Alzheimer-mutant γ-secretase complexes stall amyloid β-peptide production

Reviewer #1 (Public review):

Summary:

Arafi et al. present results of studies designed to better understand the effects of mutations in the presenilin-1 (PSEN1) gene on proteolytic processing of the amyloid precursor protein (APP). This is important because APP processing can result in the production of the amyloid β-protein (Aβ), a key pathologic protein in Alzheimer's disease (AD). Aβ exists in various forms that differ in amino acid sequence and assembly state. The predominant forms of Aβ are Aβ40 and Aβ42, which are 40 and 42 amino acids in length, respectively. Shorter and longer forms derive from processive proteolysis of the Aβ region of APP by the heterotetramer β-secretase, within which presenilin 1 possesses the active site of the enzyme. Each form may become toxic if it assembles into non-natively folded, oligomeric, or fibrillar structures. A deep mechanistic understanding of enzyme-substrate interactions is a first step toward the design and successful use of small-molecule therapeutics for AD.

The key finding of Arafi et al. is that three PSEN mutations display unusual profiles of effects on Aβ production that have novel implications for the stalled E-S complex hypothesis. PSEN1 F386S is unique in that initial ε cleavage is not reduced compared with WT PSEN1; only certain trimming steps are deficient, results consistent with FLIM experiments that reveal stabilized E-S complexes only in Aβ-rich regions in the cell. In contrast, PSEN1 A431E and A434T display very little ε cleavage and therefore very little overall Aβ production, suggesting a limited role of Aβ in the pathogenesis of these two mutants and pointing to stalled E-S complexes as the common factor. For the biochemist, this may not be surprising, but in the context of understanding and treating AD, it is immense because it shifts the paradigm from targeting the results of γ-secretase action, viz., Aβ oligomers and fibrils, to targeting initial Aβ production at the molecular level. It is the equivalent of taking cancer treatment from simple removal of tumorous tissue to prevention of tumor formation and growth. Arafi et al. have provided us with a blueprint for the design of small-molecule inhibitors of γ-secretase. The significance of this achievement cannot be overstated.

Strengths and weaknesses:

The comprehensiveness and rigor of the study are notable. Rarely have I reviewed a manuscript reporting the results of so many orthogonal experiments, all of which support the authors' hypotheses, and of so many excellent controls. In addition, as found in clinical trial reports, the limitations of the study were discussed explicitly. None of these significantly affected the conclusions of the study.

Reviewer #2 (Public review):

Summary:

The work by Arafi et al. shows the effect of Familial Alzheimer's Disease presenilin-1 mutants on endoproteinase and carboxylase activity. They have elegantly demonstrated how some mutants alter each step of processing. Together with FLIM experiments, this study provides additional evidence to support their 'stalled complex hypotheses'.

Strengths:

This is a beautiful biochemical work. The approach is comprehensive.

Weaknesses:

(1) It appears that the purified g-secretase complex generates the same amount of Ab40 and Ab42, which is quite different in cellular and biochemical studies. Is there any explanation for this?

(2) It has been reported the Ab production lines from Ab49 and Ab48 can be crossed with various combinations (PMID: 23291095 and PMID: 38843321). How does the production line crossing impact the interpretation of this work?

(3) In Figure 5, did the authors look at the protein levels of PS1 mutations and C99-720, as well as secreted Ab species? Do the different amounts of PS1 full-length and PS1-NTF/CTF influence FILM results?

(4) It is interesting that both Ab40 and Ab42 Elisa kits detect Ab43. Have the authors tested other kits in the market? It might change the interpretation of some published work.

Author response:

Reviewer 2:

(1) It appears that the purified γ-secretase complex generates the same amount of Aβ40 and Aβ42, which is quite different in cellular and biochemical studies. Is there any explanation for this?

Roughly equal production of Aβ40 and Aβ42 is a phenomenon seen with purified enzyme assays, and the reason for this has not been identified. However, we suggest that what is meaningful in our studies is the relative difference between the effects of FAD-mutant vs. WT PSEN1 on each proteolytic processing step. All FAD mutations are deficient in multiple cleavage steps in γ-secretase processing of APP substrate, and these deficiencies correlate with stabilization of E-S complexes.

(2) It has been reported the Aβ production lines from Aβ49 and Aβ48 can be crossed with various combinations (PMID: 23291095 and PMID: 38843321). How does the production line crossing impact the interpretation of this work?

In the cited reports, such crossover was observed when using synthetic Aβ intermediates as substrate. In PMID 2391095 (Okochi M et al, Cell Rep, 2013), Aβ43 is primarily converted to Aβ40, but also to some extent to Aβ38. In PMID: 38843321 (Guo X et al, Science, 2024), Aβ48 is ultimately converted to Aβ42, but also to a minor degree to Aβ40. We have likewise reported such product line “crossover” with synthetic Aβ intermediates (PMID: 25239621; Fernandez MA et al, JBC, 2014). However, when using APP C99-based substrate, we did not detect any noncanonical tri- and tetrapeptide co-products of Aβ trimming events in the LC-MS/MS analyses (PMID: 33450230; Devkota S et al, JBC, 2021). In the original report on identification of the small peptide coproducts for C99 processing by γ-secretase using LC-MS/MS (PMID: 19828817; Takami M et al, J Neurosci, 2009), only very low levels of noncanonical peptides were observed. In the present study, we did not search for such noncanonical trimming coproducts, so we cannot rule out some degree of product line crossover.

(3) In Figure 5, did the authors look at the protein levels of PS1 mutations and C99-720, as well as secreted Aβ species? Do the different amounts of PS1 full-length and PS1-NTF/CTF influence FILM results?

This is a good question. Our preliminary investigation by Western Blot shows no correlation between C99 and PSEN1 expressions and FLIM results, but we will fully address the concern in our point-by-point responses submitted with a revised manuscript.

(4) It is interesting that both Aβ40 and Aβ42 Elisa kits detect Aβ43. Have the authors tested other kits in the market? It might change the interpretation of some published work.

We have not tested other ELISA kits. In light of our findings, it would be a good idea for other investigators to test whatever ELISAs they use for specificity vis-à-vis Aβ43.