We have shown that Aβ peptides displaying a relatively low affinity for γ-secretase, when present at high concentrations, can compete with the higher affinity APP_C99 substrate for binding to the enzyme (Szaruga et al., 2017). Based on this observation, we hypothesized that an increase in the concentration of Aβ42 may promote the establishment of an inhibitory mechanism that involves the formation of ‘non-productive’ enzyme-Aβ42 complexes. The Aβ42-mediated inhibition would result in the accumulation of unprocessed γ-secretase substrates and reduced production of their intracellular domains, both of which may contribute to dysregulated downstream signaling.

As a first step, we investigated the effects of human Aβ1–42 (from now on referred to as Aβ42) on the processing of APP_C99 in well-controlled kinetic analyses (Figure 1B; Szaruga et al., 2017). We incubated purified (wild-type) γ-secretase enzyme with purified APP_C99-3xFLAG substrate at the K_M or saturating concentrations (0.4 μM and 1.2 μM, respectively) in the presence of human Aβ42 peptides at concentrations ranging from 0.5 μM to 10 μM. We then analyzed the de novo generation of AICD-3xFLAG by quantitative western blotting (Figure 1—figure supplement 1A). Methanol:chloroform extraction was performed to remove the excess of unprocessed substrates, the high levels of which could preclude the quantitative analysis of ICDs. The analysis demonstrated that human Aβ42 inhibited γ-secretase-mediated proteolysis of APP_C99 in a dose-dependent manner. The more pronounced peptide-driven inhibition at the lower substrate concentration (IC₅₀=1.3 μM vs 3.6 μM for 0.4 and 1.2 μM APP_C99, respectively) is consistent with a mechanism in which Aβ42 competes with the substrate for the enzyme. In addition, mass spectrometry analyses of the proteolytic reactions showed that Aβ42 partially inhibited the generation of both AICD product types (AICD_50-99 and AICD_49-99) (Figure 1C), indicating that Aβ42 inhibits both γ-secretase product lines. Next, we tested whether human Aβ42 exerted inhibition on all members of the γ-secretase family – i.e., irrespective of the type of PSEN (1 vs 2) and APH1 (A vs B) subunits (Figure 1D). Quantitative western blotting analysis revealed a marked inhibition of total AICD production by all types of γ-secretases in the presence of 3 µM human Aβ42. These findings support a competitive mechanism wherein low-affinity substrates (acting also as products) are able to re-associate with the protease and inhibit the processing of transmembrane substrates when present at relatively high concentrations.

To gain further insights, we investigated γ-secretase mediated processing of Aβ42 to Aβ38 under the conditions used to examine APP_C99, using the latter as a positive control (Figure 1E). Despite the use of relatively high concentrations of Aβ42 (10 µM), this peptide was not converted into Aβ38. In contrast, proteolytic reactions using APP_C99 (1.5 μM) resulted in the generation of Aβ38 (0.5–1 nM). We also tested whether Aβ42 served as a substrate in conditions that mimic a native-like environment, i.e., detergent-resistant membranes (DRMs) (Figure 1E; Szaruga et al., 2017; Matsumura et al., 2014). As in the detergent conditions, Aβ42 was barely converted into Aβ38. We note that γ-secretase processes Aβ43 into Aβ40 under similar conditions, even when this peptide was added at much lower concentrations (0.5–1 µM) (Szaruga et al., 2017). Taken together, these observations indicate that exogenous Aβ42 does interact with γ-secretases but, unlike Aβ43, does not act as a substrate (at least under these conditions), supporting the notion that Aβ42-driven inhibition of γ-secretases is mediated via the formation of non-productive enzyme-substrate (E-S) like complexes. However, a scenario wherein Aβ42 interacts with APP_C99 to reduce the amount of free APP_C99 substrate available for the enzymatic cleavage is not excluded by these data.

We also investigated whether the inhibitory effects of Aβ42 on γ-secretase were reversible. To this end, we conjugated purified γ-secretase complexes to beads using a high-affinity anti-NCSTN nanobody and incubated the enzyme-conjugated beads with 0.4 µM APP_C99, in the absence or presence of 3 µM Aβ42, for 40 min at 37 °C. Note that this concentration of peptide substantially inhibited AICD generation (Figure 1B). As a control, 10 µM γ-secretase inhibitor X (GSI, Inh X) was included. After the incubation, we collected the supernatants, washed the beads in assay buffer, and re-incubated them with 0.4 µM APP_C99 for 40 min at 37 °C. Analysis of the levels of the de novo generated AICD products in the supernatant fractions collected before (reaction 1) and after washes (reaction 2) indicated that Aβ42 inhibition of γ-secretase is fully reversible (Figure 1F). Collectively, our analyses support a model wherein Aβ42 forms a non-productive E-S-like complex with γ-secretase and its binding is reversible.

We then investigated the structure-function relationships relevant to the Aβ42-driven inhibitory mechanism. The effects of mouse/rat (murine) Aβ42 and N-terminally truncated human Aβx-42 (11–42 and 17–42) peptides on γ-secretase activity were examined in cell-free assays using peptide concentrations ranging from 0.5 μM to 10 μM (Figure 2A–C). Quantification of the de novo AICD product levels showed that murine Aβ42 maximally inhibited γ-secretase activity by ~20% (Figure 2A). As three amino acids in the N-terminal domain (R5G, Y10F and H13R) differentiate human and murine Aβ1–42 peptides (Figure 1A), the differences in the inhibition thus defined the N-terminal domain of Aβ as contributing to the inhibitory mechanism. It is noteworthy that similar to human Aβ1–42, murine Aβ1–42 was not processed to Aβ1–38 (Figure 1—figure supplement 1B). The analyses of other naturally occurring N-terminally truncated Aβx-42 peptides, generated by β-secretase (alternative) cleavage at the position 11 or by α-secretase cut at the position 17 in the Aβ sequence, showed that the truncated peptides exhibited reduced inhibitory potencies relative to Aβ42. The IC₅₀ values for Aβ11–42 were reduced 1.79- and 1.31-fold (K_M and saturating substrate concentrations, respectively), relative to Aβ42 (Figure 2B, Supplementary file 1a), while the larger N-terminal truncation (of residues 1–16) even further reduced the inhibitory effect to the level seen with murine Aβ42 (Figure 2C). Collectively, these data assign a defining role to the N-terminal region of Aβ in the inhibition of γ-secretase activity.

We next tested whether longer Aβ peptides (>Aβ42), which form more stable interactions with the protease (Szaruga et al., 2017), also inhibit γ-secretase activity. In addition, we investigated whether shortening of the C-terminus diminishes the inhibitory properties. To this end, we evaluated a series of naturally occurring Aβ1-x peptides, ranging from 37 to 43 amino acids in length, at 1 µM final concentration for their effect on γ-secretase proteolysis of APP_C99 (Figure 2D). In these experiments, the reduction in DMSO (vehicle) concentration to 0.2% was accompanied by ~1.7 fold enhanced Aβ-mediated inhibition, relative to the conditions shown in Figure 1B. We speculate that increased Aβ potency under these conditions is explained by a modest but measurable reduction in the proteolysis by the higher concentration of DMSO used in Figure 1 assays (Shelton et al., 2009). Relative to Aβ42, shorter Aβ species exerted progressively less or no inhibition on the de novo generation of AICD. It is noteworthy that peptides longer than Aβ42 serve as substrates for γ-secretases under these conditions, and their shortening potentially converts them into less potent inhibitory species (Szaruga et al., 2017). Taken together, these data strengthen the conclusion that human Aβ42 inhibits γ-secretases and indicate that both Aβ C- and N-termini modulate the inhibitory mechanism.

We reasoned that human Aβ42-driven inhibition of γ-secretase activity would lead to the accumulation of unprocessed γ-secretase substrates in cells (e.g. APP-CTFs in the case of APP) as well as reduced generation of products (Aβ peptides in the case of APP). To test these possibilities, we treated human neuroblastoma SH-SY5Y, rat pheochromocytoma PC12, and human neural progenitor cells (ReNcell VM) with recombinant human Aβ42 or p3 17–42 peptides at 1 μM or 2.5 μM final concentrations, and analyzed the levels of endogenous APP-CTFs and full-length APP (APP-FL) by western blotting (Figure 3A–C). Treatment with GSI (InhX) at 2 μM was included as a positive control. Human Aβ42 treatment increased the APP-CTF/FL ratio, a read-out of γ-secretase activity. In contrast, treatment with p3 peptide had no effect. The increments in the APP-CTF/FL ratio suggested that Aβ42 (partially) inhibits the global γ-secretase activity. To further investigate this, we measured the direct products of the γ-secretase-mediated proteolysis of APP. Since the detection of the endogenous Aβ products via standard ELISA methods was precluded by the presence of exogenous human Aβ42 (treatment), we used an N-terminally tagged version of APP_C99 and quantified the amount of total secreted Aβ, which is a proxy for the global γ-secretase activity. Briefly, we overexpressed human APP_C99 N-terminally tagged with the short 11 amino acid long HiBiT tag in human embryonic kidney (HEK) cells, treated these cultures with human Aβ42 or p3 17–42 peptides at 1 μM, or DAPT (GSI) at 10 μM, and determined total HiBiT-Aβ levels in conditioned media (CM). DAPT fully inhibits γ-secretase at the concentration used, and hence the values measured in DAPT-treated conditions were used for the background subtraction. We found a ~50% reduction in luminescence signal, directly linked to HiBiT-Aβ levels, in CM of cells treated with human Aβ42 and no effect of p3 peptide treatment, relative to the DMSO control (Figure 3D). The observed reduction in the total Aβ products is consistent with the partial inhibition of γ-secretase by Aβ42.

In addition, to controlling for the overall cellular toxicity of Aβ42, we performed two cell toxicity assays that rely on different biological principles (Figure 3E). In the first one, we quantified the activity of lactate dehydrogenase (LDH) released by cells into the conditioned medium upon plasma membrane damage (Figure 3F). In the second, we detected cellular ATP as a reporter of viability. There was no significant change in these measures in response to Aβ42 (1 μM), p3 17–42 (1 μM) or GSI (2 μM) (Figure 3G).

We next asked if the effects of Aβ42 would be also registered in wild-type human neurons. We thus examined neurons derived from induced pluripotent stem cells (iPSC) and treated with human or murine Aβ42, p3 17–42, or human Aβ40 (all at 2.5 μM). Only treatment with human Aβ42 resulted in a significant increase of APP-CTFs (Figure 3H), supporting the inhibitory role of Aβ42.

Next, we examined the effects of a broader spectrum of Aβ peptides, presenting different N- and C-termini, on APP-CTF levels in SH-SY5Y cells. In addition, we tested murine Aβ42, which failed to inhibit γ-secretase activity in cell-free assays (Figure 4A). The analysis confirmed a significant ~2.5 fold increase in the APP-CTF/APP-FL ratio in cells treated with human Aβ42 but, intriguingly, none of the other investigated peptides significantly changed APP-CTF levels.

These observations provided evidence that Aβ42 differs from other tested peptides in one or more features that are critical for γ-secretase inhibition in cells. Previous studies have shown that selective cellular uptake of human Aβ42, relative to Aβ40, leading to its concentration in the acidic endolysosomal network (ELN), promotes peptide aggregation into soluble toxic Aβ species (Hu et al., 2009; Su and Chang, 2001; Schützmann et al., 2021; Liu et al., 2010; Esbjörner et al., 2014; Friedrich et al., 2010). We reasoned that since this compartment is the main locus of γ-secretase activity (Maesako et al., 2020), the concentration of Aβ42 in ELN may not only promote its aggregation but also facilitate its inhibitory actions on γ-secretases. To investigate whether the selective accumulation of Aβ42 in ELN, relative to the other tested peptides, could explain the differential (cellular) inhibitory profiles (Figure 4B), we treated PC12 cells with human Aβ40, Aβ42, Aβ43, or p3 17–42, or murine Aβ42 peptides at 1 µM final concentration for 24 h, and examined their intracellular pools by immunostaining with an anti-Aβ/p3 antibody, 4G8 (epitope: 17–23 aa). We found that, unlike other Aβ or p3 peptides, human Aβ42 accumulated in cells. The pattern of the accumulation appeared distinct, punctate, and largely perinuclear, suggestive of its presence in the endolysosomal compartment. The data are consistent with a model in which intracellular accumulation of Aβ42, due to a selective cellular uptake or reduced peptide degradation, distinguishes it from the other peptides studied and explains its apparently unique inhibitory properties in the cellular context.

We next asked whether the accumulation of APP-CTFs in cells stems from direct inhibition of γ-secretases. To answer this question, we applied an established cell-based γ-secretase activity assay to test in situ the protease activity in primary mouse neurons (Maesako et al., 2020; Houser et al., 2020; Figure 5A). This assay uses an APP_C99-based fluorescent substrate (the C99 Y-T biosensor) to probe γ-secretase activity in living cells. This biosensor comprises APP_C99 fused at the C-terminus with YPet, followed by a linker, Turquoise-GL, and a membrane-anchoring domain to stabilize the probe in the membrane. The cleavage of the APP_C99-based probe by endogenous γ-secretases extends the distance between YPet and Turquoise-GL, and therefore results in a decrease in the efficiency of Förster resonance energy transfer (FRET) for this pair. The ratiometric nature of the reporter, and its independence of α- and β-secretase activity and cellular degradation mechanisms, allow quantitative analysis of γ-secretase activity in situ in living cells.

We treated mouse primary neurons with human Aβ42, p3, GSI (1 μM DAPT), or vehicle (DMSO) for 24 h and performed FRET analysis (Figure 5B–C). While p3 peptides did not affect the FRET signal, significant increases in FRET efficiency (i.e. increased proximity between YPet and Turquoise-GL) in cells treated with human Aβ42 or GSI, relative to vehicle-treated cultures, indicated increments in the levels of the full-length (uncleaved) fluorescent substrate, and thus reduced γ-secretase activity. These results provided quantitative evidence in a cellular context for the inhibition of endogenous γ-secretases by human Aβ42, but not p3 17–42 peptides.

Our data support a direct effect of Aβ42 peptides on the activity of γ-secretase in well-controlled, cell-free systems and in cell-based assays. Although the cell-free and FRET-based systems are independent of α- and β-secretase activity and cellular degradation pathways, we elected to further pursue the possibility that changes in the activity of these enzymes or degradation of CTFs could contribute to the observed increase in APP-CTFs in cellular systems. We investigated the impact of Aβ42 on α-secretase (ADAM10) and β-secretase (BACE1) levels, alterations in which could result in increased APP-CTF levels, by analyzing the expression of ADAM10 and BACE1 in total cell lysates from SH-SY5Y cells treated with Aβ40 (1 μM), Aβ42 (1 μM), p3 17–42 (1 μM), GSI (InhX, 2 μM), or vehicle DMSO control (Figure 6—figure supplement 1). We found no evidence of increased either α-secretase or β-secretase levels. In addition, we explored possible differences in the activities of these secretases by examining total soluble APP (sAPP) in a conditioned medium collected from SH-SY5Y cells treated under the same conditions. Our analysis showed a reduction in total sAPP levels in the conditioned medium collected from cells treated with Aβ42. This finding is inconsistent with an Aβ42-mediated effect that would increase APP-CTF levels as a result of activating α-secretase or β-secretase activity. Further studies are required to define whether the decrease in total sAPP is a consequence of sheddase inhibition or relates to an effect of Aβ42 on the rate of recycling and release of sAPP fragments.

We also evaluated potential γ-secretase independent changes in the degradation of APP-CTF by assessing the impact of Aβ42 peptides on γ-secretase independent APP-CTF half-life using the well-established cycloheximide (CHX)-based assay. CHX inhibits protein synthesis by blocking translation elongation (Schneider-Poetsch et al., 2010). In these experiments, we pre-treated SH-SY5Y cells with either GSI (DAPT, 10 μM) or GSI + Aβ42 (1 μM) for 24 h. Importantly, the GSI treatment allowed for complete γ-secretase inhibition in both conditions, resulting in the maximum accumulation of APP-CTFs that can possibly be achieved by blocking γ-secretase processing. We then added CHX (50 μg/ml) while maintaining GSI and vehicle versus GSI plus Aβ42. In addition, we treated with bafilomycin A1 (200 nM), which de-acidifies lysosomes and compromises degradation pathways, as a positive control (Figure 6A). We collected cells thereafter at 0 h, 1 h, 2.5 h and 5 h.

Quantitative analysis of the levels of both APP-CTFs and APP-FL over the 5 h time-course showed that bafilomycin A1 treatment markedly prolonged the half-life of both proteins but did not reveal significant differences in their levels between GSI versus GSI plus Aβ42-treated cells (Figure 6B–C). The lack of a significant effects of Aβ42 on the levels of APP-CTFs under these conditions (full γ-secretase inhibition) suggests that the peptide does not induce major defects in cellular degradation pathways, at least at this concentration. However, we noted an increased APP-CTF/FL ratio in GSI + Aβ42 treated cells vs GSI + DMSO treated ones at time zero (Figure 6D), suggesting that the Aβ42-induced increase in APP-CTFs is mediated by both inhibition of γ-secretase and one or more additional mechanisms. Nevertheless, the greater relative accumulation of APP-CTFs in the absence (138%) (Figure 3A) versus the presence (48%) of the GSI (Figure 6D) is evidence that Aβ42-mediated γ-secretase inhibition plays a more prominent role.

We next asked whether Aβ from disease-relevant biological sources would recapitulate the effects of recombinant Aβ42. For these studies, we used a culture medium conditioned by human iPSC-derived neurons expressing APP wild-type or the KM670/671 NL (SWE) variant. The APP SWE mutation increases total Aβ production by promoting the amyloidogenic processing of APP. ELISA-based quantification of Aβ content estimated a total concentration in the low nanomolar range (0.5–1 nM). Conditioned medium (WT or SWE) was applied to PC12 cells either directly or after Aβ depletion (delSWE) using the anti-APP 3D6 (epitope Aβ 1–5) (Figure 7). After a 24 h incubation at 37°C, the APP-CTF/FL ratio was measured. Cells incubated in control, unconditioned media were used as a reference control. We observed significant increments in the APP-CTF/FL ratio in cells treated with SWE-conditioned media, relative to control cells. Aβ immunodepletion from the SWE medium (delSWE-CM) lowered the APP-CTF/FL ratio to the levels observed in the control cells. These data provide evidence that Aβ peptides derived from biological sources induce the accumulation of APP-CTFs even when present at low nM concentrations, and thus point to this biological source of Aβ as being far more potent than the recombinant Aβ42.

The Aβ42-mediated inhibition of endogenous γ-secretase activity in cells raises the possibility that Aβ42 would inhibit the processing of γ-secretase substrates beyond APP-CTFs. To address this, we analyzed the effects of human Aβ42 on the processing of a purified NOTCH1-based substrate in cell-free conditions (Figure 8A). We incubated purified γ-secretase and the NOTCH1-based substrate (at 0.4 μM or 1.2 μM concentrations) in the presence of increasing amounts of human Aβ1–42 (ranging from 0.5 μM to 10 μM). Quantification of the de novo generated NICD-3xFLAG showed a dose-dependent inhibition of γ-secretase proteolysis of NOTCH1 by Aβ42. As for APP_C99, the derived IC₅₀ values showed that the degree of the inhibition depended on substrate concentration, consistent with a competitive mechanism (Supplementary file 1a). We extended this analysis by assessing the γ-secretase-mediated proteolysis of other substrates (ERBB4-, neurexin-, and p75-based) in the presence of 3 μM human Aβ42 (Figure 8B). Quantification of the respective de novo generated ICDs revealed that Aβ42 reduced γ-secretase proteolysis of each of these substrates. Mass spectrometry-based analyses of the respective ICDs confirmed the inhibition (Figure 8—figure supplement 1).

We next investigated the effects of human Aβ42 on γ-secretase-mediated processing of pan-cadherin in ReNcell VM cells. Cleavage of cadherins by α-secretase generates a membrane-bound CTF, which is a direct substrate of γ-secretases (Uemura et al., 2006). As before, we used the PanCad-CTF/FL ratio to estimate the efficiency of the γ-secretase-mediated proteolytic processing (Figure 8C). Treatment with human Aβ42 and InhX, but not p3, resulted in significant increases in the PanCad-CTF/FL ratio, demonstrating that Aβ42 inhibits the processing of several different γ-secretase substrates. These results suggest that this peptide can inhibit the proteolysis of γ-secretases substrates in general.

Finally, we measured the direct N-terminal products generated by γ-secretase proteolysis from a HiBiT-tagged NOTCH1-based substrate, an estimate of the global γ-secretase activity. We quantified the Aβ-like peptides secreted by HEK 293 cells stably expressing this HiBiT-tagged substrate upon treatment with 1 µM Aβ1–42, p3 17–42 peptide or 10 µM DAPT (GSI) as control for background subtraction (Figure 8D). A~20% significant reduction in the amount of secreted N-terminal HiBiT-tagged peptides derived from the NOTCH1-based substrates in cells treated with Aβ1–42 supports the inhibitory action of Aβ1–42 on γ-secretase mediated proteolysis.

By demonstrating that Aβ42 inhibits γ-secretase proteolysis of substrates that are structurally distinct from APP-CTFs, these data indicate that Aβ42 inhibits enzyme activity independent of substrate structure. This renders much less likely the mechanistic possibility that inhibition is due to an interaction with the substrate.

The p75 neurotrophin receptor plays a prominent role in neurotrophin signaling. Under certain conditions, its C-terminal fragment (p75-CTF, a γ-secretase substrate) modulates cell death (Wong et al., 2022; Conroy and Coulson, 2022). Previous studies have shown that accumulation of p75-CTF in basal forebrain cholinergic neurons (BFCNs), due to pharmacological inhibition of γ-secretases, triggers apoptosis in a TrkA activity-dependent manner (Franco et al., 2021). The proapoptotic action of p75-CTF is prevented by the expression of the TrkA receptor for nerve growth factor (NGF), which serves as a co-receptor with p75 (Franco et al., 2021). Similar observations have been made in PC12 cells, which model sympathetic neurons. In this case, γ-secretase inhibitors increased apoptosis in TrkA-deficient (PC12nnr5) (Loeb et al., 1991), but not in wild-type, TrkA-expressing cells. These findings provided an experimental system to investigate the effects of Aβ42-mediated inhibition on γ-secretase-mediated signaling in neurons and neuron-like cells.

We tested the impact of Aβ on p75 signaling in wild-type and PC12nnr5 cells. We exposed the cells to 1 µM human Aβ42, 1 µM p3, GSI (10 µM compound E), or vehicle (DMSO) for 3 days, and measured apoptosis via immunofluorescence staining for cleaved caspase 3 (Figure 9A). In addition, to assess γ-secretase proteolysis of p75, we measured the p75-CTF/FL ratio in the treated cultures (Figure 9B). Cleaved caspase 3 staining was increased in PC12nnr5 cells treated with GSI or human Aβ42, but not in those exposed to p3 or vehicle (DMSO). Of note, and consistent with our previous findings (Franco et al., 2021), none of the treatments elicited significant increases in cleaved caspase 3 in wild-type PC12 cells that express physiological levels of TrkA. In addition, analysis of the p75-CTF/FL ratio demonstrated higher p75-CTF levels in GSI and Aβ42 treated cells. These results, together with our previous observations, strongly suggest that Aβ42-mediated accumulation of p75-CTFs results in cell death signaling in the absence or near absence of TrkA expression. Our similar studies using BFCNs recapitulated the reported increased cell death under conditions in which both γ-secretase and TrkA activities were inhibited (CE and K252α inhibitor) (Franco et al., 2021), and showed that treatment with 1 µM human Aβ42 mimics the increase in cleaved caspase 3 driven by the GSI (Figure 9C). In conclusion, these results are evidence of a novel role for human Aβ42 peptide in the global inhibition of γ-secretase activity and dysregulation of cellular homeostasis.

Since both the amyloidogenic processing of APP and the accumulation of Aβ occur at the synapse (Pickett et al., 2016; Schedin-Weiss et al., 2016), we reasoned that synaptosomes are potentially a cellular locus where elevated levels of Aβ could be acting on γ-secretase. We thus tested whether γ-secretase-mediated processing of APP at the synapse is inhibited by Aβ42 (Figure 10A). To this end, we prepared synaptosomes from wild-type mouse brains, treated these fractions with 2.5 µM Aβ42 or vehicle, and then measured APP-FL and APP-CTF levels by western blotting. An increased APP-CTF/FL ratio in Aβ42 treated samples, compared to vehicle control, provided evidence of γ-secretase inhibition.

We next investigated the levels of Aβ42 in synaptosomes derived from frontal cortices of post-mortem AD and age-matched non-demented (ND) control individuals (Figure 10B). Towards this, we prepared synaptosomes from frozen brain tissues using the Percoll gradient procedure (Dunkley et al., 2008; Fonseca-Ornelas et al., 2021). Intact synaptosomes were spun to obtain a pellet which was resuspended in the minimum amount of PBS, allowing us to estimate the volume containing the resuspended synaptosome sample. This is likely an overestimate of the actual synaptosome volume. Finally, synaptosomes were lysed in RIPA buffer and Aβ peptide concentrations were measured using ELISA (MSD). We observed that the concentration of Aβ42 in the synaptosomes from (end-stage) AD tissues was significantly higher (10.7 nM) than those isolated from non-demented tissues (0.7 nM), ***p<0.0005. These data provide evidence for accumulation at nM concentrations of endogenous Aβ42 in synaptosomes in end-stage AD brains. Given that we measured Aβ42 concentration in synaptosomes, we speculate that even higher concentrations of this peptide may be present in the endolysosome vesicle system, and therein inhibit the endogenous processing of APP-CTF at the synapse. Of note treatment of PC12 cells with conditioned medium containing even lower amounts of Aβ (low nanomolar range (0.5–1 nM)) resulted in the accumulation of APP-CTFs.

Aβ peptides were purchased from rPeptide, resuspended in DMSO at 500 μM, aliquoted into single use 10 μl aliquots and stored at –80 °C. For Aβ42 the following lots were used: 4261242T, 06021342T and 02092242T. Γ-secretase inhibitors (Inhibitor X (InhX, L-685,458), DAPT and compound E (CE)) were purchased from Bioconnect, Sigma-Aldrich and Millipore, respectively. TrkA inhibitor K252α, cycloheximide and Bafilomycin A1 were purchased from Sigma Aldrich. The following antibodies were used: mouse anti-FLAG M2 (Sigma-Aldrich, F3165), rabbit anti-ADAM10 antibody (EPR5622, Abcam, ab124695), rabbit anti-APP (gift from Prof. Wim Annaert (B63)), rabbit anti-APP (Y188, Abcam, ab32136), mouse anti-APP (22C11, Thermo Fisher Scientific, 14-9749-82), rabbit anti-BACE1 (EPR19523, Abcam, ab183612), rabbit anti-pan-cadherin (Thermo Fisher Scientific, 71–7100), anti-p75 NTR (Millipore, 07–476), anti-Aβ (clone 4G8, Biolegend), anti-choline acetyltransferase (Millipore, AB144P), anti-cleaved caspase-3 (Cell Signaling, 9661 S), HRP-conjugated goat anti-rabbit (BioRad), Alexa Fluor 790-conjugated goat anti-mouse (Thermo Fisher Scientific), Cy3-conjugated donkey anti-rabbit (Jackson ImmunoResearch Laboratories), Alexa Fluor Plus 488-conjugated donkey anti-mouse (Thermo Fisher Scientific), biotinylated rabbit anti-goat (Jackson ImmunoResearch Laboratories) and Cy2-conjugated streptavidin (Jackson Immunoresearch Laboratories).

Preparation of the AAV-hSyn1-C99 Y-T biosensor was performed as described previously (Maesako et al., 2020). Briefly, the cDNA of the C99 Y-T probe was subcloned into a pAAV2/8 vector containing human synapsin 1 promoter and WPRE sequences (Maesako et al., 2017). The packaging of viruses was performed at the University of Pennsylvania Gene Therapy Program vector core (Philadelphia, PA). The virus titer was 4.95E+13 GC/ml.

High Five insect cells were transduced with baculoviruses carrying cDNA encoding all γ-secretase subunits (wild-type PSEN1 or PSEN2, NCSTN, APH1A or APH1B, PEN2) and 72 h later the cells were collected for protein purification, as described before (Szaruga et al., 2017).

Recombinant γ-secretase substrates were expressed and purified using a mammalian cell expression system or baculovirus-mediated expression system in High Five insect cells as before (Szaruga et al., 2017; Franco et al., 2021). COS cells were transfected with pSG5 plasmid encoding NOTCH1- or P75-based γ-secretase substrate, tagged at the C-terminus with 3xFLAG. High Five insect cells were transduced with baculoviruses carrying cDNA encoding APP_C99, ERBB4ΔECT, and neurexinΔECT-tagged with 3xFLAG-prescission protease (PPS) cleavage site-GFP tandem at the C-terminus. The purity of the protein was analyzed by SDS-PAGE and Coomassie staining (InstantBlue Protein Stain, Expedeon).

CHAPSO detergent-resistant membranes (DRMs) were prepared from High Five insect cells overexpressing wild-type γ-secretase complexes containing NCSTN, APH1B, PSEN1 and PEN2 subunits, as reported before (Szaruga et al., 2017).

Purified γ-secretases (~25 nM) were mixed with respective, purified 3xFLAG-tagged γ-secretase substrates (0.4 or 1.2 μM) in 150 mM NaCl, 25 mM PIPES, 0.25% CHAPSO, 0.03% DDM, 0.1% phosphatidylcholine buffer and the reactions were incubated for 40 min at 37°C. Non-incubated reactions or reactions supplemented with 10 μM InhX served as negative controls. In the experiments testing for the reversibility of the inhibition, γ-secretases were first immobilized on sepharose beads covalently coupled to anti-γ-secretase nanobody. Following the first reaction containing Aβ or GSI, the beads were washed 3 x for a total duration of 30 min with 150 mM NaCl, 25 mM PIPES, 0.25% CHAPSO buffer, and a fresh substrate was added. For γ-secretase activity assays in membrane-like conditions (25 μl total reaction volume), 6.25 μl DRMs, re-suspended in 20 mM PIPES, 250 mM sucrose, 1 M EGTA, pH7 at the concentration of 1 μg/μl, were mixed with 6.25 μl APP_C99-3xFLAG or Aβ1–42 substrate in 150 mM NaCl, 25 mM PIPES, 0.03% DDM, 2.5 μl 10xMBS, and H₂O supplemented with DDM and CHAPSO, to achieve final concentration of the detergents 0.03% and 0.1%, respectively, and final concentration of the substrates 1.5 μM and 10 μM, respectively. The reaction mixes were incubated for 40 min at 37°C. Reactions supplemented with 10 μM InhX served as negative controls.

De novo ICD generation was determined by western blotting. Briefly, the reaction mixtures were subjected to methanol:chloroform (1:2 v/v) extraction to remove the excess of unprocessed substrates, and the upper fractions (containing mainly the generated ICDs) were subjected to SDS-PAGE. Otherwise, the high levels of substrate could preclude quantitative analysis of ICDs (Figure 1—figure supplement 1A).

ICD-3xFLAG was detected by western blotting with anti-FLAG, followed by anti-mouse Alexa Fluor 790 conjugated antibodies. Fluorescent signals were developed using Typhoon (GE Healthcare). The intensity of the bands was quantified by densitometry using ImageQuant software.

Alternatively, MALDI-TOF MS was applied to determine the relative de novo ICD-3xFLAG levels generated in cell-free conditions, as reported before (Szaruga et al., 2017). The samples were spiked with Aβ1–28 as an internal standard. The mass spectra were acquired on a RapiFleX TOF mass spectrometer (Bruker Daltonics) equipped with a 10 kHz Smartbeam laser using the AutoXecute function of the FlexControl 4.2 acquisition software.

HEK293 stably expressing APP-CTF (C99) or a NOTCH1-based substrate (similar in size as APP- C99) both N-terminally tagged with the HiBiT tag were plated at the density of 10,000 cells per 96-well, and 24 h after plating treated with Aβ or p3 peptides diluted in OPTIMEM (Thermo Fisher Scientific) supplemented with 5% FBS (Gibco). Conditioned media was collected and subjected to analysis using Nano-Glo HiBiT Extracellular Detection System (Promega). Briefly, 50 µl of the medium was mixed with 50 µl of the reaction mixture containing LgBiT Protein (1:100) and Nano-Glo HiBiT Extracellular Substrate (1:50) in Nano-Glo HiBiT Extracellular Buffer, and the reaction was incubated for 10 min at room temperature. Luminescence signal corresponding to the amount of the extracellular HiBiT-Aβ or -Aβ-like peptides was measured using a victor plate reader with default luminescence measurement settings.

SH-SY5Y, PC12 and PC12nnr5 (Loeb et al., 1991) cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F-12 (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Gibco) at 37°C, 5% CO₂. ReNCell VM cells were cultured in Corning Matrigel hESC-Qualified Matrix, LDEV-free-coated flasks in DMEM/F-12 medium supplemented with B27 (Thermo Fisher Scientific), 2 μg/ml heparin (Stem Cell Technologies), 20 ng/ml EGF-1 (Cell Guidance) and 25 ng/ml FGF (Cell Guidance). Human iPSC line was derived from a cognitively unaffected male individual, previously established and characterized (CVB) (RRID: CVCL_1N86, GM25430) (Gore et al., 2011). The two neuronal progenitor cell (NPC) lines – CV4a (derived from CVB iPSC line that carries a wild-type APP, herein termed as WT) and APPSwe (homozygous for APP Swedish mutations that were genome-edited from CVB parental iPSC line) were characterized and reported (Young et al., 2015; Fong et al., 2018). NPCs were plated on 20 μg/ml poly-L-ornithine (Sigma Aldrich) and 5 μg/ml laminin (Thermo Fisher Scientific)-coated plates in NPC media: DMEM/F12/Glutamax supplemented with N2, B27, penicillin, and streptomycin (Thermo Fisher Scientific) and 20 ng/ml FGF-2 (Millipore). For neuronal differentiation, confluent NPCs were cultured in NPC media without FGF-2 for three weeks. All cells were maintained in a humidified incubator at 37 °C, 5% CO₂, and regularly tested for mycoplasma infection.

For the cellular assays, cells were plated into 6-well plates at the density of 300,000 cells per well and 24 h after plating treated with Aβ or p3 peptides diluted in OPTIMEM (Thermo Fisher Scientific) supplemented with 5% FBS (Gibco). In the experiments set to analyze sAPP in the conditioned medium, FBS was replaced with 1% knock-out serum replacement (KOSR) (Thermo Fisher Scientific) due to the interference of the antibodies present in the serum with western blotting. After 8 h, the medium was refreshed, using peptide-supplemented media, and the cultures were incubated at 37°C for 16 hr. Cell lysates were prepared in radioimmunoprecipitation (RIPA) buffer (25 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.4) supplemented with protease inhibitors. Equal amounts of protein or conditioned medium were subjected to SDS-PAGE on 4–12% Bis-Tris gels in MES or MOPS buffer or 4–16% Tris-Tricine gels (Das et al., 2013) and western blot analysis. Chemiluminescent signals were developed using Fujifilm LAS-3000 Imager or ChemiDoc XRS + imaging apparatus (BioRad). The optical density of the bands was quantified using ImageQuant or Image J software.

Cytotoxicity was assessed using LDH-Glo Cytotoxicity Assay (Promega), which is a bioluminescent plate-based assay for quantifying LDH release into the culture medium upon plasma membrane damage. The assay was performed following the manufacturer’s recommendation. Briefly, the culture medium was collected upon respective treatment and diluted 1 in 100 in LDH storage buffer (200 mM Tris-HCl (pH 7.3), 10% glycerol, 1% BSA). Medium from cells treated for 15 min with 2% Triton X-100 was used as a maximum LDH release control. 12.5 µl of the diluted medium was mixed with 12.5 µl LDH detection enzyme mix supplemented with reductase substrate in a 384-well plate. The reactions were incubated for 1 h and luminescence read using Promega GloMax Discoverer.

Cytotoxicity was also assessed using CellTiter-Glo 2.0 Assay (Promega), which is a homogeneous method used to determine the number of viable cells based on the quantitation of the ATP, an indicator of metabolically active cells. Briefly, cells were plated in a 96-well plate in 100 µl medium, treated following the treatment regime, mixed with CellTiter-Glo reagent, and luminescence was read using Promega GloMax Discoverer.

The conditioned media from the WT (WT-CM) and APPSwe (SWE-CM) human neurons were used as a biological source of Aβ. Following three weeks of differentiation, neurons cultured on 10 cm diameter dishes were incubated in NPC media without FGF-2 for five days. After five days, conditioned media were collected in a sterile atmosphere. Immunodepletion of Aβ from APPSwe medium was done following a modified protocol (Shankar et al., 2011). Briefly, 5 ml of SWE-CM were incubated with 3D6 antibody (1 µg/ml), 15 µl protein A, and 15 µl protein G resin for 3 hr at 4°C with gentle shaking on a nutator (Johnson-Wood et al., 1997). The supernatant was collected after centrifuging at 3500×g and used as immunodepleted media (delSWE-CM) for cell culture experiments. To test for the APP-CTF accumulation in the presence of different conditioned media, PC12 cells, cultured on six-well plates, were treated with WT-CM, SWE-CM, or delSWE-CM for 24 h along with regular feeding media (control). Following incubation, cells were harvested and processed to detect APP-FL and APP-CTF.

Primary neuronal cultures were obtained from the cerebral cortex of mouse embryos at gestation days 14–16 (Charles River Laboratories). The neurons were dissociated using the Papain Dissociation System (Worthington Biochemical Corporation, Lakewood, NJ) and maintained for 13–15 days in vitro (DIV) in a neurobasal medium containing 2% B27, 1% GlutaMAX Supplement and 1% penicillin/streptomycin (Thermo Fisher Scientific). A laser at 405 nm wavelength was used to excite TurquoiseGL in the C99 Y-T biosensor (Maesako et al., 2020). The emitted fluorescence from the donors (TurquoiseGL) and the acceptors (YPet) was detected at 470±10 nm and 530±10 nm, respectively, by the TruSpectral detectors on an Olympus FV3000RS confocal microscope equipped with CO₂/heating units. 10/0.25x objective was used for the imaging. The Average pixel fluorescence intensity for the cell body after subtraction of the background fluorescence was measured using Image J. The emission intensity of YPet over that of the TurquoiseGL (Y/T) ratio was used as a readout of the FRET efficiency, which reflects the relative proximity between the donor and the acceptor. Pseudo-colored images were generated in MATLAB (MathWorks, Natick, MA). The neuronal preparation procedure followed the NIH guidelines for the use of animals in experiments and was approved by the Massachusetts General Hospital Animal Care and Use Committee (2003N000243).

Animals of CD1 genetic background were housed in an animal care facility with a 12 h dark/light cycle and had free access to food and water. All experiments were performed according to the animal care guidelines of the European Community Council (86 ⁄ 609 ⁄ EEC) and to Spanish regulations (RD1201 ⁄ 2005), following protocols approved by the ethics committees of the Consejo Superior Investigaciones Científicas (CSIC). Septal areas were dissected from CD1 E17-18 embryos in chilled Hanks’ balanced salt solution (HBSS, 137 mM NaCl, 5.4 mM KCl, 0.17 mM Na₂HPO₄, 0.22 mM KH₂PO₄, 9.9 mM HEPES, 8.3 mM glucose, and 11 mM sucrose). The septa were pooled and digested with 1 ml of 0.25% trypsin (GE Healthcare) and 0.5 ml of 100 kU DNase I (GE healthcare) for 10 min at 37°C. The tissue was further dissociated by aspiration with progressively narrower tips in neurobasal medium (Thermo Fisher Scientific) supplemented with 4% bovine serum albumin (BSA), 2% B27 (Thermo Fisher Scientific), 1% L-glutamine (Thermo Fisher Scientific) and 0.5% penicillin/streptomycin (GE Healthcare) (NB/B27). After tissue dissociation, an equal volume of 4% BSA was added and samples were centrifuged at 300×g for 5 min at 4 °C. The supernatant was discarded and the cell pellet was resuspended in 5 ml of 0.2% BSA. The suspension was filtered through a 40 µm nylon filter (Sysmex) and cells were counted in the Neubauer chamber, centrifuged again and resuspended in NB/B27. Cells were seeded in 24-well plates (2×10⁵ cells/well) containing 12 mm diameter pre-coated coverslips (VWR) for immunocytochemical analysis or in pre-coated p-100 plates for western blotting. The surfaces were coated with 50 µg/ml poly-D-lysine (Sigma-Aldrich) overnight at 4°C and 5 µg/ml laminin (Sigma-Aldrich) for 2 h at 37 °C. The next day, half of the medium was exchanged and the concentration of B27 was reduced to 0.2%. The medium was supplemented with 2 µM of the antimitotic agent (1-β-D-Arabinofuranosylcytosine (Sigma Aldrich)) and 100 ng/ml NGF (Alomone labs). Neurons were cultured until DIV11. The respective treatments were applied at DIV8 and the culture continued for 72 h until DIV11.

For immunocytochemistry (ICC) cells were fixed in 2% paraformaldehyde and permeabilized with 0.1% Triton X-100. After that, they were incubated with 0.5% SDS for 5 min for antigen retrieval. Non-specific binding of antibodies was blocked using 2% BSA, 0.1% Triton X-100 in 0.1 M phosphate buffer (PB). The cells were incubated overnight with anti-choline acetyltransferase (Millipore) (only for BFCN) and anti-cleaved caspase-3 (Cell Signaling) antibodies at 4°C. The next day the samples were incubated with corresponding secondary antibodies: Cy3-conjugated donkey anti-rabbit, biotinylated rabbit anti-goat and Cy2-conjugated streptavidin (all purchased from Jackson Immunoresearch Laboratories). Nuclei were stained with DAPI and samples were mounted on glass slides. For quantitative analysis, for BFCNs total number of choline acetyltransferase positive neurons and the number of double positive neurons for choline acetyltransferase and cleaved caspase-3 was determined, while for PC12 a fraction of cleaved caspase 3 positive cells among all cells (determined by DAPI staining) was determined.

PC12 cells were plated on glass coverslips and treated for 24 h with respective Aβ peptides at 1 μM. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in TBST (0.2% Tween 20 in TBS). Blocking was performed by 1 h incubation in a solution containing 1% bovine serum albumin, 0.1% gelatin, 300 mM glycine, and 4% normal donkey serum in TBST. Primary antibodies were diluted in the blocking solution and incubated overnight. This was followed by incubation with respective Alexa Fluor Plus 488 conjugated secondary antibody and Alexa Fluor Plus 555 conjugated phalloidin. The samples were then incubated with nuclear stain DAPI for 5 min and mounted using Prolong glass. Images were acquired at 63x magnification using a Zeiss LSM 900 confocal microscope at VIB Bioimaging Core.

The post-mortem human brain tissues from the frontal cortex were obtained from the University of California San Diego–Alzheimer’s Disease Research Center (UCSD-ADRC; La Jolla, CA, USA), and the University of California Irvine–Alzheimer’s Disease Research Center (UCI-ADRC) brain tissue repository (Irvine, CA, USA; Supplementary file 1b), and stored at -80 °C prior to use. Synaptosomes from the cortices were isolated following published protocol using a discontinuous Percoll gradient (Sigma-Aldrich, USA) (Dunkley et al., 2008; Fonseca-Ornelas et al., 2021) with some modifications. All the steps were performed on ice or at 4°C unless mentioned otherwise. Briefly, 1200 mg of tissue was homogenized using a handhold Dounce homogenizer in five volumes of homogenization buffer (HB: 0.32 M sucrose, 5 mM magnesium chloride, 5 mM Tris-Cl, pH 7.4 and EDTA-free protease inhibitor cocktail). After centrifuging at 1000xg for 10 min, post-nuclear supernatant (PNS) was collected. The PNS was further centrifuged at 10800×g for 10 min to isolate the pellet fractions (P2, crude synaptosome). The P2 fraction was resuspended in HB and loaded on top of a three-step (3%, 10%, and 23%) Percoll gradient, and centrifuged at 14,500×g for 12 min at 4°C in a Fiberlite F21-8×50 y Fixed-Angle Rotor (Thermo Fisher, USA). The synaptosome-rich interface between 10% and 23% Percoll layers was collected and resuspended in 30 volumes of HB. The diluted material was centrifuged at 18,500×g for 30 min at 4°C, and the synaptosome-enriched pellet was resuspended in 500 μl of HB. To measure the concentration of Aβ42, 25 μl of synaptosome suspension was centrifuged at 18500xg for 10 min at 4°C. Supernatant was discarded and the volume of the synaptosome pellet was estimated by adding a minimum volume of liquid (2 μl of PBS) to the synaptosome pellet. Synaptosomes were dissolved in 23 μl RIPA buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). After centrifugation for 15 min at 13,800×g at 4°C, the supernatant was collected for MSD-ELISA (Meso Scale Diagnostics, USA). Concentrations of Aβ were measured using the VPLEXAβ Peptide Panel (4G8: MesoScaleDiscovery) following the manufacturer’s Instructions. Data were obtained using the MESO QuickPlex SQ 120 and analyzed using DISCOVERY WORKBENCH 3.0 (Meso Scale Diagnostics). The pg concentration of Aβ in 25 μL was converted to pg/mL and eventually to nM values using the volume of synaptosomes.

The cortical tissues from two-months old wild-type mice (n=3) were collected and stored at -80 °C. Synaptosomes from the 60–70 mg of cortices were isolated following the protocol described above. The synaptosome-rich interface between 10% and 23% Percoll layers were collected and resuspended in 30 volumes of HB. The diluted material was centrifuged at 18500×g for 30 min at 4°C, and the synaptosome-enriched pellet was resuspended in HB supplemented with 10 mM glucose. 10–15 μg of synaptosome was incubated with Aβ42 peptide at 2.5 μM final concentration at 37 °C for 18 hr. DMSO was used as a vehicle control. One synaptosomal sample was treated with 200 nM of Compound E. Following incubation, samples were resolved on SDS-PAGE, and western blotting was performed using anti-APP Y188 and anti-GAPDH antibodies. All densitometric analyses were performed using NIH ImageJ software. The animal experiments were approved by the Institutional Animal Care and Use Committee of the University of California San Diego.

Statistical analysis was performed using Excel, GraphPad Prism, R 4.2.2. and R Studio software. The following R packages were used for the analysis: readxl, ggplot2, plyr, dplyr, DescTools, gridExtra and reshape2 (Wickham, 2016; Wickham, 2007; Wickham, 2011). p<0.05 was considered as a predetermined threshold for statistical significance. One-way or two-way ANOVA, or Kruskal-Wallis test followed by Dunnett’s, Tukey’s, or Dunn multiple comparison test or unpaired Student’s t-test were used, as described in the legends.