. Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes. 2023 Sep 09 10.1101/2023.09.08.556905 (version 1) bioRxiv.

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  1. This work describes a well-designed series of studies testing the hypothesis that the pathological accumulation of Aβ1-42 competes with other substrates for γ-secretase processing, resulting in the accumulation of unprocessed γ-secretase substrates, including their intracellular C-terminal domain fragments (CTFs) and reduced levels of further processed, soluble intracellular domains (ICDs). Given the intracellular signaling roles of CTFs and ICDs, this accumulation could lead to abnormal and potentially deleterious changes in downstream signaling. Some γ-secretase substrates of note include the amyloid precursor protein (APP), the p75 neurotrophin receptor (p75NTR), and pan-cadherins. Aβ1-42-induced accumulation p75NTR CTF is of interest given its capacity to promote degenerative signaling, particularly in the absence of the p75NTR co-receptor, TrkA.

    These observations, suggesting that one mechanism by which Aβ42 promotes neuronal degeneration is through p75NTR mechanisms, are consistent with studies demonstrating that Aβ-induced neuronal degeneration is dependent on the presence of p75NTR (Sotthibundhu et al, 2008; Knowles et al., 2009; Wilcock et al., 2015; Andrade-Talavera et al., 2021; Wei et al., 2023).

    In the PC12nnr5 cell line (absent TrkA receptor), Aβ1-42- induced accumulation of CTF was associated with an increase in cleaved (activated) caspase 3, a signaling element known to trigger apoptosis of neurons and other cell types. I also note the activation of caspase-3 through p75NTR mechanisms, given the role of caspase-3 in enabling the “eat me” signaling thought to promote microglial mediated pruning of synapses (Nonaka and Nakanishi, 2019).

    References:

    . Beta-amyloid(1-42) induces neuronal death through the p75 neurotrophin receptor. J Neurosci. 2008 Apr 9;28(15):3941-6. PubMed.

    . The p75 neurotrophin receptor promotes amyloid-beta(1-42)-induced neuritic dystrophy in vitro and in vivo. J Neurosci. 2009 Aug 26;29(34):10627-37. PubMed.

    . Down syndrome individuals with Alzheimer's disease have a distinct neuroinflammatory phenotype compared to sporadic Alzheimer's disease. Neurobiol Aging. 2015 Sep;36(9):2468-74. Epub 2015 May 30 PubMed.

    . Ablation of p75NTR signaling strengthens gamma-theta rhythm interaction and counteracts Aβ-induced degradation of neuronal dynamics in mouse hippocampus in vitro. Transl Psychiatry. 2021 Apr 9;11(1):212. PubMed.

    . p75NTR enhances cognitive dysfunction in a mouse Alzheimer's disease model by inhibiting microRNA-210-3p-mediated PCYT2 through activation of NF-κB. Int J Biol Macromol. 2023 Jan 15;225:404-415. Epub 2022 Nov 12 PubMed.

    . Microglial clearance of focal apoptotic synapses. Neurosci Lett. 2019 Aug 10;707:134317. Epub 2019 Jun 5 PubMed.

    View all comments by Frank Longo
  2. Zoltowska et al. propose that Aβ42 inhibition of γ-secretase is the principal basis for lysosome dysfunction and the rise of APP-βCTF levels seen in AD brain. We welcome their appreciation of APP-βCTF’s pathogenic potential in AD and the new investigations on its roles. While we question the physiological relevance of the report’s in vitro evidence, which is based on endocytosis of a high (3 uM) human Aβ42 concentration, we view contributions to APP-βCTF build-up in neurons by any means to be compatible with our findings that the early accumulation of APP-βCTF in neuronal autolysosomes, starting well before extracellular amyloid deposition, is the primary cause of lysosomal dysfunction that drives AD pathogenesis (Lee et al., 2022). 

    This APP-βCTF accumulation in lysosomes disrupts their acidification in varied AD models (Jiang et al., 2019) via a specific and direct binding of APP-βCTF within the vATPase complex, which disrupts its assembly and impedes its proton pumping activity (Im et al., 2023). The resulting lysosomal pH rise in vivo broadly impairs proteolysis and causes massive accumulation of varied substrates, including partially inactivated cathepsins and many other soluble lysosomal components that are not γ-secretase substrates (Lee et al., 2022). 

    The authors could consider experimentally excluding the likelihood that elevated APP-βCTF levels are the more relevant basis for lysosomal deacidification and dysfunction in AD and FAD models than the proposed partial γ-secretase inhibition by exogenously applied human Aβ42. Supporting our mechanistic framework, we find that vATPase assembly/activity is disrupted in human FAD APP or APP/PSEN1 models and similarly in a Down's syndrome (DS) mouse model where elevated mouse βCTF is responsible for vATPase disruption (Im et al., 2023).

    In Zoltowska et al., mouse Aβ42 is inactive as a γ-secretase inhibitor. Inhibiting γ-secretase in DS fibroblasts exacerbates lysosome de-acidification in an APP-βCTF-dependent manner, while at the same time, it lowers Aβ42 levels (Im et al., 2023). Moreover, even though Aβ42 levels are vanishingly low in PSEN1-deleted cells, lysosomal deacidification caused by disrupted vATPase assembly causes extreme lysosomal dysfunction and substrate build-up, which can be reversed by restoring normal lysosomal pH (Lee et al., 2015Lee et al., 2020).  

    Finally, in the authors’ studies, vATPase inhibition by bafilomycin prolongs APP-βCTF half-life and raises its levels more than does exogenously delivered micromolar Aβ42, which does not change APP-βCTF half-life. If APP-βCTF's half-life is not changed by the degree of γ-secretase blockage, then the mechanism underlying an APP-βCTF increase is obscure. 

    In our proposed framework of primary lysosomal impairment in AD pathogenesis, we view lysosomal Aβ accumulation and intraneuronal Aβ formation as contributors to neuronal cell death leading to extracellular cored plaques. These events are consequences of the earlier lysosomal de-acidification induced by APP-βCTF. A possible additional effect of γ-secretase inhibition by Aβ42 on APP-βCTF, if confirmed in vivo, would be compatible with our framework.

    A pathogenic role of APP-βCTF in driving the endosomal dysfunction that underlies cholinergic neurodegeneration is established and independent of Aβ (refs. in Jiang et al. 2022). Notably, mice expressing rab5 or APPL1 (Kim et al., 2015) to model the immediate downstream signaling by pathological APP- βCTF on endosomes do not raise APP-βCTF or Aβ levels above baseline, yet they phenocopy the basal forebrain cholinergic neurodegeneration seen in DS mouse model, which is dependent on APP-βCTF elevation (Pensalfini et al., 2020; and ms in preparation).   

    References:

    . Faulty autolysosome acidification in Alzheimer's disease mouse models induces autophagic build-up of Aβ in neurons, yielding senile plaques. Nat Neurosci. 2022 Jun;25(6):688-701. Epub 2022 Jun 2 PubMed.

    . Lysosomal Dysfunction in Down Syndrome Is APP-Dependent and Mediated by APP-βCTF (C99). J Neurosci. 2019 Jul 3;39(27):5255-5268. Epub 2019 May 1 PubMed.

    . Lysosomal dysfunction in Down syndrome and Alzheimer mouse models is caused by v-ATPase inhibition by Tyr682-phosphorylated APP βCTF. Sci Adv. 2023 Jul 28;9(30):eadg1925. Epub 2023 Jul 26 PubMed.

    . Presenilin 1 Maintains Lysosomal Ca(2+) Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification. Cell Rep. 2015 Sep 1;12(9):1430-44. Epub 2015 Aug 20 PubMed.

    . β2-adrenergic Agonists Rescue Lysosome Acidification and Function in PSEN1 Deficiency by Reversing Defective ER-to-lysosome Delivery of ClC-7. J Mol Biol. 2020 Apr 3;432(8):2633-2650. Epub 2020 Feb 24 PubMed.

    . Partial BACE1 reduction in a Down syndrome mouse model blocks Alzheimer-related endosomal anomalies and cholinergic neurodegeneration: role of APP-CTF. Neurobiol Aging. 2016 Mar;39:90-8. Epub 2015 Dec 2 PubMed.

    . Preclinical and randomized clinical evaluation of the p38α kinase inhibitor neflamapimod for basal forebrain cholinergic degeneration. Nat Commun. 2022 Sep 21;13(1):5308. PubMed.

    . Evidence that the rab5 effector APPL1 mediates APP-βCTF-induced dysfunction of endosomes in Down syndrome and Alzheimer's disease. Mol Psychiatry. 2015 Jul 21; PubMed.

    . Endosomal Dysfunction Induced by Directly Overactivating Rab5 Recapitulates Prodromal and Neurodegenerative Features of Alzheimer's Disease. Cell Rep. 2020 Nov 24;33(8):108420. PubMed.

    View all comments by Ralph Nixon
  3. This is an interesting follow-up study to Szaruga et al., 2017, from the Chávez-Gutiérrez lab, in which it was first suggested that FAD mutations lead to destabilization of γ-secretase-C99 enzyme-substrate (E-S) complexes. However, neither the present EMBO Journal report nor the 2017 Cell study contain experiments that directly address E-S complex stability. Instead, the papers offer temperature-dependent activity assays measuring the relative proportions of Aβ peptide variants. The finding of increased proportions of longer Aβ peptides with increased temperature is taken as evidence of E-S destabilization. Lower temperatures are sufficient for longer-Aβ proportions from FAD-mutant E-S complexes versus wild-type E-S complexes (Szaruga et al., 2017).

    In contrast, even with higher temperatures, the proportion of longer Aβ from E-S complexes with artificial mutations in the C99 extracellular domain remains low, suggesting greater E-S stability and more efficient processing (Koch et al., 2023). However, other explanations are possible, and some direct means of detecting E-S complex stability is needed to address what remains a speculation based on activity assays.

    We had conducted the same temperature-dependent activity assays 12 years ago (Quintero-Monzon et al., 2011) and refrained from interpreting the results to speculate that increased temperature results in destabilization of E-S complexes. Such speculation was difficult to reconcile with the substantial (threefold) increased proteolytic activity with increasing enzyme reaction temperature from 35 to 45oC in our hands, similar to what Szaruga et al. observed.

    Having said that, the new findings that hydrophobic mutations in the C99 extracellular domain allow trimming to very short Aβ peptide products is remarkable. These mutations may, as the authors suggest, facilitate the repositioning of Aβ intermediates to set up for subsequent trimming. The “tug-of-war” model to explain effects of FAD mutations and the artificial C99 ectodomain mutations, however, incorrectly suggests that a negatively charged C-terminal carboxylate of Aβ intermediates (e.g., Aβ49) is necessary for the transition, and that this leads to movement toward positively charged R377 and K380 on presenilin-1. This is incorrect for two reasons. First, the authors cite Bhattarai et al., 2022, to suggest that the negatively charged C-terminus of Aβ49 intermediate is needed to set up for the Aβ49-to-Aβ46 trimming step. In that molecular dynamics study, we found that the presence of protonated, positively charged N-terminus of APP intracellular domain (AICD, coproduct of Aβ49 formation) was essential to set up the Aβ49-to-Aβ46 trimming step. As long as this positively charged AICD was present during the molecular dynamics simulations, either unprotonated, charged Aβ49 or protonated, uncharged Aβ49 worked equally well. Second, we did the biochemical experiment in Fernandez et al., 2016: Synthetic Aβ49 and synthetic Aβ49 with a C-terminal amide led to similar levels and proportions of Aβ40 and Aβ42 upon incubation with γ-secretase. As the Aβ49 C-amide at the C-terminus cannot be deprotonated at biological pH, this demonstrated that a C-terminal negative charge (i.e., carboxylate) plays no role in setting up tripeptide trimming.

    One final point: We now have evidence to the contrary of Szaruga et al. Working with Masato Maesako, Massachusetts General Hospital, Boston, we find that FAD mutations lead to stabilization, not destabilization, of E-S complexes, observing this stabilization through fluorescence lifetime imaging microscopy in whole cells (Devkota et al., 2023). This is consistent with our findings from molecular dynamics simulations (with Yinglong Miao) that FAD-mutant E-S complexes have reduced conformational flexibility.

    Such reduced flexibility explains the reduced proteolytic activity observed with FAD mutations: In general, conformational flexibility is critical for enzyme activity. Moreover, the reduced flexibility would make it more difficult for bound C99 substrate or Aβ intermediates to dissociate from the γ-secretase complex, as the substrate or intermediate is surrounded by presenilin subunits. We also developed a C. elegans model system for FAD and showed that stabilization of E-S complexes is sufficient for age-dependent synaptic loss and reduced lifespan. Production of neither Aβ42 specifically, nor Aβ in general, is necessary.  We suggest that stalled and stabilized γ-secretase E-S complexes can trigger synaptic degeneration, independently of Aβ production.

    References:

    . Alzheimer's-Causing Mutations Shift Aβ Length by Destabilizing γ-Secretase-Aβn Interactions. Cell. 2017 Jul 27;170(3):443-456.e14. PubMed. Correction.

    . Dissociation between the processivity and total activity of γ-secretase: implications for the mechanism of Alzheimer's disease-causing presenilin mutations. Biochemistry. 2011 Oct 25;50(42):9023-35. Epub 2011 Sep 30 PubMed.

    . Mechanism of Tripeptide Trimming of Amyloid β-Peptide 49 by γ-Secretase. J Am Chem Soc. 2022 Apr 13;144(14):6215-6226. Epub 2022 Apr 4 PubMed. Correction.

    . Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFβ. Biochemistry. 2016 Oct 11;55(40):5675-5688. Epub 2016 Sep 30 PubMed.

    . Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes. 2023 Sep 09 10.1101/2023.09.08.556905 (version 1) bioRxiv.

    View all comments by Michael Wolfe
  4. I would like to start by highlighting that genetic GSEC inactivation in the adult brain leads to age-dependent neurodegeneration (Saura et al., 2004; Tabuchi et al., 2009; Acx et al., 2017; Bi et al., 2021). These studies provide key evidence supporting that GSEC-mediated proteolysis plays essential roles in neuronal physiology.

    The bottom-line question that has sparked debate in recent decades is whether mutation-driven inactivation of GSECs underlies FAD, or whether it is an impaired enzyme processivity (leading to altered Aβ peptide profiles) that causes neurodegeneration in FAD.

    To my mind, this debate is settled by the following three observations:

    1. Most, but not all FAD-causing mutations in the GSECs inhibit overall proteolytic activity, but they consistently impair processivity affecting Aβ length.

    These conclusions derived from rigorous kinetic analyses reported in EMBO J a decade ago (Chavez-Gutierrez et al., 2012). Follow-up reports reiterate this finding and multiple groups have reported consistent effects of pathogenic mutations on GSEC processivity, including Fernandez et al., 2014.

    1. In the heterozygous FAD brain, the detrimental effects exerted by (most) FAD pathogenic mutations on overall GSEC proteolytic activity are rescued by the wild-type allele. However, impaired GSEC processivity prevails (Szaruga et al., 2015).

    This is a key piece of evidence given that the vast majority of FAD-linked mutation carriers are heterozygous for the mutation. In contrast to rescue of the total GSEC activity, the (mutation-driven) enhanced production of longer Aβ peptides prevails in the heterozygous FAD brains (Szaruga et al., 2015). This is likely explained by the fact that longer peptides are released from the membrane and thus out of reach of the membrane-embedded WT enzyme. This study has been recognized as one of key pieces of evidence resolving the debate regarding FAD pathogenesis in this comment (Wolfe, 2015).

    1. The (FAD-linked) mutation-driven effects on processivity, leading to enhanced production of longer and aggregation-prone Aβs, credibly explain the accumulation of longer peptides in the brains of carriers of PSEN variants.

    If FAD mutations in the enzyme reduced overall proteolytic activity (as primary mechanism arising from stalled E-S complexes, as suggested by Devkota et al.), how would this (substrate-driven) poisoning of the enzyme cause the characteristic amyloid pathology seen in FAD PSEN1/2? And in APP mutation carriers, would this mechanism be still relevant?

    Furthermore, assuming that 50 percent of the total GSEC pool in the FAD brain is mutant, and as suggested by Devkota et al., mutant enzymes are stalled in E-S complexes:

    • the mutation would lead to a kind of haploinsufficiency. To my knowledge, GSEC haploinsufficiency causes acne inversa, not FAD;
    • homozygous carriers of FAD-mutations in the enzyme would not be viable, as GSEC activity is essential during development. This is not the case, as shown by homozygous carriers of the well-established E280A PSEN1 variants (Kosik et al., 2015). 

    Regarding the molecular mechanism(s) underlying the mutation-driven impairments on GSEC function:

    A. Our thermoactivity assays, functionally assessing enzyme-substrate (GSEC-APP/Aβ) complexes during proteolysis in Szaruga et al., 2017, demonstrated that:

    • The thermal E-S complex stability decreases with every GSEC cut during sequential proteolysis, making E-S complexes with shorter (Aβ) substrates less stable (Figure 1 B-E);
    • Pathogenic mutations in PSEN1 lower the thermal stability of E-S interactions, with the less-destabilizing mutations affecting the “labile” E-S complexes (with shortened Aβ) and the more-destabilizing ones affecting E-S interactions with both Aβ and the longer endopeptidase substrate (APP C99) (Figure 3).
    • These findings are consistent with mutation-driven impairments on processivity ONLY and on processivity AND overall (endopeptidase) activity;
    • A linear relationship exists between the degree of mutation-driven destabilization and clinical onset (Figure 3). Our more recent studies show that this correlation holds true at the level of the molecular composition of Aβ peptides, with the Aβ (37+ 38 +40)/ (42+34) ratio displaying a linear correlation with age at AD onset (AA0) (Petit et al., 2022). 
    • Pathogenic mutations in the transmembrane domain of APP consistently destabilize E-S complexes formed during Aβ production by GSEC (Figure 4), which unifies the mechanism of release of longer Aβ peptides in FAD.
    • GSEC modulators stabilize E-S interactions; thus, acting in opposite mechanistic ways.

    B. Our investigations showed that pathogenic PSEN1 variants had a direct impact on the stability of the GSEC complex (Figure S4).

    C. Finally, the comprehensive and rigorous studies by Okochi et al. (2013) demonstrate that FAD-mutations in PSEN promote dissociation of mutant E-S complexes, while GSMs lower their dissociation. These data are in line with detrimental effects of pathogenic PSEN1 variants on the stability of E-S interactions.

    Regarding the preprint by Devkota et al.:

    Theoretical studies, using GaMD simulations to analyze how pathogenic PSEN1 mutations affect GSEC APP endopeptidase cleavage show that most of the six PSEN1 mutations visited the active conformation less frequently. This intriguing observation, however, does not directly assess E-S stability, nor is connected to the thermal stability of E-S interactions during proteolysis that we measured in Szaruga et al, 2020. It seems that our respective studies choose distinct semantics to define "E-S stability." The experimental data behind this term is not connected in Devkota et al and Szaruga et al, hence we believe the two do not contradict each other.

    The data showing age-dependent synaptic loss in Caenorhabditis elegans are intriguing and are in line with previous findings pointing the essential role of GSEC function in neuronal physiology Saura et al., 2004; Tabuchi et al., 2009; Acx et al., 2017; Bi et al., 2021). Moreover, the data support C. elegans as a model to study the mechanism that underlies the described pathogenesis. However, I find no evidence to conclude that FAD pathogenesis in patients is independent of Aβ production.

    References:

    . Loss of presenilin function causes impairments of memory and synaptic plasticity followed by age-dependent neurodegeneration. Neuron. 2004 Apr 8;42(1):23-36. PubMed.

    . Conditional forebrain inactivation of nicastrin causes progressive memory impairment and age-related neurodegeneration. J Neurosci. 2009 Jun 3;29(22):7290-301. PubMed.

    . Inactivation of γ-secretases leads to accumulation of substrates and non-Alzheimer neurodegeneration. EMBO Mol Med. 2017 Aug;9(8):1088-1099. PubMed.

    . Neuron-specific deletion of presenilin enhancer2 causes progressive astrogliosis and age-related neurodegeneration in the cortex independent of the Notch signaling. CNS Neurosci Ther. 2021 Feb;27(2):174-185. Epub 2020 Sep 22 PubMed.

    . The mechanism of γ-Secretase dysfunction in familial Alzheimer disease. EMBO J. 2012 May 16;31(10):2261-74. Epub 2012 Apr 13 PubMed.

    . Alzheimer presenilin-1 mutations dramatically reduce trimming of long amyloid β-peptides (Aβ) by γ-secretase to increase 42-to-40-residue Aβ. J Biol Chem. 2014 Nov 7;289(45):31043-52. Epub 2014 Sep 19 PubMed.

    . Qualitative changes in human γ-secretase underlie familial Alzheimer's disease. J Exp Med. 2015 Nov 16;212(12):2003-13. Epub 2015 Oct 19 PubMed.

    . Cutting to the chase: How pathogenic mutations cause Alzheimer's. J Exp Med. 2015 Nov 16;212(12):1991. PubMed.

    . Homozygosity of the autosomal dominant Alzheimer disease presenilin 1 E280A mutation. Neurology. 2015 Jan 13;84(2):206-8. Epub 2014 Dec 3 PubMed.

    . Alzheimer's-Causing Mutations Shift Aβ Length by Destabilizing γ-Secretase-Aβn Interactions. Cell. 2017 Jul 27;170(3):443-456.e14. PubMed. Correction.

    . Aβ profiles generated by Alzheimer's disease causing PSEN1 variants determine the pathogenicity of the mutation and predict age at disease onset. Mol Psychiatry. 2022 Jun;27(6):2821-2832. Epub 2022 Apr 1 PubMed.

    . γ-secretase modulators and presenilin 1 mutants act differently on presenilin/γ-secretase function to cleave Aβ42 and Aβ43. Cell Rep. 2013 Jan 31;3(1):42-51. PubMed.

    View all comments by Lucia Chavez-Gutierrez
  5. The manuscript from Lucia Chavez-Guttierez and colleagues may prove to be a milestone in the reconciliation of the idea that Aβ is the pathological agent driving Alzheimer’s disease and the alternative view that, instead, APP’s βCTF is critical for the disease. They present evidence of a negative feedback mechanism whereby production of Aβ by γ-secretase cleavage of βCTF is, itself, inhibited by Aβ. Interestingly, they show that the 42 amino acid residue form of Aβ, Aβ42, is most inhibitory toward γ-secretase while shorter or longer forms appear less potent.

    The murine form of Aβ42 was considerably less inhibitory than human Aβ42, consistent with the rapid evolutionary divergence of the murine form relative to the Aβs of other tetrapods (Sharman et al., 2013; Moore et al., 2014). One implication of this is that it is less likely that βCTF-equivalent fragments of γ-secretase’s other >100 substrates will have similar inhibitory effects while, conversely, the authors showed that Aβ42 can suppress γ-secretase cleavage of those other substrates, such as NOTCH1 and cadherins.

    Another interesting aspect of the work: Cell culture medium conditioned by human iPSC-derived neurons homozygous for the Swedish mutant form of APP (which has increased β-secretase cleavage of APP leading to increased Aβ production [Cai et al., 1993]) was, when applied to PC12 cells, measurably more potent in apparent inhibition of γ-secretase than medium conditioned by equivalent non-mutant neurons. This supports that the γ-secretase-inhibitory action of Aβ occurs at physiologically relevant concentrations.

    In the discussion, the authors suggest that the inhibitory action of Aβ42 on γ-secretase may explain how fAD mutations in presenilin genes cause γ-secretase loss-of-function phenotypes according to Shen and Kelleher’s “presenilin hypothesis of Alzheimer's disease” (Shen et al., 2007). Here I disagree, since not all fAD mutations in the presenilin genes caused increased production of Aβ42 (Sun et al., 2017) and the human genetic evidence supports that decreased γ-secretase activity causes the skin disease hidradenitis suppurativa/acne inversa but not fAD (Wang et al., 2010). 

    This message I take from this work: High Aβ levels can inhibit γ-secretase in a physiologically relevant manner to increase levels of βCTF, with predicted impacts on the function of the lysosome (Jiang et al., 2019; Im et al., 2023) and on mitochondria via mitochondria-associated endoplasmic reticulum membrane-related changes (Pera et al., 2017). 

    References:

    . The Guinea Pig as a Model for Sporadic Alzheimer's Disease (AD): The Impact of Cholesterol Intake on Expression of AD-Related Genes. PLoS One. 2013;8(6):e66235. PubMed.

    . Asynchronous evolutionary origins of Aβ and BACE1. Mol Biol Evol. 2014 Mar;31(3):696-702. Epub 2013 Dec 19 PubMed.

    . Release of excess amyloid beta protein from a mutant amyloid beta protein precursor. Science. 1993 Jan 22;259(5094):514-6. PubMed.

    . The presenilin hypothesis of Alzheimer's disease: evidence for a loss-of-function pathogenic mechanism. Proc Natl Acad Sci U S A. 2007 Jan 9;104(2):403-9. PubMed.

    . Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production of Aβ42 and Aβ40 peptides by γ-secretase. Proc Natl Acad Sci U S A. 2017 Jan 24;114(4):E476-E485. Epub 2016 Dec 5 PubMed.

    . Gamma-secretase gene mutations in familial acne inversa. Science. 2010 Nov 19;330(6007):1065. PubMed.

    . Lysosomal Dysfunction in Down Syndrome Is APP-Dependent and Mediated by APP-βCTF (C99). J Neurosci. 2019 Jul 3;39(27):5255-5268. Epub 2019 May 1 PubMed.

    . Lysosomal dysfunction in Down syndrome and Alzheimer mouse models is caused by v-ATPase inhibition by Tyr682-phosphorylated APP βCTF. Sci Adv. 2023 Jul 28;9(30):eadg1925. Epub 2023 Jul 26 PubMed.

    . Increased localization of APP-C99 in mitochondria-associated ER membranes causes mitochondrial dysfunction in Alzheimer disease. EMBO J. 2017 Nov 15;36(22):3356-3371. Epub 2017 Oct 10 PubMed.

    View all comments by Michael Lardelli
  6. My remarks below are in reply to Lucia Chávez-Gutiérrez's comments on Devkota et al. The term “enzyme-substrate (E-S) complex” has a specific biochemical meaning, as does the term “stability” of E-S complexes. The Chávez-Gutiérrez lab clearly claims that FAD mutations destabilize γ-secretase E-S complexes to release long Aβ peptide intermediate substrates: This is in the very title of the Szaruga et al., 2017, report. In contrast, our new report, posted in bioRxiv, shows that FAD mutations stabilize E-S complexes, (Devkota et al., 2023), the opposite of the Chávez-Gutiérrez lab findings. This is not a mere difference in semantics, as suggested by Dr. Chávez-Gutiérrez in her comment above.

    Both these claims cannot be true. In neither Szaruga et al., 2017, nor in Koch et al., 2023, was E-S complex stability tested in any way. In our new BioRxiv report, we find, working with Masato Maesako at Massachusetts General Hospital, Boston, that FAD mutations lead to stabilization of E-S complexes, observing this stabilization through fluorescence lifetime imaging microscopy in whole cells. It is incorrect to state that we only conducted molecular dynamics simulations as evidence for E-S stabilization. Only one side in this discussion has conducted experiments to test the stability of E-S complexes, and the evidence supports FAD mutations stabilizing these complexes.

    I respectfully dispute another assertion in Chávez-Gutiérrez's comments: We are not claiming that the stabilized FAD-mutant E-S complexes simply lead to reduction of overall γ-secretase activity. All our studies on the effects of FAD mutations have shown reduction in one or more proteolytic functions of γ-secretase on APP substrate (Quintero-Monzon et al., 2011; Fernandez et al., 2014; Devkota et al., 2021). In some cases, total Aβ production is quite low, but never zero. However, we have consistently observed that FAD mutations skew the proportion of Aβ products to longer forms that are >45 residues due to reduced carboxypeptidase function. We first reported this phenomenon in 2011 using long, hand-cast gels for SDS-PAGE and western blotting (Quintero-Monzon et al., 2011), and more recently used liquid chromatography with tandem mass spectrometry (a method first reported by the lab of Yasuo Ihara) to conduct comprehensive and quantitative analysis of all proteolytic processing steps by γ-secretase on APP substrate (Devkota et al., 2023). We have cross-validated this method in multiple ways to ensure that the effects we observe due to FAD mutations are accurate.

    Typically, the reduced carboxypeptidase function caused by FAD mutations also leads to the increased ratios of Aβ42/Aβ40 widely considered critical to AD pathogenesis. We suggest that elevated Aβ42/Aβ40 is a consequence of stalled, stabilized E-S complexes, but that this elevation may not be the trigger of synaptic loss (i.e., increased Aβ42/Aβ40 may be an epiphenomenon). Reduced conformational flexibility of FAD-mutant E-S complexes, which we observe in our molecular dynamic simulations, leads to decreased proteolysis, particularly carboxypeptidase trimming of long Aβ peptides to shorter forms, thereby increasing Aβ42/Aβ40.

    However, in our new transgenic C. elegans model system for FAD, where we co-express APP substrate and PSEN1 in neurons, we find that production of Aβ42 specifically, or Aβ generally, is not required for age-dependent synaptic degeneration, only stabilization of E-S complexes. We suggest that the stalled E-S complexes per se, not inhibition of γ-secretase cleavage of other substrates, triggers this synaptic loss. We suggest this in our new report precisely because of the genetic evidence in humans that dominant mutations leading to nonsense-mediated decay of γ-secretase components (true loss-of-function mutations) cause a hereditary skin disease, not neurodegeneration (Wang et al., 2010).

    As such, we favor a gain of toxic function due to stabilized E-S complexes. We provide an alternative to the amyloid hypothesis that accounts for FAD mutations in both APP and presenilins, without requiring amyloid production and without invoking loss of γ-secretase activity per se for synaptic degeneration. Aggregation of Aβ42 (or Aβ43) is not a good thing: Evidence suggests that it can, for instance, lead to overactivation of microglia and neuroinflammation. However, after more than 30 years of the amyloid hypothesis, the cumulative evidence that Aβ is the primary driver of the disease remains unconvincing. Perhaps this is why almost all anti-Aβ therapeutic strategies failed in the clinic, and the recently approved anti-amyloid antibodies only modestly slow the rate of cognitive decline. Ours is a fundamentally new way of thinking about how FAD mutations trigger neurodegeneration and deserves serious consideration, after decades of controversy over the role of amyloid and the unimpressive clinical results with anti-amyloid drug candidates.

    References:

    . Alzheimer's-Causing Mutations Shift Aβ Length by Destabilizing γ-Secretase-Aβn Interactions. Cell. 2017 Jul 27;170(3):443-456.e14. PubMed. Correction.

    . Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes. 2023 Sep 09 10.1101/2023.09.08.556905 (version 1) bioRxiv.

    . APP substrate ectodomain defines amyloid-β peptide length by restraining γ-secretase processivity and facilitating product release. EMBO J. 2023 Dec 1;42(23):e114372. Epub 2023 Oct 18 PubMed.

    . Dissociation between the processivity and total activity of γ-secretase: implications for the mechanism of Alzheimer's disease-causing presenilin mutations. Biochemistry. 2011 Oct 25;50(42):9023-35. Epub 2011 Sep 30 PubMed.

    . Alzheimer presenilin-1 mutations dramatically reduce trimming of long amyloid β-peptides (Aβ) by γ-secretase to increase 42-to-40-residue Aβ. J Biol Chem. 2014 Nov 7;289(45):31043-52. Epub 2014 Sep 19 PubMed.

    . Familial Alzheimer's disease mutations in amyloid protein precursor alter proteolysis by γ-secretase to increase amyloid β-peptides of ≥45 residues. J Biol Chem. 2021;296:100281. Epub 2021 Jan 12 PubMed.

    . gamma-Secretase: successive tripeptide and tetrapeptide release from the transmembrane domain of beta-carboxyl terminal fragment. J Neurosci. 2009 Oct 14;29(41):13042-52. PubMed.

    . Gamma-secretase gene mutations in familial acne inversa. Science. 2010 Nov 19;330(6007):1065. PubMed.

    View all comments by Michael Wolfe
  7. Here, I respectfully avoid commenting on data as those papers are in the preprint stage. On the other hand, I would like to comment from the viewpoint of one of the very few junior faculties (people sometimes call us one of the “endangered species”) who seek to better understand the biology of γ-secretase and its role in AD:

    The enormous amount of hard work that γ-secretase researchers have done for the past three decades has given us a massive quantity of knowledge and research materials to study γ-secretase, which I greatly appreciate. Moreover, these extraordinary studies have allowed delivery of medication(s) in the clinic, which may not be perfect yet but could provide hope and a next step to move up.

    Still, I believe the entire picture regarding why genetic mutations in presenilins (PSEN1/2), the catalytic subunit of γ-secretase, and APP, one of the substrates of γ-secretase, cause early onset FAD neurodegeneration has not been drawn. We all know that the γ-secretase complex with mutations in PSEN1/2 is “inefficient.” It is still unclear to me how much FAD neurodegeneration caused by inefficient γ-secretase is dependent on, and/or independent of, altered Aβ peptide profiles. I.e., is it either or both? If the answer is both, how much is via dependent and how much via independent mechanisms?

    γ-Secretase has a wide variety of substrates. Genetic and pharmacological inhibition of γ-secretase cause detrimental effects, but APP is only the one in which FAD mutations have been discovered so far, and the mutation sites are clustered around the Aβ region; therefore, I currently believe the mechanism(s) dependent on altered Aβ peptide profiles to a certain degree play a role in FAD neurodegeneration (but that may change in the future). We all know that various species of Aβ peptides are generated by γ-secretase, long Aβ (e.g., Aβ42, 43) is accumulated in the AD brain, and their removal can slow the progression of the disease in a statistically significant manner.

    However, I cannot find any clear answers in previous publications regarding what role(s) longer Aβ species (i.e., Aβ45, 46, 48, 49), which are supposed to be bound to the γ-secretase within the membrane, play, and whether they are associated with FAD neurodegeneration and, if so, how much. It is also unclear whether shorter Aβ species (i.e., Aβ37, 38, 40) and 3-4 amino acids intermediate peptides, generated in the process of the longer Aβ species trimming by γ-secretase, may play some roles. Thus, my lab is currently seeking to determine the role of shorter Aβ species and the 3-4 amino acids peptides inside the cells. I also very much appreciate the recent progress the field has made since we now know the pivotal roles of C99 and p75CTF.

    Another challenge I face (as do others) is understanding how γ-secretase functions in intact/live cells and human disease. I guess this challenge could be in part because of the unique environment in which γ-secretase is located and plays its proteolytic role: the lipid membrane bilayer. In other words, γ-secretase can be dynamically affected by changes in lipid membrane properties in living neurons (and likely in glial cells), which seems to be critical in the process of aging and the disease.

    I hope our ongoing, healthy, collaborative, and competitive research approach enables us to better understand γ-secretase biology, and that this will soon help treat people with FAD mutations and some sporadic cases!

    View all comments by Masato Maesako

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