Too Basic: APP β-CTF's YENTPY Motif Binds Proton Pump, Thwarts Lysosomes

The rap sheet for β-CTF is growing longer. The C-terminal fragment, produced when β-secretase cleaves APP, binds to—and blocks assembly of—vacuolar ATPase, a proton pump that fuels acidification of lysosomes. This is according to a paper published today in Science Advances. This only happens when β-CTF is phosphorylated at tyrosine-682 of APP's YENPTY internalization motif, suggesting the interaction is highly specific and part of APP’s physiological function.

  • In fibroblasts from people with DS, the pH of lysosomes rises.
  • A specific phosphorylation in APP’s YENTPY C terminal internalization motif interacts directly with v-ATPase.
  • Blocking β-CTF production or phosphorylation restores lysosome acidification.
  • Human DS, AD brain, and mouse models show evidence of same pathway.

In situations where β-CTF was in excess, such as in cells from people with Down's syndrome and mouse models of DS or Alzheimer's disease, lysosomes became less acidic and lost their ability to process their many substrates. Dampening production or phosphorylation of β-CTF restored lysosomes to their full refuse-disposing prowess. Building on previous studies, the new data argue that excess β-CTF drives endolysosomal breakdown early in AD pathogenesis. The study was led by Ralph Nixon of New York University in Orangeburg.

“By elucidating this mechanism, this paper readily identifies new therapeutic targets: phosphorylation sites on APP β-CTF and molecules to improve lysosomal acidification,” wrote Jessica Young of the University of Washington in Seattle. To Nixon’s mind, the findings also support using low-dose BACE inhibitors to diminish production of β-CTF.

Stefan Lichtenthaler of the German Center for Neurodegenerative Diseases in Munich agreed. “This work has a major translational implication for the use of BACE inhibitors,” he wrote, noting that in addition to reducing Aβ and boosting the production of the neuroprotective sAPPa fragment, BACE inhibitors come with the third benefit of lowering β-CTF (full comment below).

The study dovetails with other recent work implicating the YENTPY motif in APP function. Researchers led by Subhojit Roy of the University of California, San Diego, are taking aim at the motif by way of CRISPR-based gene therapy, presenting preclinical work at the Alzheimer’s Association International Conference, held July 16-20 in Amsterdam. Carmela Matrone at Aarhus University, Denmark, who previously reported phosphorylation of the APP tyrosine-682 in blood cells of AD patients, noted that it would be interesting to look for interactions between v-ATPase and the APP YENPTY motif in blood mononuclear cells from people with AD and DS (full comment below).

A backed-up endolysosomal system—as evidenced by bloated endosomes, lysosomes choked with unprocessed substrates, and a slowdown in autophagy—arises early in AD (Cataldo et al., 2000). AD risk genes found in GWAS, as well as all four genes tied to familial AD, have the lion’s share of their impact within these intracellular compartments, casting the vesicular system as ground zero in the AD cascade.

Nothing is more fundamental to proper lysosomal function than acidification, Nixon argues, and v-ATPase is the multimeric enzyme for that. This proton pump's 14 subunits fall into two major sectors: the membrane-associated “V0” sector forms the proton channel, while the cytosolic "V1" sector fuels the pump via ATP hydrolysis. For the pump to work, the two must come together.

How might this go awry in AD? Previously, Nixon's group reported that familial AD mutations in PS1 disrupt delivery of v-ATPase's V0a1 subunit to lysosomes, thwarting their acidification (Jun 2010 news on Lee et al., 2010). Later, the scientists linked lysosomal dysfunction seen in cells from people with Down’s to APP’s β-CTF fragment (Jiang et al., 2019).

In today’s paper, first author Eunju Im and colleagues investigated how that happens. They studied fibroblasts from people with DS, who develop early onset AD due to their extra copy of APP. Compared to control fibroblasts, DS fibroblasts had lysosomes with a higher pH, and their pH-dependent proteases no longer worked. The scientists tied this high pH to flagging v-ATPase function. In purified lysosomes, they found a dearth of cytosolic V0 associated with V1, suggesting that the two key components of v-ATPase hadn’t assembled. Knocking down APP expression in the DS cells to normal levels restored this assembly, and v-ATPase worked again.

Suspecting that the β-CTF arising from the extra APP was meddling with putting together v-ATPase, the scientists treated the cells with a BACE inhibitor to squelch production of this APP fragment. This restored v-ATPase assembly and lysosomal acidification in the DS cells, and even improved lysosomal function in control cells.

β-CTF Disables v-ATPase. Untreated Down’s cells (left) have low levels of V1 (red) on their lysosomes (green). γ-Secretase inhibition (middle) has no effect, while β-secretase inhibition (right) restores V1. [Courtesy of Im et al., Science Advances, 2023.]

The opposite happened when the scientists treated the cells with a γ-secretase inhibitor, which leads to a buildup of β-CTF by blocking its subsequent processing into Aβ peptides. In control cells, the γ-secretase inhibitor nixed v-ATPase function and ramped up lysosomal pH, whereas in DS cells, which already had these problems, the γ-secretase inhibitor had no further effect. Notably, neither Aβ, nor α-CTF, the product of α-secretase cleavage of APP, influenced v-ATPase activity. The findings suggested that β-CTF imparted a specific, tonic effect on v-ATPase activity.

How could β-CTF be putting the kibosh on the pump? The researchers found that β-CTF bound to the enzyme's V0a1 subunit, preventing its association with the enzyme's V1 component. Specifically, phosphorylation of β-CTF at tyrosine-682, the first residue of the YENTPY membrane internalization sequence of APP, was required for this inhibitory binding. Preventing this phosphorylation with the Fyn kinase inhibitor saracatinib restored v-ATPase function, and lowered lysosomal pH in DS cells.

Beta Block. The YENPTY motif of β-CTF binds to the V0a1 subunit of v-ATPase on the cytoplasmic side of the lysosomal membrane (bottom left). The phosphorylation stabilizes the interaction within the V0a1 binding pocket (right), preventing V1 docking and derailing assembly of the v-ATPase. [Courtesy of Im et al., Science Advances, 2023.]

To see if these molecular liaisons played out in neurons in the brain, the researchers crossed Ts2 mice, which express three copies of APP, with TRGL mice, a transgenic line that facilitates visualization of lysosomal and autophagosomal acidification. In the brains of these crosses, they saw a glut of poorly acidified lysosomes and autophagosomes, and a dearth of fully assembled v-ATPase within these vesicles. Treating the mice with the kinase inhibitor saracatinib corrected these deficits. Saracatinib has been studied in a repurposing trial in AD.

These same two abnormalities were seen in 5xFAD and Tg2576 mouse models of AD, both of which also harbored an excess of Tyr-682-phosphorylated β-CTF. Importantly, the researchers also found disrupted v-ATPase assembly, and elevated levels of β-CTF, in membrane fractions from postmortem brain samples of people with DS.

Acid Comeback. Autolysosomes are insufficiently acidified in neurons of untreated Ts2xTRGL Down's mouse models (top) (deep purple). Treatment with saracatinib to inhibit -CTF phosphorylation (bottom) restored acidification (bright magenta). [Courtesy of Im et al., Science Advances, 2023.]

The study supports the idea that lysosomal dysfunction, particularly through interference with v-ATPase, is an upstream pathogenic cellular mechanism in neurological conditions such as AD and DS, commented Grace Stutzmann of Rosalind Franklin University in Chicago. She added that the possible role of APP as a “tonic” regulator of lysosomal function could have far broader implications, as several neurological conditions, including AD and traumatic brain injury, result in elevated APP production.

Previously, researchers led by UCSD's Roy reported the use of a CRISPR/Cas9 gene-editing approach that targets the YENTPY motif, limiting APP's internalization from the cell surface and thus preventing its cleavage by BACE1 (Sun et al., 2019). At AAIC, Brent Aulstun from Roy’s lab presented preclinical findings on the strategy. He reported that in APPNL-G-F knock-in mice, editing APP relieved lysosomal dysfunction, reduced Aβ plaques, calmed neuroinflammation, and improved memory.

Nixon believes that YENPTY phosphorylation may also facilitate β-CTF’s sway over an earlier part of the vesicular pathway. Previously, he reported that the motif fuels β-CTF’s interaction with the PTB domain of APPL1, which leads to the activation of Rab5, revving endocytosis (Jul 2015 news). Although he has yet to formally test whether tyr-682 phosphorylation enhances the β-CTF/APPL1 partnership, other studies have shown that this phosphorylation enhances APP binding to the PTB domains of other proteins (Tarr et al., 2002; Tamayev et al., 2009).

Nixon considers β-CTF a key regulator of the endolysosomal system. He believes that when phosphorylated β-CTF accumulates, dysfunction ensues, noting that BACE is known to be highly active in familial and sporadic forms AD. He sees the resulting uptick in β-CTF as a tipping point toward the endolysosomal meltdown that drives the disease. This cascade also triggers accumulation of Aβ, which might in some cases stem from faulty lysosomal acidification (Jun 2022 news).—Jessica Shugart

Comments

  1. This thorough study pinpoints a mechanism describing lysosomal dysfunction in AD. In both DS and FAD mouse models, the Nixon group show that a phosphorylated form of β-CTF of APP directly interacts with and inhibits a v-ATPase subunit that is involved in proper lysosomal acidification.

    Lysosomal dysfunction is an early and universal hallmark of AD. By elucidating this mechanism, this paper readily identifies new therapeutic targets: phosphorylation sites on APP β -CTF and molecules to improve lysosomal acidification. It will be interesting for future studies to determine the extent of this dysfunction in the various cell types of the CNS.

    View all comments by Jessica Young

  2. This study is elegant and intriguing. It supports a crucial role of increased phosphorylation levels of the APP Tyr682 residue in the neurons of patients with AD and DS. The study provides a mechanism through which this increased phosphorylation leads to dysfunctional and defective lysosomes by preventing correct lysosomal v-ATPase assembly, compromising its activity.

    Furthermore, it suggests an interaction between lysosomal v-ATPase and the APP/YENPTY motif via the Tyr682 residue, implying that targeting v-ATPase, specifically its subunit V0a1, may have clinical implications, particularly in individuals with elevated phosphorylation levels of the Try682 residue.

    Further research is necessary to explore how changes in APP Tyr682 phosphorylation affect its adaptor interactions in patients with AD throughout various stages of the disease in order to predict clinical outcomes and progression.

    It would be interesting to ascertain whether the same interaction between v-ATPAse and the APP/YENPTY motif also occurs in blood mononuclear cells from patients. This is relevant because we have previously reported that APP Tyr682 phosphorylation levels are increased in the blood mononuclear cells of patients with AD (Ferretti et al., 2023), and suggested that the Tyr682 phosphorylation might have diagnostic and therapeutic implications, using inhibitors of Fyn tyrosine kinase (Iannuzzi et al., 2020, 2021). Translation of these findings to peripheral blood cells could pave the way for new diagnostic strategies and allow the design of personalized therapeutic approaches for patients with increased APP Tyr682 phosphorylation.

    References:

    Ferretti G, Serafini S, Angiolillo A, Monterosso P, Di Costanzo A, Matrone C. Advances in peripheral blood biomarkers of patients with Alzheimer's disease: Moving closer to personalized therapies. Biomed Pharmacother. 2023 Sep;165:115094. Epub 2023 Jun 29 PubMed.

    Iannuzzi F, Sirabella R, Canu N, Maier TJ, Annunziato L, Matrone C. Fyn Tyrosine Kinase Elicits Amyloid Precursor Protein Tyr682 Phosphorylation in Neurons from Alzheimer's Disease Patients. Cells. 2020 Jul 30;9(8) PubMed. Correction.

    Iannuzzi F, Frisardi V, Annunziato L, Matrone C. Might Fibroblasts from Patients with Alzheimer's Disease Reflect the Brain Pathology? A Focus on the Increased Phosphorylation of Amyloid Precursor Protein Tyr682 Residue. Brain Sci. 2021 Jan 14;11(1) PubMed. Correction.

    View all comments by Carmela Matrone
    • User Profile Image Grace Stutzmann
      Rosalind Franklin University/The Chicago Medical School
    • Posted:

    This study expands the extent to which lysosome dysfunction—particularly through interference of the vATPase proton pump—is implicated as an upstream pathogenic, cellular mechanism in neurological conditions such as AD and in DS, which share many proteinopathy features.

    The role of specific APP fragments in driving lysosome dysfunction is particularly notable in light of Randy Nixon's previous work demonstrating the role of mutant PS1 similarly interfering with proton pump assembly and lysosome functions. Thus, it appears that these two protein products of fAD-causing genes share a common pathological endpoint resulting in aberrant protein and cellular debris accumulation. 

    The possible contribution of APP to tonic lysosome regulation is also a novel finding with far broader implications, as several neurological conditions, such as brain injury, result in increased APP generation. Notably, TBI and AD also share many histopathological phenotypes. Revealing the central role of lysosome dysfunction across many brain conditions will be of great interest and clinical relevance. 

    View all comments by Grace Stutzmann

  4. This is an exciting study implicating the BACE1-generated APP C-terminal fragment as a key player in lysosomal dysfunction in Down’s syndrome fibroblasts and animal models. This work has a major translational implication for the use of BACE inhibitors. These inhibitors not only have two beneficial effects, namely reduced Aβ generation and increased production of the neuroprotective sAPPα, but also a third beneficial effect, namely lowered abundance of the BACE1-generated APP C-terminal fragment.

    Thus, BACE inhibitors may correct lysosomal dysfunctions, both in AD and in Down’s syndrome. Yet, at high doses, BACE inhibitors also have an undesired side effect and induce a mild cognitive worsening in patients. A dose reduction to clearly less than 50 percent BACE inhibition is assumed to prevent the cognitive worsening and is considered for forthcoming clinical trials. Future work needs to determine how strongly such a reduced dose of a BACE inhibitor may correct lysosomal dysfunctions in vivo.

    View all comments by Stefan Lichtenthaler

  5. This work is thrilling and interesting as the authors continue to report deficits of the endo-lysosomal system in aging and neurodegeneration. In both Down's and AD brains, the phosphorylated APP-β-CTF fragment selectively binds to the V0a1 subunit of the v-ATPase and inhibits lysosomal acidification and subsequent degradative activity, which eventually leads to abnormal protein accumulation and neuronal death. This paper and their previously published work (Lee et al., 2022), show a direct link of maladaptive APP fragments to lysosomal deficits, furthering the hypothesis that disrupted intracellular organelle function is an early mechanism for Alzheimer’s pathogenesis.

    Early calcium dysregulation can also feed into this process by increasing BACE1 activity and altering phosphotase/kinase activity, both pathways that encourage phosphorylated-APP-BCTF production (Stutzmann, 2007; Chami and Checler, 2012). Additionally, calcium dysregulation via ryanodine receptors was shown to disrupt lysosomal acidfication and lead to increased hyperphosphoryated tau accumulation in autolysosomes (Mustaly-Kalimi et al., 2022). 

    Hyperphosphorylated protein targets, hence, are being sequestered into these compartments and disrupting lysosomal degradative activity; therefore targeting mechanisms that reduce maladaptive protein accumulation and increase lysosomal acidification can be an early, beneficial therapeutic for Alzheimer’s disease. This study, and others, have shown that reducing APP levels, reacidfication of lysosomes, and decreasing calcium dysregulation can improve lysosomal function in AD (Lee et al., 2015; Mustaly-Kalimi et al., 2022). 

    References:

    Chami L, Checler F. BACE1 is at the crossroad of a toxic vicious cycle involving cellular stress and β-amyloid production in Alzheimer's disease. Mol Neurodegener. 2012;7:52. 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.

    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.

    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.

    Stutzmann GE. The pathogenesis of Alzheimers disease is it a lifelong "calciumopathy"?. Neuroscientist. 2007 Oct;13(5):546-59. PubMed.

    View all comments by Sarah Mustaly

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References

News Citations

  1. Death of the Neatnik: Neurons Perish When Trash Clutters Their Space?
  2. Partners in Crime: APP Fragment and Endosomal Protein Impair Endocytosis
  3. Behold PANTHOS, a Toxic Wreath of Perinuclear Aβ That Kills Neurons

Mutations Citations

  1. Trisomy 21

Therapeutics Citations

  1. Saracatinib

Research Models Citations

  1. APP NL-G-F Knock-in

Paper Citations

  1. Cataldo AM, Peterhoff CM, Troncoso JC, Gomez-Isla T, Hyman BT, Nixon RA. Endocytic pathway abnormalities precede amyloid beta deposition in sporadic Alzheimer's disease and Down syndrome: differential effects of APOE genotype and presenilin mutations. Am J Pathol. 2000 Jul;157(1):277-86. PubMed.
  2. 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.
  3. 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.
  4. Sun J, Carlson-Stevermer J, Das U, Shen M, Delenclos M, Snead AM, Koo SY, Wang L, Qiao D, Loi J, Petersen AJ, Stockton M, Bhattacharyya A, Jones MV, Zhao X, McLean PJ, Sproul AA, Saha K, Roy S. CRISPR/Cas9 editing of APP C-terminus attenuates β-cleavage and promotes α-cleavage. Nat Commun. 2019 Jan 3;10(1):53. PubMed.
  5. Tarr PE, Roncarati R, Pelicci G, Pelicci PG, D'Adamio L. Tyrosine phosphorylation of the beta-amyloid precursor protein cytoplasmic tail promotes interaction with Shc. J Biol Chem. 2002 May 10;277(19):16798-804. PubMed.
  6. Tamayev R, Zhou D, D'Adamio L. The interactome of the amyloid beta precursor protein family members is shaped by phosphorylation of their intracellular domains. Mol Neurodegener. 2009;4:28. PubMed.

Further Reading

Primary Papers

  1. 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.

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