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Cited by 167Alzheimer's disease research and treatmentsAutophagy in Disease and TherapyDown syndrome and intellectual disability research

Lysosomal Dysfunction in Down Syndrome Is APP-Dependent and Mediated by APP-βCTF (C99)

Open access
Jiang, Ying, Sato, Yutaka, Im, Eunju, Berg, Martin, Bordi, Matteo, Darji, Sandipkumar, Kumar, Asok, Mohan, Panaiyur S. +4 more·2019·The Journal of Neuroscience
DOI 10.1523/jneurosci.0578-19.2019PMC PMC6607756
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Abstract

Lysosomal failure underlies pathogenesis of numerous congenital neurodegenerative disorders and is an early and progressive feature of Alzheimer9s disease (AD) pathogenesis. Here, we report that lysosomal dysfunction in Down ayndrome (trisomy 21), a neurodevelopmental disorder and form of early onset AD, requires the extra gene copy of amyloid precursor protein (APP) and is specifically mediated by the β cleaved carboxy terminal fragment of APP (APP-βCTF, C99). In primary fibroblasts from individuals with DS, lysosomal degradation of autophagic and endocytic substrates is selectively impaired, causing them to accumulate in enlarged autolysosomes/lysosomes. Direct measurements of lysosomal pH uncovered a significant elevation (0.6 units) as a basis for slowed LC3 turnover and the inactivation of cathepsin D and other lysosomal hydrolases known to be unstable or less active when lysosomal pH is persistently elevated. Normalizing lysosome pH by delivering acidic nanoparticles to lysosomes ameliorated lysosomal deficits, whereas RNA sequencing analysis excluded a transcriptional contribution to hydrolase declines. Cortical neurons cultured from the Ts2 mouse model of DS exhibited lysosomal deficits similar to those in DS cells. Lowering APP expression with siRNA or BACE1 inhibition reversed cathepsin deficits in both fibroblasts and neurons. Deleting one Bace1 allele from adult Ts2 mice had similar rescue effects in vivo. The modest elevation of endogenous APP-βCTF needed to disrupt lysosomal function in DS is relevant to sporadic AD where APP-βCTF, but not APP, is also elevated. Our results extend evidence that impaired lysosomal acidification drives progressive lysosomal failure in multiple forms of AD. SIGNIFICANCE STATEMENT Down syndrome (trisomy 21) (DS) is a neurodevelopmental disorder invariably leading to early-onset Alzheimer9s disease (AD). We showed in cells from DS individuals and neurons of DS models that one extra copy of a normal amyloid precursor protein (APP) gene impairs lysosomal acidification, thereby depressing lysosomal hydrolytic activities and turnover of autophagic and endocytic substrates, processes vital to neuronal survival. These deficits, which were reversible by correcting lysosomal pH, are mediated by elevated levels of endogenous β-cleaved carboxy-terminal fragment of APP (APP-βCTF). Notably, similar endosomal-lysosomal pathobiology emerges early in sporadic AD, where neuronal APP-βCTF is also elevated, underscoring its importance as a therapeutic target and underscoring the functional and pathogenic interrelationships between the endosomal-lysosomal pathway and genes causing AD.

AI snapshot

Written from the paper’s full text.

TL;DR

Lysosomal dysfunction in Down syndrome (DS) fibroblasts and Ts2 mouse neurons requires an extra APP gene copy and is mediated by APP-βCTF (C99). The defect is driven by impaired lysosomal acidification, which inactivates acid hydrolases such as cathepsin D and slows autophagic/endocytic substrate degradation.

Key findings

  • DS fibroblasts showed elevated lysosomal pH (5.03 ± 0.08 vs 4.66 ± 0.06 in 2N; ~0.6 units higher) and more than eightfold more poorly acidified autolysosomes (9.24 ± 1.23% vs 1.13 ± 0.41% of total autolysosomes).
  • Lysosomal cathepsin D (CTSD) activity was reduced by 45% in situ in DS fibroblasts, and CTSD specific activity was less than half that in 2N cells, despite 2.5-fold more mature 32 kDa CTSD protein.
  • Lowering APP with siRNA or inhibiting BACE1 rescued lysosomal pH and CTSD deficits in DS fibroblasts and Ts2 neurons; deleting one Bace1 allele in Ts2 mice restored brain CTSD activity.
  • Raising APP-βCTF in 2N fibroblasts with γ-secretase inhibition or mCherry-C99 transfection reproduced lysosomal pH elevation and reduced CTSD activity, while BACE1 inhibition ameliorated the DS phenotype.

Methods

  • Primary human foreskin fibroblasts from individuals with DS and age-matched diploid controls, plus primary cortical neurons from Ts2 DS model mice and adult Ts2 mouse brain homogenates.
  • Lysosomal function was assessed by [³H]-leucine long-lived protein degradation, LysoSensor Yellow/Blue dextran pH measurement, mRFP-eGFP-LC3 autophagic flux imaging, BODIPY FL-Pepstatin A and Magic Red CatB activity assays, in vitro enzyme activity assays, EM, immunoblotting, immunocytochemistry, and RNA-Seq.
  • Interventions included APP siRNA, BACE1 inhibitor IV, γ-secretase inhibitor L-685,458, acidic nanoparticles (NP3), and BACE1 heterozygous crossing; statistics used two-tailed unpaired t tests or one-way ANOVA with Tukey’s post hoc analysis.

Limitations

  • The paper states that its conclusion does not exclude possible additional influences of other triplicated chromosome 21 genes or metabolic factors such as oxidative stress, lipid peroxidation, and protein nitration.
  • Several mechanistic claims about APP-βCTF interaction with the vATPase V0a1 subunit and impaired vATPase assembly are described as unpublished observations, not fully reported in this paper.
  • Evident from the text: the main human evidence comes from primary fibroblasts rather than human brain tissue, and much of the neuronal evidence comes from cultured Ts2 mouse neurons or mouse brain homogenates, so direct human neuronal in vivo validation is not provided.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1523/jneurosci.0578-19.2019

Cites

110 of 110 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

26 of 67 cited sources checked · 9 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    L685,458 boosts APP-βCTF levels by inhibiting its cleavage.

    “which boosts APP-␤CTF levels by inhibiting its cleavage (Fig. 5 A, B) (Jiang et al., 2010;Lauritzen et al., 2016;Hung and Livesey, 2018).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Jiang et al., 2010

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Lauritzen et al., 2016 source

      The Results section (Fig. 5a; page unavailable) reports that the γ-secretase inhibitor ELND006 increased C99, an APP-βCTF. This supports the general effect of inhibiting its cleavage, but the paper does not identify or test L685,458, so it does not establish the claim for that specific compound. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites Hung and Livesey, 2018 source

      The Results section (‘Gamma-Secretase Inhibition Disrupts Function of the Lysosomal-Autophagic System in Human Neurons’; page not provided) reports that inhibiting γ-secretase prevents β-CTF turnover and markedly increases β-CTF levels. But the Methods name DAPT as the γ-secretase inhibitor used, not L685,458, so this paper does not establish the claim for that specific compound. (LLM, conf=0.89)

      Experimental — accuracy not yet measured
  • Findingown data

    Transcript levels for the analyzed acid hydrolases are comparable in 2N and DS fibroblasts.

    “our RNA-Seq analyses, combined with previously published RNA-Seq data (Letourneau et al., 2014;Sullivan et al., 2016), showed transcript levels for the analyzed acid hydrolases to be comparable in 2N and DS fibroblasts”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Letourneau et al., 2014 source
    • cites Sullivan et al., 2016 source
    • Rests on an assumption: The 2N and DS fibroblasts are comparable except for trisomy 21.
  • Premisecitation

    Extensive characterization of adult Ts2 mice has documented neuropathological abnormalities equivalent in type and onset to those in the Ts65Dn trisomy model of DS.

    “Extensive characterization of adult Ts2 mice (Kaur et al., 2014;Hamlett et al., 2016;Jiang et al., 2016) has documented neuropathological abnormalities equivalent in type and onset to those in the Ts65Dn trisomy model of DS”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Kaur et al., 2014 source

      The Animals section reports that Ts2 mice were studied at 8 months, and the Discussion documents hippocampal glutamatergic and synaptic abnormalities. The Introduction describes synaptic plasticity and neurotransmitter abnormalities in Ts65Dn mice, but the paper does not establish that the two models’ neuropathological abnormalities are equivalent in type or onset; it compares Ts2 with disomic controls, not Ts65Dn. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites Hamlett et al., 2016

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Jiang et al., 2016

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Premisecitation

    Increased APP-βCTF has been linked to lysosomal degradation defect in neurons derived from individuals with DS (Ts21) or familial AD with APP mutation.

    “Increased APP-␤CTF has been linked to lysosomal degradation defect seen in the neurons derived from individuals with DS (Ts21) or familial AD with APP mutation (Hung and Livesey, 2018).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Hung and Livesey, 2018 source
  • Interpretationcitation

    The findings support an emerging concept that acidification impairment may be a proximal biochemical event in AD, common to multiple genetic risk factors that cause or promote AD.

    “The findings support an emerging concept that acidification impairment may be a proximal biochemical event in AD, common to multiple genetic risk factors that cause or promote AD (Lee et al., 2010;Xue et al., 2014;Vidoni et al., 2016).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Lee et al., 2010 source

      The Discussion (page not available in our copy) links impaired lysosomal acidification to PS1 mutations that cause familial AD and suggests lysosomal dysfunction may also contribute to sporadic AD. It does not establish that acidification impairment is common to multiple distinct genetic risk factors for AD; its genetic findings concern PS1. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites Xue et al., 2014 source

      The Abstract and the section “CRND8 Glia Exhibit Lysosomal Dysfunction, Which Is Reversed by SWNT” link mutant APP in an AD mouse model to lysosomal dysfunction, and the Figure 1 description suggests that restoring lysosomal acidification may improve proteolysis (page numbers unavailable). The paper does not establish acidification impairment as a proximal event in AD or show that it is common to multiple genetic risk factors; it studies glia from one mutant-APP mouse model. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites Vidoni et al., 2016 source
  • May combine two claims. 16
  • Classified as probably not a substantial claim. 23

  • May combine two claims.

    Premisecitation

    Early onset AD invariably develops in Down syndrome due mainly to a third copy of APP present on trisomic chromosome 21.

    “a disorder in which an early onset form of AD invariably develops due mainly to a third copy of APP present on trisomic chromosome 21 (Korbel et al., 2009;Doran et al., 2017;Colacurcio et al., 2018).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Korbel et al., 2009 source

      The Results and Discussion links increased Alzheimer’s disease risk in Down syndrome to increased APP copy number, but it does not say the disease develops invariably. It describes a 65-year-old subject with Down syndrome who had neither dementia nor amyloid accumulation, contrary to the claim of inevitable early-onset disease. Page numbers are not supplied in our copy. (LLM, conf=0.86)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites Doran et al., 2017 source

      The Introduction links APP triplication in Down syndrome to increased brain amyloid, and the Discussion describes increased APP copy number as central to AD pathogenesis. But the Introduction distinguishes ubiquitous AD-like neuropathology from dementia: dementia prevalence varies from 30–75% at age 65. The paper therefore does not support the claim that early-onset AD invariably develops. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites Colacurcio et al., 2018 source

      Queued — the background check reaches it, usually within a day.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Emerging genetic, neuropathological, and biological evidence implicates dysfunction of the endosomal-lysosomal pathway in AD pathogenesis at early and late stages of disease and in other late-onset neurodegenerative diseases.

    “Emerging genetic, neuropathological, and biological evidence implicates dysfunction of the endosomal-lysosomal pathway in AD pathogenesis at early and late stages of disease (Nixon, 2017) as well as in other late-onset neurodegenerative diseases (Nixon, 2013;Stoka et al., 2016;Vidoni et al., 2016;Colacurcio et al., 2018).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Nixon, 2017 source
    • cites Nixon, 2013 source
    • cites Stoka et al., 2016 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Lysosomal protein constituents such as progranulin and NPC1 interact directly with CTSD, influencing its activity, stability, and conceivably turnover rate.

    “Protein constituents of lysosomes, such as progranulin (Beel et al., 2017) and NPC1 (Liao et al., 2007;Macías-Vidal et al., 2016), also interact directly with CTSD, influencing its activity, stability, and conceivably turnover rate.”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites Beel et al., 2017 source
    • cites Liao et al., 2007 source

      The sections on increased cathepsin D levels and early-onset lysosomal abnormality report higher cathepsin D levels and activity in Npc1−/− mouse brains (page not provided). They do not show that NPC1 interacts directly with CTSD or affects its stability or turnover. Progranulin is supported by a separate citation, not the one audited here. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Macías-Vidal et al., 2016

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CTSD protein levels are elevated in CTSB/CTSL double knockout mice.

    “CTSD protein levels are elevated in CTSB/CTSL double KO mice (Felbor et al., 2002), suggesting intralysosomal CTSD proteolysis, even though the involved protease(s) are unclear.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Felbor et al., 2002 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In familial AD caused by presenilin 1 (PSEN1) mutations, failed lysosomal delivery of the V0a1 subunit markedly impairs lysosomal acidification, inducing acid hydrolase inhibition and lysosomal substrate buildup in patient primary fibroblasts.

    “in familial AD caused by presenilin 1 (PSEN1) mutations, failed lysosomal delivery of the V0a1 subunit markedly impairs lysosomal acidification, inducing acid hydrolase inhibition and lysosomal substrate buildup in patient primary fibroblasts ( Lee et al., 2010 ( Avrahami et al., 2013).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Lee et al., 2010 source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites Avrahami et al., 2013

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    CTSD is a ubiquitous protease, but its unique importance in brain is illustrated by the relatively selective neurodevelopmental and progressive neurodegenerative phenotype induced by loss-of-function CTSD mutations causing CLN10.

    “CTSD is a ubiquitous protease, but its unique importance in brain is illustrated by the relatively selective neurodevelopmental and progressive neurodegenerative phenotype induced by the loss-of-function CTSD mutations causing the congenital disorder neuronal ceroid lipofuscinosis CLN10 (Tyynela ¨et al., 2000;Siintola et al., 2006).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Tyynela ¨et al., 2000 source

      The Results describe a loss-of-function cathepsin D mutation in sheep with severe brain pathology at birth, while other examined organs appeared macroscopically unaffected; the Discussion calls brain pathology the most striking feature. But this paper does not establish that CTSD mutations cause human CLN10: its Discussion says that whether congenital human NCL represents cathepsin D deficiency remains to be determined. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites Siintola et al., 2006

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    mTOR activation is abnormally high in the brains of DS mouse models and in DS fibroblasts.

    “the abnormally high level of mTOR activation seen in the brains of DS mouse models (Iyer et al., 2014;Perluigi et al., 2014) and in DS fibroblasts (Bordi et al., manuscript submitted)”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Iyer et al., 2014 source
    • cites Perluigi et al., 2014 source

      The Results section (mTOR activation and inhibition of autophagosome formation; page unavailable) reports increased mTOR phosphorylation in Down syndrome brain tissue compared with age-matched controls. The Methods identify the tissue as frontal cortex from people with Down syndrome, not mouse models. The Discussion mentions altered Akt phosphorylation in DS mouse models, but does not establish increased mTOR activation in their brains. The fibroblast claim is attached to a different citation. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites Bordi et al., manuscript submitted

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Mutations or imbalance of the subunits or accessory proteins composing the proton pump impair acidification and lead to neurodegeneration in numerous congenital diseases.

    “Mutations or imbalance of the subunits or accessory proteins composing the proton pump impair acidification and lead to neurodegeneration in numerous congenital diseases (Colacurcio and Nixon, 2016).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Colacurcio and Nixon, 2016 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    CTSD activation is deficient in various neuropathologic states and as a result of genetic mutations.

    “CTSD activation is deficient in various neuropathologic states and as a result of genetic mutations (Colacurcio and Nixon, 2016;Vidoni et al., 2016).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Colacurcio and Nixon, 2016 source

      Section 2.2 (page unavailable) reports impaired cathepsin activation in glial cells expressing mutant APP and impaired cathepsin function or maturation in other genetic models of neurological disease. Section 1.2 identifies cathepsin D as an acid-dependent protease, but the disease passages do not establish that the impaired activation is specifically CTSD activation. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites Vidoni et al., 2016 source

  • Classified as probably not a substantial claim.

    Premisecitation

    APP-βCTF has been previously linked to lysosomal dysfunction in AD models involving overexpressed forms of FAD-mutated APP with or without additional overexpression of other mutated human AD genes, and in iPSC generated Ts21 neurons.

    “APP-␤CTF has also been previously linked to lysosomal dysfunction in AD models involving overexpressed forms of FAD-mutated APP with or without additional overexpression of other mutated human AD genes (Lauritzen et al., 2012(Lauritzen et al., , 2016;;Yang et al., 2014;Kaur et al., 2017;Bourgeois et al., 2018), and in iPSC generated Ts21 neurons (Hung and Livesey, 2018).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Lauritzen et al., 2012 source
    • cites Yang et al., 2014 source

      The Introduction describes lysosomal dysfunction in TgCRND8 mice overexpressing FAD-mutated human APP. The section on prevention of lysosomal lipid accumulation discusses impaired lysosomal degradation and accumulation of APP-CTFs, but does not identify APP-βCTF specifically as a cause of that dysfunction. The Ts21-neuron clause is attributed to a different citation, not Yang et al. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites Hung and Livesey, 2018 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Endosomal anomalies in DS are APP-dependent and mediated by APP-βCTF.

    “Lysosomal deficits in DS add to the known endosomal anomalies known to develop in DS within the same vesicular trafficking pathway, which include some signature features of early AD and are also APP-dependent and mediated by APP-␤CTF (Jiang et al., 2010(Jiang et al., , 2016;;Hung and Livesey, 2018).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Jiang et al., 2010

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Jiang et al., 2016

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Hung and Livesey, 2018 source

      In the Results section on early endosomes (page unavailable), the paper reports enlarged early endosomes in trisomy 21 (DS) neurons, alongside neurons with APP duplication or mutation. That supports the presence of endosomal anomalies and an association with APP, but does not establish that the DS endosomal anomalies are APP-dependent or mediated by APP-βCTF. Its β-secretase inhibition and APP-knockout results address lysosomal-autophagy defects, not the early-endosome anomalies. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CTSD is considered a key protease in the lysosomal degradation of many pathogenic proteins in addition to APP metabolites, including α-synuclein and huntingtin.

    “CTSD is considered as a key protease in the lysosomal degradation of many pathogenic proteins in addition to APP metabolites, including ␣-synuclein and huntingtin (Kegel et al., 2000;Sevlever et al., 2008).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Kegel et al., 2000 source

      The Abstract reports that huntingtin-containing vacuoles incorporated cathepsin D, and the Discussion proposes the endosomal–lysosomal pathway as a route for huntingtin removal. The paper does not establish that CTSD is a key protease responsible for degrading huntingtin, and it does not address CTSD-mediated degradation of α-synuclein or APP metabolites. Page numbers are unavailable in our copy. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites Sevlever et al., 2008 source

  • Classified as probably not a substantial claim.

    Premisecitation

    The level of V1 sector association with the V0 sector regulates ATPase and proton pump activities of the vATPase complex on lysosomes.

    “The level of V1 sector association with the V0 sector regulates ATPase and proton pump activities of the vATPase complex on lysosomes (Cotter et al., 2015).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Cotter et al., 2015 source

  • May combine two claims.

    Findingown data

    The study uncovered AD-related lysosomal functional deficits in primary fibroblasts from individuals with DS and in neurons from Ts2 mice modeling DS.

    “In this investigation, we uncovered AD-related lysosomal functional deficits in primary fibroblasts from individuals with DS, representing the authentic trisomy 21 disorder, and in neurons from Ts2 mice modeling DS.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Ts2 mice are a valid model of Down syndrome.

  • Probably not a substantial claim: may not pass S4 (based).

    Findingnone given

    Impaired vATPase assembly, like the deficits reported in the present study, is rescued by specifically lowering APP-βCTF levels.

    “Impaired vATPase assembly, like the deficits reported in the present study, is rescued by specifically lowering APP-␤CTF levels (E.I., Y.J., R.A.N., unpublished observations).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Ts2 mice are a valid model of Down syndrome.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    BACE1 activity and APP-βCTF levels are elevated in AD brain in the absence of detectable APP elevation.

    “Notably, BACE1 activity and APP-␤CTF levels are elevated in AD brain in the absence of detectable APP elevation (Holsinger et al., 2002;Pera et al., 2013;Kim et al., 2016).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Holsinger et al., 2002 source
    • cites Pera et al., 2013

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Kim et al., 2016

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisecitation

    Lysosomal alterations develop months before β-amyloid deposition in AD mouse models with late-onset amyloidosis.

    “lysosomal alterations develop months before ␤-amyloid deposition in AD mouse models with late-onset amyloidosis (J.H.L. and R.A.N., manuscript in preparation).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites J.H.L. and R.A.N., manuscript in preparation

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    APP-βCTF levels are higher in AD brain, even though APP levels are not detectably elevated.

    “Given that APP-␤CTF levels are also higher in AD brain, even though APP levels are not detectably elevated (Pera et al., 2013;Kim et al., 2016)”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Pera et al., 2013

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Kim et al., 2016

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Similar CTSD changes in fibroblasts carrying the ATP13A2 mutation in Parkinson disease were reversed by normalizing lysosomal pH with acidic nanoparticles.

    “Similar CTSD changes seen in fibroblasts carrying the ATP13A2 mutation in Parkinson disease (Bourdenx and Dehay, 2016) were reversed by normalizing lysosomal pH with acidic nanoparticles”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Bourdenx and Dehay, 2016 source
    • cites Bourdenx et al., 2016

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S4 (based).

    Study design and conductnone given

    Primary DS fibroblasts represent authentic human DS genetically unmodified.

    “Importantly, primary DS fibroblasts represent authentic human DS genetically unmodified, in contrast to previously used genetically modified models of AD engineered by the transgenic overexpression of APP carrying FAD mutations together in some cases with other mutated AD-related human genes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Posttranslational modifications of CTSD, such as oxidation or nitration, have been reported for CTSD in DS.

    “Additionally, posttranslational modifications of CTSD, such as oxidation or nitration, which have been reported for CTSD in DS (Butterfield et al., 2014b), could alter its activity and increase its resistance to proteolysis, thereby promoting accumulation.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Butterfield et al., 2014b

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationreasoning

    Functional lysosomal abnormalities precede flagrant lysosomal pathology and accumulation of lysosomal content, implying a proximal link to the gene most strongly implicated in AD.

    “The detection of functional lysosomal abnormalities preceding the flagrant lysosomal pathology and accumulation of lysosomal content imply a proximal link to the gene most strongly implicated in AD.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    DS fibroblasts exhibit markedly lowered baseline AP formation and mitophagy, which can be restored by selectively inhibiting TORC1 and TORC2.

    “also exhibiting markedly lowered baseline AP formation and mitophagy, which can be restored by selectively inhibiting TORC1 and TORC2 (M.Bordi, manuscript submitted)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites M.Bordi, manuscript submitted

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    CTSB is a lysosomal exopeptidase with a pH optimum in the mildly acidic range.

    “Notably, CTSB, a lysosomal exopeptidase with a pH optimum in the mildly acidic range (Barrett and Kirschke, 1981), was more active in DS lysosomes.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Barrett and Kirschke, 1981 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Even a partial loss of CTSD function is associated with CNS deficits in humans.

    “Notably, even a partial loss of CTSD function is associated with CNS deficits in humans (Mamo et al., 2012;Tatti et al., 2013).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Mamo et al., 2012 source
    • cites Tatti et al., 2013

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Premisecitation

    Lysosomal acidification is maintained principally by the vacuolar-type H+-ATPase complex (vATPase), which imports hydrogen ions into the lysosome.

    “Lysosomal acidification is maintained principally by the vacuolar-type H ϩ -ATPase complex (vATPase), which imports hydrogen ions into the lysosome (Mindell, 2012).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Mindell, 2012 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Other triplicated chromosome 21 genes or metabolic factors (oxidative stress, lipid peroxidation, protein nitration) are likely additive factors in corrupting lysosome functions in DS.

    “This conclusion, however, does not exclude the possible additional influences of other triplicated chromosome 21 genes or metabolic factors, such as oxidative stress, lipid peroxidation, and protein nitration (Perluigi and Butterfield, 2012;Butterfield et al., 2014a), which are likely additive factors in corrupting lysosome functions.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Perluigi and Butterfield, 2012

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Butterfield et al., 2014a

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    CTSD gene transcription in neurons is upregulated at early stages of AD and is accompanied by the accumulation of CTSD forms with decreased activity.

    “CTSD gene transcription in neurons is upregulated at early stages of AD (Cataldo et al., 1995) and is accompanied by the accumulation of CTSD forms with decreased activity (P.S.M. and R.A.N., manuscript in preparation).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Cataldo et al., 1995 source
    • cites P.S.M. and R.A.N., manuscript in preparation

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Study design and conductown data

    Deficient acidification was established by direct ratiometric measurement in lysosomes in situ and confirmed that ALs were incompletely acidified using an mRFP-eGFP-LC3 construct transfected into DS cells in conjunction with dextran-AlexaFluor-647 uptake.

    “We established deficient acidification by direct ratiometric measurement in lysosomes in situ and confirmed that ALs were incompletely acidified using an mRFP-eGFP-LC3 construct transfected into DS cells in conjunction with dextran-AlexaFluor-647 uptake.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The identification of a lysosomal acidification defect in DS and DS model neurons implicates APP via its BACE1-cleaved metabolite as a second key etiologic factor in AD, together with PSEN1, in directly dysregulating vATPase.

    “The identification of a lysosomal acidification defect in DS and DS model neurons therefore implicates APP via its BACE1-cleaved metabolite as a second key etiologic factor in AD, together with PSEN1, in directly dysregulating vATPase.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Ts2 mice are a valid model of Down syndrome.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ventricular CSF obtained postmortem from AD patients has substantially elevated levels of CTSD with markedly lowered specific activity compared with CSF from individuals with Huntington's disease or from non-neurologic disease controls.

    “we previously reported that ventricular CSF obtained postmortem from AD patients has substantially elevated levels of CTSD with markedly lowered specific activity compared with CSF from individuals with Huntington's disease or from non-neurologic disease controls (Schwagerl et al., 1995).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Schwagerl et al., 1995 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Impaired autolysosomal acidification is the earliest autophagy alteration seen in AD mouse models expressing a neuronal mRFP-eGFP-LC3 transgene and accompanies the appearance of intraneuronal APP-βCTF.

    “impaired autolysosomal acidification is the earliest autophagy alteration seen in AD mouse models expressing a neuronal mRFP-eGFP-LC3 transgene and accompanies the appearance of intraneuronal APP-␤CTF (Lee et al., 2019) (J.H.L. and R.A.N., manuscript in preparation).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Lee et al., 2019

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites J.H.L. and R.A.N., manuscript in preparation

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

32 of 32

  • The paper relies on this without support.

    The extra copy of APP is the only trisomic gene necessary for the lysosomal dysfunction in Down syndrome.

    “Here, we report that lysosomal dysfunction in Down ayndrome (trisomy 21), a neurodevelopmental disorder and form of early onset AD, requires the extra gene copy of amyloid precursor protein (APP)”

  • The paper relies on this without support.

    BACE1 inhibition reduces cathepsin deficits specifically by lowering APP-βCTF, not through other BACE1 substrates.

    “Lowering APP expression with siRNA or BACE1 inhibition reversed cathepsin deficits in both fibroblasts and neurons.”

  • The paper relies on this without support.

    The Ts2 mouse model's lysosomal deficits are representative of human Down syndrome.

    “Cortical neurons cultured from the Ts2 mouse model of DS exhibited lysosomal deficits similar to those in DS cells.”

  • The paper relies on this without support.

    The 2N and DS fibroblasts are comparable except for trisomy 21.

    “Human foreskin fibroblasts from DS patients and diploid age-matched controls (2N) were purchased from the Coriell Cell Repositories (AG07096, AG04823, GM04617, GM08680, AG07095, AG06922, GM08429, GM5381) and maintained according to the distributor's protocols”

  • The paper relies on this without support.

    Lysosomal abnormalities in DS fibroblasts are representative of the toxic effects of APP triplication on lysosomes in DS.

    “The toxic effects of APP triplication on lysosomes in DS are due mainly to APP-␤CTF”

  • The paper relies on this without support.

    BACE1 inhibitor IV at 10 µM for 24 h specifically blocks APP-βCTF production without affecting lysosomal pH through off-target mechanisms.

    “blocking APP-␤CTF production using BACE1 inhibitor IV (10 M) for 24 h had no effect on the lysosomal pH of 2N fibroblasts (Fig. 5E), but as expected, the lysosomal pH deficit in DS fibroblasts was ameliorated (Fig. 5E; F (3,34) ϭ 15, p Ͻ 0.0001; one-way ANOVA).”

  • The paper relies on this without support.

    Transient transfection of mCherry-C99 raises APP-βCTF levels without itself causing lysosomal defects independent of βCTF.

    “transient transfection of a fluorescence-tagged ␤CTF (mCherry-C99) significantly reduced in situ CTSD activity in 2N cells and further lowered CSTD activity in DS fibroblasts”

  • The paper relies on this without support.

    Ts2 mice are a valid model of Down syndrome.

    “neurons from Ts2 mice modeling DS”
    “primary cortical neurons from Ts2 mice”
8 of 8

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Identical t statistics have different reported p values

    Minor

    The lysosome-number and lysosome-size comparisons report the same t statistic and degrees of freedom but different p values. Both p values cannot correspond to that statistic under the stated unpaired t test.

  • Gamma-secretase inhibitor identifier changes

    Minor

    The inhibitor used to raise APP-βCTF is identified as L-685,485 in the results, but as L-685,458 in the methods.

  • Figure 4G test is labeled APP although the panel concerns TFE3 and TFEB

    Minor

    The results attribute a comparison cited to Figure 4G to APP, whereas the figure caption assigns APP to panel F and TFE3 and TFEB to panel G.

3 of 3

Cites

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Related

Authors
Jiang, Y., Sato, Y., Im, E., Berg, M., Bordi, M., Darji, S., Kumar, A., Mohan, P. S., Bandyopadhyay, U., Diaz, A., Cuervo, A. M., & Nixon, R. A.
Affiliations
  • Nathan Kline Institute for Psychiatric Research (US)
  • University of Rome Tor Vergata (IT)
  • Albert Einstein College of Medicine (US)
ORCID
Venue
The Journal of Neuroscience
Publisher
Society for Neuroscience
Published
2019-05-01
Volume
39
Issue
27
Pages
5255-5268
ISSN
  • 0270-6474 (ISSN-L)
  • 1529-2401
DOI
10.1523/jneurosci.0578-19.2019
PMC
PMC6607756
OpenAlex
W2943621977
Cited by
166
References
110
Citations by year
2026: 10 · 2025: 21 · 2024: 15 · 2023: 23 · 2022: 20 · 2021: 36 · 2020: 33 · 2019: 8
FWCI
13.96
Type
journal article
Language
en
Open access
Locations
  • Journal of Neuroscience (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion)
  • PubMed Central (submittedVersion)
Funders
National Institute on Aging (P01AG017617)
Topics
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
  • Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Down syndrome and intellectual disability research
Keywords
Down syndrome, lysosomal dysfunction, lysosomal acidification, amyloid precursor protein, C99, BACE1, cathepsin D, autophagy, lysosomal enzymes, sporadic Alzheimer's disease, neurodegeneration
MeSH terms
Alzheimer Disease, Cells, Cultured, Down Syndrome, Fibroblasts, Humans, Lysosomes, Peptide Fragments, Amyloid beta-Protein Precursor, Proteolysis

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-27.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-27.

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Statistics and arithmetic are recomputed, not judged.

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