APP C-Terminal Fragments Stifle Calcium Flow from ER to Lysosomes

C-terminal fragments of amyloid precursor protein have been blamed for endolysosomal dysfunction in Alzheimer’s disease. Now, scientists led by Wim Annaert at KU Leuven, Belgium, claim that’s because these fragments disrupt calcium flux. In the April 15 Developmental Cell, they reported that knocking out presenilin 1 and 2 in mouse fibroblasts causes APP-CTFs to accumulate in lysosomes, especially at junctions with the ER. This prevents the flow of calcium ions between the two compartments, slowing endolysosomal maturation. Reducing the APP-CTFs restored lysosome function.

  • APP CTFs accumulate in lysosomes, especially at junctions with the endoplasmic reticulum.
  • This stunts calcium flow from the ER to lysosomes, stalling the cellular waste disposal system.
  • Knocking out APP normalizes endolysosome function.

Scientists praised the research. “This is beautiful cell biology work addressing important questions,” Gunnar Gouras of Lund University, Sweden, told Alzforum. “This study provides exciting mechanistic insights into how APP-CTFs, and not Aβ, may contribute to the lysosomal dysfunction in Alzheimer’s disease,” wrote Stefan Lichtenthaler at the German Center for Neurodegenerative Diseases, Munich (comments below). Aβ has previously been tied to bloated, neurotoxic lysosomes (Jun 2022 news).

APP-CTFs also have been incriminated in lysosome malfunction. Scientists led by Marc Tessier-Lavigne of Stanford University found that familial AD mutations in APP or presenilin 1 caused endosomal dysfunction due to accumulation of β-CTFs (Aug 2019 news). Randy Nixon at New York University in Orangeburg reported that β-CTFs, created when β-secretase cleaves APP, block the assembly of vacuolar ATPase that fuels lysosome acidification and that reducing β-CTF production revived the organelles (Jul 2023 news). Others have reported poor acidification caused by presenilin mutations as well (Chou et al., 2023; Martin-Maestro et al., 2017; see Nixon comment below).

Annaert has long been a proponent of a different reason for β-CTF lysosomal dysfunction: calcium imbalance. He has reported that presenilins, better known as catalytic components of γ-secretase, also regulate lysosomal calcium levels (Jul 2012 news). Without presenilin, calcium levels in the lysosome falls and the cellular waste system skids to a halt. In the latest twist, he implicates APP-CTFs in this signaling snafu.

With or Without Presenilin. In healthy cells (left), calcium flows from the endoplasmic reticulum (purple) to the lysosomes (red circle in center). Early endosomes (EEA1+), recycling endosomes (VPS35+), and late endosomes/lysosomes (LAMP1+) function normally. When APP-CTFs accumulate in the lysosomal membrane (right), lysosomal calcium drops and endolysosomes swell. Mitochondria (green, bottom left) made less ATP (yellow, bottom right) though it’s unclear why. [Courtesy of Bretou et al., Developmental Cell, 2024.]

To better understand the role of presenilins in the endolysosomal system, first author Marine Bretou turned to mouse embryonic fibroblasts. “We wanted to work with a simple model, then validate findings in neurons later,” Annaert told Alzforum.

First, Bretou treated the fibroblasts with the γ-secretase inhibitor DAPT to prevent proteolytic processing of APP-CTFs. Within a day, the fragments had accumulated in lysosomes and late endosomes, which had swollen. By days three and four, recycling endosomes and early endosomes, respectively, had enlarged as well. These results indicate a delay in endosome-to-lysosome maturation, with lysosomes failing first.

Calcium levels in lysosomes also fell by 30 percent in the 24 hours after DAPT. Bretou saw the same effects of DAPT in primary mouse hippocampal neurons: APP-CTF accumulation, lysosomal calcium decrease, and endolysosomal swelling.

To tease apart APP-CTF’s from Aβ’s role in endolysosomal demise, Bretou and colleagues knocked out presenilin 1 and 2 in mouse embryonic fibroblasts to completely eliminate Aβ production since some still occurs with DAPT treatment. In these knockout cells, lysosomes had about fivefold less Ca2+ than did lysosomes in wild-type fibroblasts.

This imbalance did not stem from calcium leaking out of lysosomes but from restricted calcium transport into lysosomes from the endoplasmic reticulum, the authors found. When Bretou emptied lysosomes of Ca2+ using the dipeptide gly-phe-β-naphtylamide to disrupt their membranes, those in control cells refilled quickly, while lysosomes in presenilin knockout cells did not. Control lysosomes failed to refill when Bretou added an endoplasmic reticulum Ca2+ pump inhibitor to the medium. The ER pump recycles cytosolic calcium back into the organelle. Because the lysosomes did not refill with calcium, either, the authors concluded that the ER was the source of these ions.

To directly test whether CTFs are involved in lysosomal Ca2+ flux, the scientists knocked out APP and both presenilins in fibroblasts, then reintroduced either α- or β-CTFs, fragments cleaved by α- and β-secretase, respectively. To their surprise, they found that both equally reduced lysosomal calcium and stunted endolysosomal function. Previous work suggested only the shorter β-CTFs disrupted lysosome function.

Further support for the idea that CTFs dysregulate calcium flow came from super-resolution microscopy. It showed the fragments clustering on the lysosomal membrane, right where it contacts the ER (image below). Scientists believe the ER exchanges small molecules and ions, such as Ca2+, with organelles through these membrane junctions (reviewed by Burgoyne et al., 2015). The authors suspect the CTFs keep the lysosomes tightly bound to the ER, as well, because in PS1/2 knockout lines, the lysosomes moved sluggishly around the cytoplasm.

Cozy Organelles. When APP β-CTFs were added to mouse fibroblasts lacking presenilin 1/2 and APP, the fragments (red) bound to lysosomes (blue), which swelled and became enmeshed in the endoplasmic reticulum (green). [Courtesy of Bretou et al., Developmental Cell, 2024.]

Could the stalled endolysosomes be jolted back into action? Indeed, expressing human presenilin 1 in the presenilin double knockout fibroblasts shrank endosomes and lysosomes, enabled lysosomes to move normally through the cytoplasm, and normalized lysosomal Ca2+ levels. Deleting APP in the presenilin knockout fibroblasts also ameliorated endolysosomal defects, directly implicating the precursor protein in weakening this recycling system.

These endolysosomal deficits also occurred when the triple knockout fibroblasts expressed the APP intracellular domain (AICD) carrying an N-terminal motif prone to modification with myristic acid. The lipid anchors AICD to the membrane, effectively turning it into a CTF. AICD contains the YENPTY sequence, to which multiple proteins bind, including Abl kinase and the sortilin receptor SorLA. When the researchers mutated the motif to abolish these interactions, lysosomal calcium levels and endolysosome morphology were normal. To the authors, this suggested that YENPTY interactions, in addition to accumulation of APP-CTFs, induces endolysosomal dysfunction.

As a corollary of this, γ-secretase, which releases AICD into the cytoplasm, would protect against endolysosomal defects. AICD, like the Notch intracellular domain, is believed to form complexes that regulate gene expression, but the authors think their findings dispel that notion. They claim that failure to degrade CTFs, not production of AICD, sustains downstream signaling, instigating lysosomal dysfunction. Rick Livesey, University College London, thinks this is plausible. “Proteolysis of APP-CTFs is an off switch for APP-CTF-mediated functions, rather than activating downstream signaling,” he wrote (comment below). “In this scenario, accumulation of APP-CTFs is effectively a toxic gain-of-function.”

On that note, the data offer another explanation for why γ-secretase inhibitors failed in clinical studies in AD with one, semagacestat, worsening cognition (Doody et al., 2013). “It is tempting to hypothesize that [this] effect on cognition might be mediated by APP-CTFs and/or endolysosomal dysfunction,” wrote Rik van der Kant of Vrije University in Amsterdam (comment below). As such, the findings support the use of γ-secretase modulators to encourage processing of APP-CTFs into shorter, non-toxic Aβ peptides or BACE inhibitors to prevent production of β-CTFs to begin with (Jul 2023 conference news; Aug 2022 conference news).—Chelsea Weidman Burke

Comments

  1. I really enjoy reading papers from the Annaert lab because of their high level of cell biology, which is so needed for our field to better understand the cellular mechanisms underlying AD. This paper by Bretou et al. is a tour de force with diverse and elegant methods, and provides numerous novel insights, such as on the importance of APP CTFs at membrane contact sites between the ER and late endosome/lysosomes (LE/Lys) relating to γ-secretase inhibition/loss of presenilins and lysosomal calcium. It is also remarkable that cholesterol efflux can reverse the endolysosomal abnormalities they detect in γ-secretase deficient cells.

    There are, however, a few issues where I am less certain about their interpretation. In particular, I am not as convinced about their view that APP CTFs drive early AD cellular dysfunction, and when they write “endosomal abnormalities are observed early in AD pathology, independent from the build-up of Aβ …”  The earliest preclinical biomarker for both sporadic and familial AD is a change in Aβ42 rather than in CTFs, and as we showed, Aβ42 also accumulates early on, and prior to plaques, in late endosomal multivesicular bodies, where the current paper shows aberrant CTF accumulations.

    Nevertheless, this, as well as work by others, is increasingly swaying me to consider even more the importance of APP CTFs.

    View all comments by Gunnar Gouras

  2. This study provides exciting mechanistic insights into how APP CTFs, and not Aβ, may contribute to the lysosomal dysfunction in Alzheimer’s disease. This cell biological study is comprehensive, carefully conducted, convincing, and in good agreement with previous studies. It highlights a (patho-)physiological function for APP CTFs in controlling cross talk between ER and lysosomes and this function depends on a specific amino acid motif in the cytoplasmic domain of APP.

    I am surprised that APP CTFs, specifically, induce this effect, given that a small number of other transmembrane proteins also contain similar NPXY motifs in their cytoplasmic domains. Potentially, they can induce similar effects, at least in cell types where they are highly expressed. The toxic function of accumulating APP CTFs, as shown in this study, provides one more reason for clinical trials with BACE inhibitors in AD: Besides inhibiting Aβ production and increasing neuroprotective sAPPα, BACE inhibitors also lower APP CTF production.

    View all comments by Stefan Lichtenthaler

  3. Previous studies in human neurons have established that mutations in APP and PSEN1 causal for AD result in endolysosomal and autophagy dysfunction as a consequence of reduced turnover of C-terminal fragments of APP (APP-CTFs), and that this is probably a primary event in disease initiation (Hung and Livesey, 2018; Kwart et al., 2019). This fascinating work by the Annaert group uses γ-secretase inhibition and loss of function in mouse embryonic fibroblasts and hippocampal neurons to provide a mechanistic understanding of how accumulation of APP-CTFs disrupts endolysosomal function. In a tour de force of cell biology, the team clearly demonstrate that preventing turnover of APP-CTFs negatively impacts lysosomal calcium homeostasis, which appears to be due to accumulated APP-CTFs altering lysosome-ER membrane contact sites that mediate calcium refilling of lysosomes.

    A key finding of this study is that proteolysis of APP-CTFs is an off switch for APP-CTF-mediated function, rather than activating downstream signaling. In this scenario, accumulation of APP-CTFs is effectively a toxic gain of function. This study also adds weight to one hypothesis for why γ-secretase inhibition, which greatly reduces production of Aβ peptides, proved clinically to be not only ineffective in slowing disease progression, but to lead to worsening cognition (e.g., Doody et al., 2013): accumulation of APP-CTFs resulted in increased disruption of the endolysosomal system, potentially accelerating disease pathogenesis.

    Overall, this paper reinforces the view that lowering production and accumulation of APP-CTFs could be an effective therapeutic approach in both familial (monogenic) and late-onset AD. Notably, in this context, this report and previous studies (Hung et al., 2021; Hung and Livesey, 2018) have shown that reduction of total APP by genetic knockout or by antisense oligonucleotide (ASO) knockdown corrects lysosome dysfunction due to mutations in PSEN1 and another familial AD gene, SORL1. A number of different modalities and mechanisms targeting APP are in the early stages of development. These include siRNAs and ASOs to reduce total APP, as well as previously developed γ-secretase modulators, which increase APP-CTF turnover by γ-secretase. This paper also highlights that lysosome dysfunction is an early pathology in AD and other neurodegenerative diseases, preceding protein aggregation, and that correcting lysosome function is another attractive therapeutic strategy, for which there are several candidate targets and molecules in development.

    References:

    Doody RS, Raman R, Farlow M, Iwatsubo T, Vellas B, Joffe S, Kieburtz K, He F, Sun X, Thomas RG, Aisen PS, , Siemers E, Sethuraman G, Mohs R. A phase 3 trial of semagacestat for treatment of Alzheimer's disease. N Engl J Med. 2013 Jul 25;369(4):341-50. PubMed.

    Hung C, Tuck E, Stubbs V, van der Lee SJ, Aalfs C, van Spaendonk R, Scheltens P, Hardy J, Holstege H, Livesey FJ. SORL1 deficiency in human excitatory neurons causes APP-dependent defects in the endolysosome-autophagy network. Cell Rep. 2021 Jun 15;35(11):109259. PubMed.

    Hung CO, Livesey FJ. Altered γ-Secretase Processing of APP Disrupts Lysosome and Autophagosome Function in Monogenic Alzheimer's Disease. Cell Rep. 2018 Dec 26;25(13):3647-3660.e2. PubMed.

    Kwart D, Gregg A, Scheckel C, Murphy EA, Paquet D, Duffield M, Fak J, Olsen O, Darnell RB, Tessier-Lavigne M. A Large Panel of Isogenic APP and PSEN1 Mutant Human iPSC Neurons Reveals Shared Endosomal Abnormalities Mediated by APP β-CTFs, Not Aβ. Neuron. 2019 Oct 23;104(2):256-270.e5. Epub 2019 Aug 12 PubMed.

    View all comments by Rick Livesey

  4. I think this is a beautiful paper with a lot of high-quality cell biology on the role of APP and γ-secretase on endolysosomal function. The authors convincingly show that the inhibition, or loss, of γ-secretase activity results in endolysosomal dysfunction, such as early endosome enlargement, changes in lysosomal calcium, cholesterol, and membrane contact sites.

    Most phenotypes in the manuscript are rescued by deletion of APP, whereas C-terminal fragments induce endolysosomal dysfunction even in APP knockout cells. Overall, this presents a very convincing case for the strong role of APP C-terminal fragments in regulation of the endolysosomal system, fitting with recent observations from other labs. One experiment I found particularly interesting is the overexpression of membrane-anchored AICD, which showed that this part of APP is important for mediating the downstream effects of the C-terminal fragments.

    Performing such high-quality mechanistic research is hard, and often requires using model systems that are a bit easier to manipulate, which is why mouse embryonic fibroblasts were used in this case. As the authors note, it would be important to replicate these findings in neurons. In addition, most experiments rely on γ-secretase inhibition or full knockouts of APP and PSEN, so it would be interesting to see how strongly these phenotypes replicate in FAD mutant cell lines. However, we know that γ-secretase inhibitors in the clinic lead to poorer cognition. It is tempting to hypothesize that the effect of this inhibition on cognition might be mediated by APP-CTFs and/or endolysosomal dysfunction.

    It is also important to note that our field for many years relied heavily on mouse models of Alzheimer’s disease that carry multiple mutations in PSEN and APP. Overall, we tend to contribute all phenotypes in these mice to the accumulation of Aβ. It is time to consider that some, or possibly many, of the pathological effects in these mice might not be mediated by Aβ, but rather by downstream effects of PSEN/APP dysfunction on the endolysosomal system (this paper amongst others) and/or altered cholesterol metabolism (this paper and Essayan-Perez and Südhof, 2023). Interestingly, these pathways are also implicated in sporadic Alzheimer’s disease, and therefore might provide different ways of looking at the pathological disease cascade.

    References:

    Essayan-Perez S, Südhof TC. Neuronal γ-secretase regulates lipid metabolism, linking cholesterol to synaptic dysfunction in Alzheimer's disease. Neuron. 2023 Oct 18;111(20):3176-3194.e7. Epub 2023 Aug 4 PubMed.

    View all comments by Rik van der Kant
    • User Profile Image Ralph Nixon
      New York University School of Medicine/Nathan Kline Institute
    • User Profile Image Ju-Hyun Lee
      Nathan Kline Institute/NYU Langone Med
    • Posted:

    Bretou and the Annaert group report observations on the interplay of calcium, APP-βC-terminal fragments (βCTF), and the endolysosomal system using mainly a mouse embryonic fibroblast cell line that was CRISPR-edited to delete both presenilin 1 and PSEN2. Their data support earlier evidence that deleting either PSEN1 or 2 dysregulates endoplasmic reticulum (ER) calcium channels, while also elevating levels of APP-βCTF by blocking its degradation. PSEN1 mutations/deletion alone elevate cytosolic calcium, which activate calpains and cdk5/p25—calcium-dependent enzymes able to trigger degeneration and cell death when overactive. This more robust model of calcium overload and cytotoxicity—created by deleting both PSEN1 and PSEN2, which each dysregulate calcium by different mechanisms—has unclear relevance as an Alzheimer’s disease model. Further, informing the temporal order of pathobiological events in Alzheimer’s disease is particularly questionable. In our earlier studies of PSEN1 and 2, we had abandoned the use of the PSEN double knockout cell model because of survival instability and because of the multiple stress responses the cells activate to fend off necrotic death. While deleting both PSEN1 and PSEN2 may yield greater γ-secretase inhibition, the myriad differences in transcriptomic profiles and function of PSEN 1 and 2 likely amplify the baseline compromise of PSEN dKO models, accounting for their reduced viability in our hands. PSEN1 loss of function alone already induces substantial AD-relevant β-CTF elevation and endosomal-lysosomal dysfunction.

    Primary dysregulation of calcium seen in the dKO model is expected, given that interaction of presenilins with ER calcium channels is well accepted. Such actions, however, do not exclude parallel “primary” disease-relevant mechanisms, stemming from other direct presenilin pathological actions, or from the direct disruptions caused by APP mutations or various risk gene-driven causes of APP-βCTF and Aβ elevation in sporadic AD. In the PSEN dKO model that greatly amplifies calcium disruption, defining a “chronology” of downstream events tracked over periods of days in vitro and deeming these as secondary responses seems quite speculative. Chronology aside, it is noteworthy, however that the authors confirmed various reported findings by us and others on the pathogenicity of APP-βCTF accumulation, including even our recent identification of the tyrosine phosphorylated variant of the YENPTY motif in the C-terminal domain APP as the bioactive moiety mediating several of APP’s pathological effects (Im et al., 2023).

    The authors report little to no change in lysosome acidification although their dismissal of the observed disease-associated 0.5-unit (4.8 to 5.3) elevation of pH, a logarithmic scale, as irrelevant to the lysosomal dysfunction seen in a Down’s syndrome mouse model (Im et al., 2023) would likely be surprising to lysosomal biologists, especially given that normalizing pH in this study reversed autophagy-lysosomal dysfunction. Rather than get into the technical weeds of the evidence reported using dKO cells, we would like to point out that, as support for their conclusion regarding pH and vATPase in this  model, the authors cite only their own earlier report (Coen et al., 2012), which we have challenged on multiple technical grounds (Lee et al., 2015), and a second report (Zhang et al., 2012) with serious technical flaws in measurements of lysosomal pH and assessment of autophagy flux that have been detailed (Nixon et al., 2012). By contrast, the authors did not cite at least a dozen subsequent publications reporting lysosomal acidification deficits in PSEN1 models from at least eight different investigative groups besides ours, using conventional AD models. These have included FAD-PSEN1 patient fibroblasts (Coffey et al., 2014; Lee et al., 2020; Martin-Maestro et al., 2017) and human FAD- PSEN1 induced neurons (Chou et al., 2023), human FAD- PSEN1 IPSCs (Mustaly-Kalimi et al., 2022; Yang et al., 2019; Martin-Maestro et al., 2017), FAD-PSEN1 knock-in mice (Lie et al., 2022), as well as single PSEN 1 loss of function cell models (Sharma et al., 2019) and PS1/APP and APP mouse models of AD (Mustaly-Kalimi et al., 2022; Avrahami et al., 2013; Jiang et al., 2019; Lee et al., 2022). Additional reports have documented that direct targeting of lysosomes with acidic nanoparticles or pharmacological agents that restore acidification also reverse autophagic-lysosomal pathway pathology (Coffey et al., 2014; Lee et al., 2020; Lie et al., 2022; Avrahami et al., 2013).

    The authors acknowledge that their conclusions “are limited to PSEN deficiency and need to be extended to FAD-associated mutations in PSEN1 to extrapolate these observations to endolysosomal abnormalities as observed in the AD brain. Herein, follow-up studies in physiologically more relevant AD models, including iPSC-derived human neurons, AD mouse models, and human samples, are required to generalize our main outcomes.” As mentioned above, numerous studies of PSEN1 and APP-βCTF actions on the endolysosomal network in relevant AD models have already been published by many investigators and should be considered in relation to findings in this report and future studies. 

    References:

    Im E, Jiang Y, Stavrides PH, Darji S, Erdjument-Bromage H, Neubert TA, Choi JY, Wegiel J, Lee JH, Nixon RA. 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.

    Coen K, Flannagan RS, Baron S, Carraro-Lacroix LR, Wang D, Vermeire W, Michiels C, Munck S, Baert V, Sugita S, Wuytack F, Hiesinger PR, Grinstein S, Annaert W. Lysosomal calcium homeostasis defects, not proton pump defects, cause endo-lysosomal dysfunction in PSEN-deficient cells. J Cell Biol. 2012 Jul 9;198(1):23-35. PubMed.

    Lee JH, McBrayer MK, Wolfe DM, Haslett LJ, Kumar A, Sato Y, Lie PP, Mohan P, Coffey EE, Kompella U, Mitchell CH, Lloyd-Evans E, Nixon RA. 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.

    Tong BC, Wu AJ, Huang AS, Dong R, Malampati S, Iyaswamy A, Krishnamoorthi S, Sreenivasmurthy SG, Zhu Z, Su C, Liu J, Song J, Lu JH, Tan J, Pan W, Li M, Cheung KH. Lysosomal TPCN (two pore segment channel) inhibition ameliorates beta-amyloid pathology and mitigates memory impairment in Alzheimer disease. Autophagy. 2021 Jul 27;:1-19. PubMed.

    Zhang X, Garbett K, Veeraraghavalu K, Wilburn B, Gilmore R, Mirnics K, Sisodia SS. A role for presenilins in autophagy revisited: normal acidification of lysosomes in cells lacking PSEN1 and PSEN2. J Neurosci. 2012 Jun 20;32(25):8633-48. PubMed.

    Nixon RA, Lee JH, Wolfe D. Comments on Presenilins and Lysosome pH Revisited Again. Journal of Neuroscience, Dec 5, 2012 Comments on Presenilins and Lysosome pH Revisited Again

    Coffey EE, Beckel JM, Laties AM, Mitchell CH. Lysosomal alkalization and dysfunction in human fibroblasts with the Alzheimer's disease-linked presenilin 1 A246E mutation can be reversed with cAMP. Neuroscience. 2014 Mar 28;263:111-24. Epub 2014 Jan 10 PubMed.

    Lee JH, Wolfe DM, Darji S, McBrayer MK, Colacurcio DJ, Kumar A, Stavrides P, Mohan PS, Nixon RA. 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.

    Chou CC, Vest R, Prado MA, Wilson-Grady J, Paulo JA, Shibuya Y, Moran-Losada P, Lee TT, Luo J, Gygi SP, Kelly JW, Finley D, Wernig M, Wyss-Coray T, Frydman J. Proteostasis and lysosomal quality control deficits in Alzheimer's disease neurons. bioRxiv. 2023 Mar 27; PubMed.

    Mustaly-Kalimi S, Gallegos W, Marr RA, Gilman-Sachs A, Peterson DA, Sekler I, Stutzmann GE. Protein mishandling and impaired lysosomal proteolysis generated through calcium dysregulation in Alzheimer's disease. Proc Natl Acad Sci U S A. 2022 Dec 6;119(49):e2211999119. Epub 2022 Nov 28 PubMed.

    Yang M, Wang Y, Liang G, Xu Z, Chu CT, Wei H. Alzheimer's Disease Presenilin-1 Mutation Sensitizes Neurons to Impaired Autophagy Flux and Propofol Neurotoxicity: Role of Calcium Dysregulation. J Alzheimers Dis. 2019;67(1):137-147. PubMed.

    Martín-Maestro P, Gargini R, A Sproul A, García E, Antón LC, Noggle S, Arancio O, Avila J, García-Escudero V. Mitophagy Failure in Fibroblasts and iPSC-Derived Neurons of Alzheimer's Disease-Associated Presenilin 1 Mutation. Front Mol Neurosci. 2017;10:291. Epub 2017 Sep 14 PubMed.

    Lie PP, Yoo L, Goulbourne CN, Berg MJ, Stavrides P, Huo C, Lee JH, Nixon RA. Axonal transport of late endosomes and amphisomes is selectively modulated by local Ca2+ efflux and disrupted by PSEN1 loss of function. Sci Adv. 2022 Apr 29;8(17):eabj5716. PubMed.

    Sharma D, Otto G, Warren EC, Beesley P, King JS, Williams RS. Gamma secretase orthologs are required for lysosomal activity and autophagic degradation in Dictyostelium discoideum, independent of PSEN (presenilin) proteolytic function. Autophagy. 2019 Aug;15(8):1407-1418. Epub 2019 Mar 21 PubMed.

    Avrahami L, Farfara D, Shaham-Kol M, Vassar R, Frenkel D, Eldar-Finkelman H. Inhibition of Glycogen Synthase Kinase-3 Ameliorates β-Amyloid Pathology and Restores Lysosomal Acidification and Mammalian Target of Rapamycin Activity in the Alzheimer Disease Mouse Model: IN VIVO AND IN VITRO STUDIES. J Biol Chem. 2013 Jan 11;288(2):1295-306. PubMed.

    Jiang Y, Sato Y, Im E, Berg M, Bordi M, Darji S, Kumar A, Mohan PS, Bandyopadhyay U, Diaz A, Cuervo AM, Nixon RA. 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.

    Lee JH, Yang DS, Goulbourne CN, Im E, Stavrides P, Pensalfini A, Chan H, Bouchet-Marquis C, Bleiwas C, Berg MJ, Huo C, Peddy J, Pawlik M, Levy E, Rao M, Staufenbiel M, Nixon RA. 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.

    Wolfe DM, Lee JH, Kumar A, Lee S, Orenstein SJ, Nixon RA. Autophagy failure in Alzheimer's disease and the role of defective lysosomal acidification. Eur J Neurosci. 2013 Jun;37(12):1949-61. PubMed.

    View all comments by Ralph Nixon View all comments by Ju-Hyun Lee

  6. In this elegant study by Bretou et al., new mechanistic evidence is provided for APP-mediated endolysosomal dysfunction in AD. Altered lysosomal Ca2+ levels due to APP-CTF accumulation at the lysosome side of lysosome-ER contact sites appear to be the driver of endolysosomal collapse in this model, showing, surprisingly, that lysosome dysfunction may be first in the chronology of events that affect the endolysosomal network in AD. Interestingly, they also show that a membrane anchored AICD induces this endolysosomal collapse. Together, this work suggests that precise regulation of APP-CTF cleavage by γ-secretase is needed to promote a healthy endolysosomal network.

    An interesting further line of investigation might be to explore transcriptomic and epigenomic effects that would be caused by altered localization of APP fragments. AICD can regulate gene expression through a complex with FE65 and TIP60. Since TIP60 (or KAT5) is a histone acetyltransferase, altered localization of APP fragments due to reduced γ-secretase activity may directly affect gene-expression pathways that either contribute to, or are activated by, endolysosomal dysfunction.

    View all comments by Jessica Young

  7. I agree w Dr. Young that this is an elegant study, and with other colleagues' qualifiers related to limitations of this thought-provoking new insight.

    However, I would like to stress one issue that might bear wider relevance: Endolysosomal toxicity by APP-CTF could occur in combination with mitochondrial exposure to APP-CTF, which is mito-toxic, in keeping with the schematic that has the mitochondrion on the pathologic side being energy-impaired.

    Mitochondrial defects were demonstrated in a recent investigation that combined mouse models and human pathology (Vaillant‐Beuchot et al., 2021). In particular, Fig. 10 details the human autopsy data supporting the main conclusion, namely, accumulation of amyloid precursor protein C‐terminal fragments triggers mitochondrial structure, function, and mitophagy defects in Alzheimer’s disease models and human brains.

    Thus, APP-CTF could be a co-driver of disease pathogenesis, yet it is not affected by amyloid immunotherapy, i.e., aggregated Aβ sequestrating antibodies such as lecanemab and donanemab.

    So, could this new paper, together with previous evidence (Vaillant‐Beuchot et al., 2021), offer a simple answer as to why antibodies that effectively purge affected brains are only moderately effective? Because one of the "big guns," the CTFs, remains on the battlefield?

    Could it be that Aβ antibodies increase intracellular of APP-CTF, thus limiting an otherwise larger effect size?

    References:

    Vaillant-Beuchot L, Mary A, Pardossi-Piquard R, Bourgeois A, Lauritzen I, Eysert F, Kinoshita PF, Cazareth J, Badot C, Fragaki K, Bussiere R, Martin C, Mary R, Bauer C, Pagnotta S, Paquis-Flucklinger V, Buée-Scherrer V, Buée L, Lacas-Gervais S, Checler F, Chami M. Accumulation of amyloid precursor protein C-terminal fragments triggers mitochondrial structure, function, and mitophagy defects in Alzheimer's disease models and human brains. Acta Neuropathol. 2021 Jan;141(1):39-65. Epub 2020 Oct 20 PubMed.

    View all comments by Wolfgang Liedtke
    • User Profile Image Marçal Vilar
      Institute of Biomedicine of Valencia-CSIC
    • Posted:

    In this very interesting paper Bretou et al. described the toxic effect produced by the genetic and pharmacologic inhibition of the γ-secretase complex. The authors described that these treatments increased the levels of APP-CTF inducing a dysfunction of the endolysosomal system leading to aberrant cholesterol loading and cell toxicity. The authors suggested that the main culprit is the presence of APP-CTF, however, it is already known that the γ-secretase complex has several membrane substrates and the impact of its inhibition could be not only restricted to the APP world. One of these substrates is the p75 neurotrophin receptor, p75NTR.

    Bretou et al. use mouse embryonic fibroblasts (MEFs) that do not express p75 endogenously and the expression of p75NTR in hippocampal neurons is lower than in other CNS neurons, such as basal forebrain cholinergic neurons. We recently showed that acute inhibition of the γ -secretase complex induced an increase of the levels of p75-CTF, leading to an increase of JNK and p38 SAPK, leading to cell toxicity in PC12 cell lines that endogenously express p75 and in primary cultures of mouse cholinergic neurons, where this receptor is highly expressed for the whole life of these cells. Interestingly, the toxic effect is significantly reduced in the case of neurons from p75NTR-KO mice (Franco et al., 2021). Furthermore, overexpression of p75-CTF induced the formation of aberrant large intracellular endosomes, although the endolysosomal system was not studied. In addition, the aberrant expression in mice of a short isoform of p75, similar to p75-CTF, induced a decrease of cholinergic neuron number during aging and an increase in the transcriptional levels of some cholesterol biosynthesis genes, such as HMGRC (Comaposada et al., 2023). This effect is mimicked by overexpression of p75-CTF in PC12 cells, suggesting that the accumulation of p75-CTFs at the lipid membrane could impinge on the cholesterol biosynthetic machinery. Interestingly, it was recently described that γ-secretase inhibition leads to cholesterol biosynthesis disruption and synapse dysfunction (Essayan-Perez and Südhof, 2023).  Finally, following our collaboration with the laboratory of Dr. Lucía Chavez-Gutierrez (Zoltowska et al., 2023) we described that the feedback inhibition of the γ-secretase complex by human amyloid Aβ1-42 induced an increase in levels of p75-CTF (and other CTFs, including APP-CTF) leading to cholinergic neuron death in conditions of low pro-survival activity, as is the case with restricted NGF/TrkA signaling pathway activation--a condition found in older AD patients).

    While Bretou et al. described that the Ca2+ signaling alterations are normalized in APP KO cells, other effects caused by the accumulation in other cell types of protein substrates, such as p75-CTF, might contribute to the neurotoxicity in AD. Our working hypothesis is that p75-CTF, as well as APP-CTF, contributes to the worsening of the cognition of the AD patients who had been treated with GSIs, taking into account the role of the cholinergic system in memory and learning and the high levels of p75NTR in these neurons.

    References:

    Essayan-Perez S, Südhof TC. Neuronal γ-secretase regulates lipid metabolism, linking cholesterol to synaptic dysfunction in Alzheimer's disease. Neuron. 2023 Oct 18;111(20):3176-3194.e7. Epub 2023 Aug 4 PubMed.

    Franco ML, García-Carpio I, Comaposada-Baró R, Escribano-Saiz JJ, Chávez-Gutiérrez L, Vilar M. TrkA-mediated endocytosis of p75-CTF prevents cholinergic neuron death upon γ-secretase inhibition. Life Sci Alliance. 2021 Apr;4(4) Print 2021 Apr PubMed.

    Vilar M. Neurotrophin receptοrs, gamma-secretase inhibitors, and neurodegeneration of basal forebrain cholinergic neurons. Neural Regen Res. 2022 Jul;17(7):1493-1494. PubMed.

    Comaposada-Baró R, Benito-Martínez A, Escribano-Saiz JJ, Franco ML, Ceccarelli L, Calatayud-Baselga I, Mira H, Vilar M. Cholinergic neurodegeneration and cholesterol metabolism dysregulation by constitutive p75NTR signaling in the p75exonIII-KO mice. Front Mol Neurosci. 2023;16:1237458. Epub 2023 Oct 13 PubMed.

    Zoltowska KM, Das U, Lismont S, Enzlein T, Maesako M, Houser MC, Franco ML, Moreira DG, Karachentsev D, Becker A, Hopf C, Vilar M, Berezovska O, Mobley W, Chávez-Gutiérrez L. Alzheimer's disease linked Aβ42 exerts product feedback inhibition on γ-secretase impairing downstream cell signaling. bioRxiv. 2023 Oct 28; PubMed.

    View all comments by Marçal Vilar

  9. First and foremost, we would like to thank our colleagues for their comments. We appreciate the new insights and the interesting discussion arising from them.

    We would also like to reply more particularly to Drs. Lee and Nixon. They argue that the model we used is not relevant in the context of AD, and essentially focus on the aspect of lysosomal pH. Rather than being primarily relevant for AD, the PSENdKO (+/- APPKO) models we engineered are a tool to dissect basic mechanisms, including the cytopathological role of excess APP-derived C-terminal fragments on an isogenic background. In so doing so, and contrary to studies carried out in FAD fibroblasts or iPSCs cited by the authors (which in many cases lack isogenic controls), we only identified a small pH defect in PSENdKO clones (around 0.2 to 0.3 pH units). The pH we monitored was still compatible with optimal lysosomal enzyme activity (Butor et al., 1995; Pillay et al., 2002).

    On the other hand, we systematically observe a large deficit in lysosomal Ca2+ in all independent clones. This is fully rescued by reintroducing hPSEN1 or by knocking out APP. In our scatter plot (Fig. S2B), we show that a similar Ca2+ deficit can only be induced by fully inhibiting the proton pump using Bafilomycin, i.e., a greater than1,000-fold lowered acidification. The rather mild pH effect, i.e., a four- to fivefold lowered acidification, observed in our PSENdKO cells is thus clearly not correlated with the large Ca2+ deficit, arguing against pH defects being upstream of Ca2+ defects in these cells. To note, our study does not rule out any pH contribution, for instance in an AD context; this was not investigated here.

    In this regard, Drs. Lee and Nixon refer to several studies that indeed showed lysosomal alkalinization in an FAD context, or in trisomy 21. In such models, organelle defects may more likely be linked to aberrant production of toxic Aβ and to excess APP-CTFs, as demonstrated in their recent manuscript (Im et al., 2023), rather than directly to PSEN1 function. Hence, these models do not allow us to address whether pH defects are upstream of Ca2+ defects, or vice versa, and so cannot be used as an argument in this discussion. Obviously, restoring pH with acidic nanoparticles can alleviate defects, but restoring lysosomal Ca2+ can do so as well.

    Further, our CRISPR-engineered PSENdKO clones display no defects in the N-glycosylation of the a1 subunit, nor in v-ATPase assembly, contrary to observations and hypotheses raised by the Nixon group.

    In addition, the use of a novel compound that only recognizes active v-ATPases (Fig. S2H) indicates the presence of active proton pumps on late endosomes/lysosomes in PSENdKO cells. These data, included in the supplementary material of our manuscript, argue against a direct role for (full-length) PSEN1 alongside the oligosaccharyltransferase complex in mediating the N-glycosylation of the a1 subunit in the ER (Lee et al., 2010), and they motivate our choice to use lysosomal Ca2+ as a functional readout rather than pH.

    To note, we found no significant pH defects in the case of late-onset Alzheimer's disease-linked PLD3 deficiency, either, whether through knockout models or single nucleotide polymorphisms (Van Acker et al., 2023). SH-SY5Y cells deficient in PLD3 even exhibited a substantial increase in the number of acidified autolysosomes (Fig. 6), further supporting that pH defects are a secondary event in organellar dysfunction.

    Our paper focuses on explaining where, and how, excess APP-CTFs exert their toxic effect, questions that had gone unanswered for too long. In agreement with the commenters themselves, as well as others (Kwart et al., 2019; Hung and Livesey, 2018; Im et al., 2023), we confirmed the pathogenic role of APP-CTFs. Whether the pathological defects rely on direct modulation of the v-ATPase (which we do not observe to be defective in PSENdKO cells), or on transcriptomic and epigenomic effects, which—as suggested by Dr. Young—might differ within models, is therefore open to debate. Modulating the latter may nonetheless help restore some of the observed endolysosomal defects, similarly to what has been described by Drs. Prasad and Rao, making the link between epigenetics and endosomal pH.

    With this in mind, we would like to highlight the major novelties of our study: (i) we localized APP-CTFs in late endosomes/lysosome-ER contact sites, where their processing is required to keep inter-organellar communication in check; and (ii) we demonstrated that APP-CTF processing by γ-secretase is required to abrogate their signaling rather than to initiate a downstream cascade.

    Therefore, in lieu of the discrepancies, we would like to stress similarities between the different studies. They all underline the central role of APP-CTFs and the importance of an efficient organellar communication between LE/Lys and the ER, together bridging several parallel hypotheses in the field. The models published here give us the tools to further explore how APP-CTF-associated signaling modulates contact site dynamics, and how, exactly, this is regulated by intramembrane proteolysis, including in major brain cell types.

    References:

    Butor C, Griffiths G, Aronson NN Jr, Varki A. Co-localization of hydrolytic enzymes with widely disparate pH optima: implications for the regulation of lysosomal pH. J Cell Sci. 1995 Jun;108 ( Pt 6):2213-9. PubMed.

    Pillay CS, Elliott E, Dennison C. Endolysosomal proteolysis and its regulation. Biochem J. 2002 May 1;363(Pt 3):417-29. PubMed.

    Im E, Jiang Y, Stavrides PH, Darji S, Erdjument-Bromage H, Neubert TA, Choi JY, Wegiel J, Lee JH, Nixon RA. 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.

    Lee JH, Yu WH, Kumar A, Lee S, Mohan PS, Peterhoff CM, Wolfe DM, Martinez-Vicente M, Massey AC, Sovak G, Uchiyama Y, Westaway D, Cuervo AM, Nixon RA. Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations. Cell. 2010 Jun 25;141(7):1146-58. PubMed.

    Van Acker ZP, Perdok A, Hellemans R, North K, Vorsters I, Cappel C, Dehairs J, Swinnen JV, Sannerud R, Bretou M, Damme M, Annaert W. Phospholipase D3 degrades mitochondrial DNA to regulate nucleotide signaling and APP metabolism. Nat Commun. 2023 May 24;14(1):2847. PubMed.

    Kwart D, Gregg A, Scheckel C, Murphy EA, Paquet D, Duffield M, Fak J, Olsen O, Darnell RB, Tessier-Lavigne M. A Large Panel of Isogenic APP and PSEN1 Mutant Human iPSC Neurons Reveals Shared Endosomal Abnormalities Mediated by APP β-CTFs, Not Aβ. Neuron. 2019 Oct 23;104(2):256-270.e5. Epub 2019 Aug 12 PubMed.

    Hung CO, Livesey FJ. Altered γ-Secretase Processing of APP Disrupts Lysosome and Autophagosome Function in Monogenic Alzheimer's Disease. Cell Rep. 2018 Dec 26;25(13):3647-3660.e2. PubMed.

    View all comments by Wim Annaert

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References

News Citations

  1. Behold PANTHOS, a Toxic Wreath of Perinuclear Aβ That Kills Neurons
  2. Familial AD Mutations, β-CTF, Spell Trouble for Endosomes
  3. Too Basic: APP β-CTF's YENTPY Motif Binds Proton Pump, Thwarts Lysosomes
  4. Presenilins and Calcium: A Lysosomal Stew With Acid Controversy
  5. Give BACE Inhibitors a Second Chance?
  6. Can BACE Inhibitors Stage a Comeback?

Paper Citations

  1. Chou CC, Vest R, Prado MA, Wilson-Grady J, Paulo JA, Shibuya Y, Moran-Losada P, Lee TT, Luo J, Gygi SP, Kelly JW, Finley D, Wernig M, Wyss-Coray T, Frydman J. Proteostasis and lysosomal quality control deficits in Alzheimer's disease neurons. bioRxiv. 2023 Mar 27; PubMed.
  2. Martín-Maestro P, Gargini R, A Sproul A, García E, Antón LC, Noggle S, Arancio O, Avila J, García-Escudero V. Mitophagy Failure in Fibroblasts and iPSC-Derived Neurons of Alzheimer's Disease-Associated Presenilin 1 Mutation. Front Mol Neurosci. 2017;10:291. Epub 2017 Sep 14 PubMed.
  3. Burgoyne T, Patel S, Eden ER. Calcium signaling at ER membrane contact sites. Biochim Biophys Acta. 2015 Sep;1853(9):2012-7. Epub 2015 Feb 4 PubMed.
  4. Doody RS, Raman R, Farlow M, Iwatsubo T, Vellas B, Joffe S, Kieburtz K, He F, Sun X, Thomas RG, Aisen PS, , Siemers E, Sethuraman G, Mohs R. A phase 3 trial of semagacestat for treatment of Alzheimer's disease. N Engl J Med. 2013 Jul 25;369(4):341-50. PubMed.

Further Reading

Primary Papers

  1. Bretou M, Sannerud R, Escamilla-Ayala A, Leroy T, Vrancx C, Van Acker ZP, Perdok A, Vermeire W, Vorsters I, Van Keymolen S, Maxson M, Pavie B, Wierda K, Eskelinen EL, Annaert W. Accumulation of APP C-terminal fragments causes endolysosomal dysfunction through the dysregulation of late endosome to lysosome-ER contact sites. Dev Cell. 2024 Jun 17;59(12):1571-1592.e9. Epub 2024 Apr 15 PubMed.

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