 2 μM concentration were quantified by western blotting. The PanCad-CTF/FL ratio was calculated from the integrated density of the corresponding bands. The data are presented as mean ± SEM, n=6–8. The statistics were calculated using one-way ANOVA and multiple comparisons Dunnett’s test, with DMSO set as a reference. *p<0.05. (D) The graph presents the quantification of the HiBiT-Aβ like peptide levels in conditioned media collected from HEK cell line stably expressing the HiBiT-NOTCH1 based substrate and treated with DMSO, Aβ1–42 (1 μM), or p3 17–42 (1 μM). The data are shown as mean ± SEM, n=8–16. The statistics were calculated using one-way ANOVA and multiple comparisons Dunnett’s test, with DMSO set as a reference. ****p<0.0001.

Figure 8—figure supplement 1.. Mass spectrometry analysis confirms the inhibitory action of human Aβ1–42 peptides.

The chromatograms present the generation of intracellular domains (ICDs) from three different substrates in in vitro cell-free γ-secretase activity assays, analyzed by mass spectrometry. Solid red – vehicle; dashed red – 3 μM human Aβ1–42.

Figure 9.. Human Aβ1–42 peptides compromises p75-mediated signaling.

(A) PC12 wild-type or PC12 deficient for TrkA (PC12nnr5) were incubated with human Aβ1–42 or p3 17–42 peptides, γ-secretase (GSI) (compound E), or vehicle (DMSO) for 72 h. The images present immunocytochemical analyses of cleaved caspase 3, and the graph corresponding quantification of the percentage of cleaved caspase 3 positive cells. Scale bar 50 µm. The statistics were calculated within PC12 and PC12nnr5 groups using one-way ANOVA and multiple comparison Dunnett’s test, with DMSO set as a reference. The data are presented as mean ± SEM, n=3. **p<0.01,****p<0.0001. (B) Representative western blot demonstrates the accumulation of p75-CTFs in the cells treated with human Aβ1–42 peptide or GSI. The data are presented as mean ± SEM, n=3. The statistics were calculated within PC12 and PC12nnr5 groups using one-way ANOVA and multiple comparison Dunnett’s test, with DMSO set as a reference. *p<0.05, **p<0.01. (C) Mouse primary neurons were treated with human Aβ1–42 (1 μM), p3 17–42 (1 μM), GSI (compound E, 10 μM), or vehicle control, in the absence or presence of K252α inhibitor at 0.5 μM. Level of apoptosis in basal frontal cholinergic neurons (BFCNs) was analyzed by immunostaining for choline acetyltransferase (ChAT) and cleaved caspase 3. Representative images are shown, scale bar 25 µm. The graph presents the quantification of the percentage of cleaved caspase-3 positive cells among ChAT-positive cells. The data are presented as mean ± SEM, n=3. The statistics were calculated within -K252α and + K252α groups using one-way ANOVA and multiple comparison Dunnett’s test, with DMSO set as a reference. **p<0.01, ***p<0.001.

Figure 10.. Aβ42 accumulates in Alzheimer’s disease (AD)-linked synaptosomes and treatment with this peptide increases APP-CTFs in control, mouse-derived synaptosomes.

(A) Synaptosomes from brain cortices of three wild-type mice were isolated, treated with DMSO or Aβ1–42, and analyzed by western blotting for APP-FL and APP-CTFs. Note the increase in APP-CTF/ FL ratio in Aβ1–42 treated samples relative to the control. The data are presented as mean ± SEM, n=3. The statistics were calculated using an unpaired Student’s t-test. *p<0.05. (B) Aβ42 levels in synaptosomes derived from frontal cortices of post-mortem AD and age-matched non-demented (ND) control individuals were measured by ELISA. The data are presented as mean ± SEM, n=3. The statistics were calculated using an unpaired Student’s t-test. ***p<0.001. (C) Schematic representation of the amyloid β (Aβ)-driven γ-secretase inhibitory feedback model is shown. (D) Scheme of the inhibitory model. Pathological increments in Aβ42 and endosomal accumulation of the peptide facilitate the establishment of an inhibitory product feedback mechanism that results in impairments in γ-secretase-mediated homeostatic signaling and contributes to AD progression. The inhibitory mechanism is complex, competitive, and reversible, and hence results in pulses of γ-secretase inhibition.

Author response image 1.