Author Response
We are grateful to the editors and the reviewers for the thorough evaluation of our manuscript and their feedback, as it allows us to provide additional clarification of our findings and improve the manuscript.
In their evaluation reviewers raised a key conceptual point linked to the inhibitory mechanism that appeared to be insufficiently explained in the manuscript, leading to a misconception regarding the physiological relevance. They have also missed experimental data related to the concentrations of Aβ used and their relevance for Alzheimer’s disease (AD). We believe that our studies, although performed in vitro in model systems, provide novel conceptual framework and shed light on the unexplored mechanisms underlying AD.
We discuss these points below in a provisional response to their comments.
Reviewer #1 (Public Review):
Summary:
Human Abeta42 inhibits gamma-secretase activity in biochemical assays.
Strengths:
Determination of inhibitory concentration human Abeta42 on gamma-secretase activity in biochemical assays.
Weaknesses:
Human Abeta42 may concentrate up to microM order in endosomes.
This is correct.
If so, production of Abeta42 would be attenuated then lead to less Abeta deposition in the brain. The authors finding is interesting but does not fit the physiological condition in the brain.
We thank the reviewer for raising this key conceptual point, as this gives us the opportunity to clarify it for the future readers.
The characterized inhibitory mechanism is more complex than the reviewer’s interpretation, and a number of factors must be considered. Indeed, our data show that Aβ42 upon intracellular concentration inhibits γ-secretase activity, resulting in increased γ-secretase substrate (C-terminal fragment, CTF) levels. It is important however to highlight that this inhibition is competitive in nature, implying that it is partial, reversible, and regulated by the relative concentrations of the Aβ42 peptide (inhibitor) and the substrates. The model that we put forward is that cellular uptake and intracellular concentration of Aβ42 facilitates γ-secretase inhibition, which results in the accumulation of APP-CTFs (and γ-secretase substrates in general). However, as Aβ42 levels fall, the increased concentration of substrates shifts the equilibrium towards their processing and Aβ production. As Aβ42 concentration raises again, equilibrium is shifted back towards inhibition and so on. This inhibitory mechanism will translate into pulses of (partial) γ-secretase inhibition, which will alter γ-secretase mediated signalling (arising from increased CTF levels or decreased release of soluble intracellular domains from substrates). These alterations may affect the dynamics of systems oscillating in the brain, such as NOTCH signalling, implicated in memory formation (2), and potentially others (related to e.g. cadherins, p75 or neuregulins).
It is worth noting that oscillations in γ-secretase activity induced by treatment with a γ-secretase inhibitor (semagacestat) have been proposed to have contributed to the cognitive alterations observed in semagacestat treated patients in the failed Phase-3 IDENTITY clinical trial (2, 3); and that semagacestat, like Aβ42, acts as a high affinity competitor of substrates (Koch et al, 2023).
We will include this clarification in the discussion of the revised manuscript and create an additional figure presenting the proposed mechanism.
It is not clear whether the FRET-based assay in living cells really reflect gamma-secretase activity.
The specificity of this assay is supported by the γ-secretase inhibitor treatment included as a positive control (Figure 3). In addition, the following literature supports that this assay truthfully assesses γ-secretase activity in cellular context (4-7).
Processing of APP-CTF in living cells is not only the cleavage by gamma-secretase.
This is correct, and therefore we have analysed the contribution of other APP-CTF degradation pathways by performing cycloheximide-based stability assay in the presence of γ-secretase inhibitor. Quantitative analysis of the levels of both APP-CTFs and APP-FL over the 5h time-course failed to reveal significant differences between Aβ42 treated cells and controls. As expected, Bafilomycin A1 treatment markedly prolonged the half-life of both proteins (Figure 7B & C). The lack of a significant impact of Aβ42 on the half-life of APP-CTFs under the conditions of γ-secretase inhibition is consistent with the proposed inhibitory mechanism. Finally, we note that the inhibition will not only affect APP-CTF, but also the processing of γ-secretase substrates in general.
Reviewer #2 (Public Review):
Summary:
In the current study, the authors tested the hypothesis that Aβ42 toxicity arises from its proven affinity for γ-secretases. Specifically, the 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. They showed 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 (CTFs of APP, p75 and pan-cadherin. Moreover, Aβ42 dysregulated cellular homeostasis by inducing p75-dependent neuronal death. Because γ-secretases process many other membrane proteins, including NOTCH, ERB-B2
receptor tyrosine kinase 4 (ERBB4), N-cadherin (NCAD) and p75 neurotrophin receptor (p75-NTR), revealing a broad range of downstream signaling pathways, including those critical for neuronal structure and function. Hence, they propose to identification of a selective role for the Aβ42 peptide, and raise the intriguing possibility that compromised γ-secretase activity against the CTFs of APP and/or other neuronal substrates contributes to the pathogenesis of AD. Overall, the data are not very convincing to support the main claim.
Strengths.
Different in vitro and cellular approaches are employed to test the hypothesis.
Weaknesses.
The experimental concentrations for Aβ42 peptide in the assay are too high, which are far beyond the physiological concentrations or pathological levels. The artificial observations are not supported by any in vivo experimental evidence.
It is correct that in the majority of the experiments we used low μM concentrations of Aβ42. However, we would like to note that we also performed experiments where conditioned medium collected from human APP.Swe expressing neurons was used as a source of Aβ. In these experiments total Aβ concentration was in low nM range (0.5-1 nM) (Figure 4G). Treatment with this conditioned medium led to the increase APP-CTF levels, supporting that low nM concentrations of Aβ are sufficient for partial inhibition of γ-secretase.
We would like to underline that Aβ is estimated to be present in the brain in concentration ranging from fM to mM, depending on the pool (soluble, aggregated, fibrillar, etc) that is considered (8, 9). However, it is rather the local than the global concentration of Aβ that is critical for the disease pathogenesis. In this regard, it is proposed that as AD progresses Aβ42 slowly accumulates in the endo-lysosomal system wherein it reaches μM concentrations that are required for aggregation and seeding (1, 10, 11).
Our findings are consistent with the analysis showing that extracellular soluble Aβ42 peptide, at low nM concentrations, is taken up by cortical neurons and neuroblastoma (SH-SY5Y) cells, and concentrated in the endo-lysosomal system wherein effective peptide concentrations reach ~2.5 μM (1). Hence, a slow vesicular peptide accumulation and/or degradation imbalance (1, 11, 12) could lead to several order of magnitude increases in the effective concentration of Aβ42 over the span of years to decades in AD pathogenesis. We note that our experimental settings, using low μM concentrations of extracellular Aβ42 over 24h treatment, were designed to accelerate this 'peptide concentration’ process in vitro. As discussed in our report, a high μM Aβ peptide concentration in the endo-lysosomal system not only leads to aggregation but also facilitates γ-secretase inhibition. Of note, we are currently developing protocols and will undertake follow up studies to quantitatively define the Aβ concentration in synaptosomes and endosomes in AD brain, as well as in in vitro systems (i.e. cells treated with Aβ preparations obtained from AD brains).
Finally, we would like to highlight that analyses of the brains of the AD affected individuals have shown that APP-CTFs accumulate in both sporadic and genetic forms of the disease (13-15); and recently, Ferrer-Raventós et al have revealed a correlation between APP-CTFs and Aβ levels at the synapse (13).
To conclude, we would like to highlight that as clarified above, the Aβ peptide concentrations and the conditions tested fit well within pathophysiology, and that the data presented in our report collectively provide evidence in support of an Aβ42-mediated inhibitory effect on γ-secretase.
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