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Cited by 504Alzheimer's disease research and treatmentsAutophagy in Disease and TherapyNeuroinflammation and Neurodegeneration Mechanisms

LC3-Associated Endocytosis Facilitates β-Amyloid Clearance and Mitigates Neurodegeneration in Murine Alzheimer’s Disease

Open access
Heckmann, Bradlee L., Teubner, Brett J.W., Tummers, Bart, Boada-Romero, Emilio, Harris, Lacie, Yang, Mao, Guy, Clifford S., Zakharenko, Stanislav S. +1 more·2019·Cell
DOI 10.1016/j.cell.2019.05.056PMC PMC6689199
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Abstract

SUMMARY The expression of some proteins in the autophagy pathway decline with age, which may impact neurodegeneration in diseases, including Alzheimer’s Disease. We have identified a novel non-canonical function of several autophagy proteins in the conjugation of LC3 to Rab5+, clathrin+ endosomes containing β-amyloid in a process of LC3-associated endocytosis (LANDO). We found that LANDO in microglia is a critical regulator of immune-mediated aggregate removal and microglial activation in a murine model of AD. Mice lacking LANDO but not canonical autophagy in the myeloid compartment or specifically in microglia have a robust increase in pro-inflammatory cytokine production in the hippocampus and increased levels of neurotoxic β-amyloid. This inflammation and β-amyloid deposition was associated with reactive microgliosis and tau hyperphosphorylation. LANDO-deficient AD mice displayed accelerated neurodegeneration, impaired neuronal signaling, and memory deficits. Our data support a protective role for LANDO in microglia in neurodegenerative pathologies resulting from β-amyloid deposition.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper identifies LC3-associated endocytosis (LANDO), a non-canonical autophagy-protein-dependent process in microglia that conjugates LC3 to Rab5+/clathrin+ endosomes containing β-amyloid and supports recycling of Aβ receptors. Loss of LANDO, but not canonical autophagy, exacerbates Aβ deposition, neuroinflammation, tau pathology, neurodegeneration, and memory deficits in 5xFAD mice.

Key findings

  • LANDO requires ATG5 and Rubicon, but not FIP200, for LC3 recruitment to Aβ-containing endosomes; ATG5- or Rubicon-deficient BV2 microglia showed reduced LC3 recruitment and membrane-associated LC3, while FIP200 deficiency did not.
  • In 4-month-old 5xFAD mice, myeloid ATG5 deficiency and global or microglia-specific Rubicon deficiency increased hippocampal and cortical Aβ plaque number/size, pro-inflammatory cytokines (TNFα, IL-1β, IL-6), reactive microgliosis, and tau hyperphosphorylation; myeloid or microglial FIP200 deficiency had no effect.
  • LANDO-deficient cells showed impaired secondary Aβ uptake and abrogated recycling of CD36, TLR4, and TREM2, while primary Aβ uptake, Aβ degradation, endosome maturation, and lysosome association were unaffected.
  • LANDO-deficient 5xFAD mice displayed accelerated neurodegeneration: reduced NeuN+ hippocampal neurons, increased cleaved-caspase 3 in CA3, impaired hippocampal synaptic transmission and LTP, and behavioral/memory deficits; Rubicon-deficient mice showed anhedonia as young as 2.5 months and approximately 50% sucrose preference by 4 months.

Methods

  • Murine AD model: 5xFAD mice crossed with myeloid LysM-Cre conditional FIP200 or ATG5, germline Rubicon−/−, or CX3CR1-creERT2 conditional Rubicon/FIP200; experiments used mixed-sex cohorts at 4 months unless noted, on >97% C57BL/6J background.
  • Cell and biochemical assays: BV2 microglia, RAW264.7 macrophages, primary BMDMs and microglia; CRISPR/Cas9 knockouts, GFP-LC3, oligomeric Aβ1-42, microscopy, flow cytometry, immunoblot, qPCR, ELISA, pH-rodo maturation, receptor recycling, Aβ uptake, electrophysiology, and behavior (sucrose preference, Y-maze, novel object recognition).
  • Statistics: GraphPad Prism 7.0, powered to at least 0.8 by G*Power, significance at p < 0.05.

Limitations

  • Stated: the 5xFAD models cannot fully exclude a role for infiltrating peripheral monocytes in Aβ clearance and neuroinflammation; CX3CR1-creERT2 transient ablation may not completely exclude Rubicon-deficient peripheral monocytes entering the brain before reconstitution.
  • Stated: the receptor-recycling assay does not evaluate LANDO’s role in receptor degradation or other endosome-trafficking perturbations, and altered receptor signaling could promote cytokine expression; the authors also cannot formally exclude LAP functions in Aβ clearance/response in vivo.
  • Evident from the text: findings are based on murine 5xFAD models and cultured cell systems, with no human validation reported; the authors also note tamoxifen may reduce Aβ oligomerization as a possible confound in MG-cre animals.

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

Integrity signals

Registry

Crossref · 10.1016/j.cell.2019.05.056

Cites

89 of 89 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 59 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Primary microglia isolated from LysM-Cre+ FIP200fl/fl and ATG5fl/fl mice show a significant reduction in mRNA and protein expression compared to LysM-Cre− littermates.

    “Primary microglia isolated from LysM-Cre+ FIP200fl/fl and ATG5fl/fl mice (referred to as FIP200,cre+ and ATG5,cre+) showed a significant reduction in mRNA and protein expression when compared to LysM-Cre− littermates (Fig. S1A,B).”
    Quote in the paperSubstantial claim
  • Findingown data

    The autophagy protein FIP200 does not have the protective function against Aβ accumulation, neuroinflammation, and neurodegeneration that ATG5 has.

    “In contrast, another requisite autophagy protein, RB1-inducible coiled-coil protein 1, also called FIP200 (herein, FIP200), has no such function.”
    Quote in the paperSubstantial claim
  • Findingown data

    In the myeloid compartment (including microglia), the autophagy protein ATG5 promotes protection from Aβ accumulation, neuroinflammation, and neurodegeneration.

    “We employed an established murine model of AD and found that in the myeloid compartment (including microglia), the autophagy protein, ATG5, promoted protection from Aβ accumulation, neuroinflammation, and neurodegeneration.”
    Quote in the paperSubstantial claim
  • Findingown data

    LANDO is a novel form of receptor-mediated endocytosis and receptor recycling characterized by association of LC3/GABARAP-family proteins with endosomal membranes.

    “In dissecting these effects, we identified a novel form of RME and receptor recycling characterized by association of LC3/GABARAP-family proteins (herein, “LC3”) with endosomal membranes, a process we call LC3-Associated eNDOcytosis (LANDO).”
    Quote in the paperSubstantial claim
  • Findingown data

    Rubicon, a protein that constrains canonical autophagy, is required for the protective effects against Aβ accumulation, neuroinflammation, and neurodegeneration.

    “Strikingly, a protein that constrains canonical autophagy, Rubicon, was similarly required for the protective effects.”
    Quote in the paperSubstantial claim
  • May combine two claims. 48
  • Classified as probably not a substantial claim. 26
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data

    In 5xFAD mice, absence of Rubicon (global or microglial) or myeloid ATG5 leads to accelerated disease pathology, neurodegeneration, reactive microgliosis, tau pathology, and behavioral impairment, whereas FIP200 deficiency does not.

    “In the absence of Rubicon either globally or conditionally in microglia, or myeloid ATG5 (but not myeloid or microglial FIP200), we observed accelerated disease pathology and neurodegeneration, reactive microgliosis, tau pathology, and behavioral impairment.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In 5xFAD mice, autophagy deficiency (FIP200 LysM- or MG-cre+) does not alter Aβ deposition, whereas myeloid ATG5 deficiency and Rubicon deficiency (global or microglial) increase Aβ plaque number and size in the hippocampus and cortex.

    “Consistent with this notion and similar to mice deficient in myeloid ATG5, 5xFAD Rubicon−/− mice and 5xFAD Rubicon MG-cre+ mice displayed an increase in Aβ plaque number and plaque size compared to either Rubicon+/− or MG-cre− littermates (Fig. 1A-D and S1I).”
    • Also stated in: § ATG5 and Rubicon-deficiency exacerbate Aβ deposition and pathology · plain
      “Likewise, both 5xFAD ATG5 LysM-cre+, 5xFAD Rubicon−/−, and 5xFAD Rubicon MG-cre+ mice showed early accumulation of Aβ within the cortex, whereas mice deficient for myeloid or microglial FIP200 were unaffected compared to LysM- or MG-cre− littermates (Fig. 1E,F and Fig. S1J,K).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Conditional deletion of Rubicon or FIP200 in microglia via CX3CR1-creERT2 ablates the genes in microglia but not in peripheral splenic macrophages.

    “Primary microglia isolated from 5xFAD, Rubicon and FIP200, CX3CR1-creERT2 mice treated with tamoxifen at one month of age and allowed to reconstitute for 3 months (herein referred to as MG-cre+) showed ablation of Rubicon (Fig. S1F,G) or FIP200 (Fig. S1G), as indicated. In contrast, expression of each gene was restored in peripheral, splenic macrophages (Fig. S1H), as expected.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Ablation of Beclin1, VPS34, ATG5, ATG7, or Rubicon prevents recycling of TLR4, TREM2, and CD36, whereas ablation of ULK1, FIP200, or ATG14 has no effect.

    “Ablation of Beclin1, VPS34, ATG5, or ATG7, required for both autophagy and LAP, prevented recycling of TLR4, TREM2, and CD36, as did Rubicon, required for LAP but not autophagy (Martinez et al., 2015). In contrast, no effects were seen upon ablation of ULK1, FIP200, or ATG14, all required for autophagy, but dispensable for LAP (Martinez et al., 2015).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Martinez et al., 2015 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Loss of FIP200, ATG5, or Rubicon has no effect on primary uptake of Aβ, but secondary uptake is reduced in ATG5- and Rubicon-deficient cells and not in FIP200-deficient cells.

    “Consistent with our clearance assay, the loss of FIP200, ATG5, or Rubicon had no effect on primary uptake of Aβ (Fig. 2G). We next added TAMRA-labeled Aβ1-42 and allowed for a second round of RME to occur. Again, we quantified the amount of internalized TAMR-1-42 and observed a reduction in secondary uptake in ATG5 and Rubicon-deficient cells but not those deficient in FIP200 (Fig. 2G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Neither microglial activation nor pro-inflammatory cytokine expression were altered in mice deficient in the 5xFAD transgene.

    “Neither microglial activation nor pro-inflammatory cytokine expression were altered in mice deficient in the 5xFAD transgene (Fig. S5A,B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Over 90% of CD11b+ cells in brains from 5xFAD Rubicon+/− and −/− mice were TMEM119+, and TMEM119+ cells isolated from 5xFAD Rubicon−/− mice had higher Iba1 expression compared to Rubicon+/− animals.

    “We observed that over 90% of CD11b+ cells in brains from the 5xFAD Rubicon+/− and−/− mice were TMEM119+ (a microglia-specific marker (Bennett et al., 2016)) (Fig. S5C,D). Furthermore, the TMEM119+ cells isolated from 5xFAD Rubicon−/− mice had higher Iba1 expression compared to Rubicon+/− animals (Fig. S5E).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Bennett et al., 2016 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Significant increases in TNFα, IL-1β, IL-6, and a marginal increase in CCL5 occur within the hippocampus of 5xFAD Rubicon−/− and myeloid ATG5-deficient mice compared to control littermates.

    “Using Iba1 expression as a positive control, we observed significant increases in TNFα, IL-1β, IL-6, and a marginal increase in CCL5 within the hippocampus of 5xFAD Rubicon−/− and myeloid ATG5-deficient mice compared to control littermates (Fig. 4G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    FIP200 deficiency has no impact on cytokine expression in response to Aβ1-42, whereas loss of LANDO in ATG5- and Rubicon-deficient cells results in a robust increase in all four pro-inflammatory genes evaluated.

    “FIP200-deficiency failed to have any impact on cytokine expression in response to Aβ1-42 (Fig. 3D). However, loss of LANDO in both ATG5 and Rubicon-deficient cells resulted in a robust increase in all four pro-inflammatory genes evaluated (Fig. 3D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Myeloid or microglia-specific FIP200 deficiency has no effect on the extent of Iba1 expression in either the hippocampus or cortex of 5xFAD mice, whereas microglial hyperactivation is present in 5xFAD mice deficient in either Rubicon or myeloid ATG5.

    “Consistent with our in vitro findings, myeloid or microglia-specific FIP200-deficiency had no effect on the extent of Iba1 expression in either the hippocampus or cortex of 5xFAD mice (Fig. 4A-C). In contrast, microglial hyperactivation was present in 5xFAD mice deficient in either Rubicon or myeloid ATG5 (Fig. 4A,B).”
    Quote in the paperSubstantial claim

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

    Findingcitation

    BV2 cells treated with Aβ1-42 have elevated pro-inflammatory gene expression, including IL-1β, IL-6, CCL5, and TNFα.

    “As expected, BV2 cells treated with Aβ1-42 had elevated pro-inflammatory gene expression, including IL-1β, IL-6, CCL5, and TNFα as reported (Pan et al., 2011) and consistent with human disease.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Pan et al., 2011 source

      Not checked in the free run.

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    In cells lacking ATG5 or Rubicon, phagosome maturation and LAMP1 association are diminished, while maturation and LAMP1 association of Aβ-containing endosomes are unaffected.

    “Consistent with these reports, in the absence of Rubicon and ATG5, maturation of phagosomes was diminished as determined using pH-rodo-labeled zymosan, while maturation of endosomes containing pH-rodo-labeled Aβ was unaffected (Fig. 2E, S2E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Rubicon or myeloid ATG5 deficiency leads to significant upregulation of pro-inflammatory cytokines at the transcriptional level in the brain, whereas myeloid FIP200 deficiency has no effect.

    “Consistent with what we observed with cultured cells (Fig. 3), Rubicon- or myeloid ATG5-deficiency led to significant upregulation of pro-inflammatory cytokines at the transcriptional level in the brain, whereas myeloid FIP200-deficiency had no effect.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In RAW264.7 cells, TLR4, TREM2, and CD36 effectively recycle to the plasma membrane following antibody-induced internalization; this recycling is impaired upon Rubicon or ATG5 ablation, ATG4DN expression, or RavZ expression.

    “To further explore this phenomenon, we employed the RAW264.7 myeloid cell line, and found that TLR4, TREM2, and CD36 effectively recycled to the plasma membrane following antibody-induced internalization. This recycling was dramatically impaired upon CRISPR/Cas9-mediated ablation of Rubicon or ATG5, upon expression of a dominant negative ATG4, or upon expression of the LC3-specific protease, RavZ”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    FIP200 depletion has no effect on surface expression, internalization, or recycling of CD36, TLR4, or TREM2, whereas ATG5 or Rubicon ablation abrogates recycling of all three receptors while initial surface expression and internalization are unaffected.

    “In BV2 microglia, depletion of FIP200 had no effect on either the surface expression, internalization (Fig. S2D), or recycling of CD36, TLR4, or TREM2 (Fig. 2H,I). Strikingly, ablation of either ATG5 and Rubicon led to abrogation of recycling for all three receptors (Fig. 2H,I) while initial surface expression and internalization were unaffected (Fig. S2D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ablation of either ATG5 or Rubicon has no effect on either the endocytosis or degradation of Aβ.

    “Contrary to our assumptions, however, ablation of either ATG5 or Rubicon had no effect on either the endocytosis or degradation of Aβ (Fig. 2D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both global and microglia-specific deletion of Rubicon and myeloid-specific deletion of ATG5 in 5xFAD mice led to significant memory impairment.

    “We observed that both global and microglia-specific deletion of Rubicon and myeloid-specific deletion of ATG5 resulted in exacerbated Aβ deposition and plaque formation, reactive microgliosis, tau hyperphosphorylation, and neuronal cell death and dysfunction, leading to significant memory impairment.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The 5xFAD mouse model is a well-characterized model of Aβ aggregate deposition and Alzheimer's disease.

  • May combine two claims.

    Findingown data

    The paper observed no effects of their deletions on the rate of Aβ degradation, endosome maturation, or lysosome association.

    “However, while LAP functions to promote phagosome maturation and cargo destruction (Heckmann et al., 2017), we observed no effects of our deletions on the rate of Aβ degradation, endosome maturation, or lysosome association (Fig. 2).”
    Quote in the paperSubstantial claim

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

    Findingown data

    No variations in total fluid intake between genotypes were observed.

    “No variations in total fluid intake between genotypes was observed (Fig. 7B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    5xFAD Rubicon−/− mice showed reduced hippocampal synaptic transmission and impaired long-term potentiation (LTP) compared to littermate controls.

    “We found that 5xFAD Rubicon−/− mice showed a reduction in hippocampal synaptic transmission and, as a consequence, impaired long-term potentiation (LTP) when compared to littermate controls (Fig. 6E,F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Myeloid or microglia-specific FIP200 depletion has no impact on tau phosphorylation, whereas loss of either Rubicon (global or microglia-specific) or myeloid ATG5 promotes tau phosphorylation throughout the hippocampus and the cerebral cortex.

    “Myeloid or microglia-specific FIP200 depletion had no impact on tau phosphorylation, however loss of either Rubicon (global or microglia-specific) or myeloid ATG5 promoted tau phosphorylation throughout the hippocampus (Fig. 5A,C, S6A) and the cerebral cortex (Fig. 5B,D, S6B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both global and microglia-specific deletion of Rubicon and myeloid-specific deletion of ATG5 in 5xFAD mice resulted in reactive microgliosis.

    “We observed that both global and microglia-specific deletion of Rubicon and myeloid-specific deletion of ATG5 resulted in exacerbated Aβ deposition and plaque formation, reactive microgliosis, tau hyperphosphorylation, and neuronal cell death and dysfunction, leading to significant memory impairment.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The 5xFAD mouse model is a well-characterized model of Aβ aggregate deposition and Alzheimer's disease.

  • May combine two claims.

    Findingown data

    LC3 associates with endosomal markers Rab5 and clathrin at the membranes of Aβ-containing endosomes, and this association was not altered by inhibition of phagocytosis.

    “Because we observed the association of LC3 and the endosomal markers Rab5 and clathrin at the membranes of Aβ-containing endosomes that was not altered by inhibition of phagocytosis, we called this LANDO.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both global and microglia-specific deletion of Rubicon and myeloid-specific deletion of ATG5 in 5xFAD mice resulted in exacerbated Aβ deposition and plaque formation.

    “We observed that both global and microglia-specific deletion of Rubicon and myeloid-specific deletion of ATG5 resulted in exacerbated Aβ deposition and plaque formation, reactive microgliosis, tau hyperphosphorylation, and neuronal cell death and dysfunction, leading to significant memory impairment.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The 5xFAD mouse model is a well-characterized model of Aβ aggregate deposition and Alzheimer's disease.

  • May combine two claims.

    Findingown data

    5xFAD Rubicon−/−, microglia-specific Rubicon-deficient, and myeloid ATG5-deficient mice all display a decrease in NeuN+ hippocampal neurons, whereas FIP200 deficiency has no impact on neuronal number.

    “5xFAD Rubicon−/− microglia-specific Rubicon-deficient, and myeloid ATG5-deficient mice all displayed a decrease in NeuN+ hippocampal neurons (Fig. 6A,B). FIP200-deficiency had no impact on neuronal number.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both global and microglia-specific deletion of Rubicon and myeloid-specific deletion of ATG5 in 5xFAD mice resulted in neuronal cell death and dysfunction.

    “We observed that both global and microglia-specific deletion of Rubicon and myeloid-specific deletion of ATG5 resulted in exacerbated Aβ deposition and plaque formation, reactive microgliosis, tau hyperphosphorylation, and neuronal cell death and dysfunction, leading to significant memory impairment.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The 5xFAD mouse model is a well-characterized model of Aβ aggregate deposition and Alzheimer's disease.

  • May combine two claims.

    Findingown data

    ULK1, FIP200, and ATG14 were dispensable for receptor recycling.

    “We further found that Rubicon, ATG5, ATG7, and ATG4 were required, while ULK1, FIP200, and ATG14 were dispensable for this effect.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Loss of LANDO affected secondary uptake of Aβ, leading to increased extracellular deposition.

    “However, loss of LANDO affected secondary uptake of Aβ, leading to increased extracellular deposition, and it is plausible that these deposits signal at the cell surface via other receptors to promote inflammatory signaling.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Use of CX3CR1-creERT2 to transiently ablate Rubicon resulted in Rubicon-deficient microglia but Rubicon-sufficient splenic macrophages at 4 months of age.

    “Moreover, the use of CX3CR1- creERT2 to transiently ablate Rubicon resulted in Rubicon-deficient microglia but Rubicon-sufficient splenic macrophages at 4-months of age, but does not completely exclude the possibility that Rubicon-deficient peripheral monocytes may have entered the brain prior to reconstitution of this population from the bone marrow.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Expression of several autophagy proteins, particularly Beclin1, ATG5, and ATG7, declines with age and is exacerbated in Alzheimer's disease.

    “The expression of several autophagy proteins, in particular Beclin1, ATG5, and ATG7, decline with age, and is exacerbated in AD (Lipinski et al., 2010; Lucin et al., 2013; Pickford et al., 2008; Rubinsztein et al., 2011).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Lipinski et al., 2010 source

      Not checked in the free run.

    • cites Lucin et al., 2013 source

      Not checked in the free run.

    • cites Pickford et al., 2008

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    In response to Aβ, LANDO supports the clearance of Aβ deposits and prevents microglial activation, such that in its absence, inflammatory cytokine production and neurodegeneration proceed unchecked.

    “In response to Aβ, LANDO supports the clearance of Aβ deposits and prevents microglial activation, such that in its absence, inflammatory cytokine production and neurodegeneration proceed unchecked.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Pro-inflammatory cytokines and chemokines secreted into the immediate neurological environment accelerate neuronal injury and eventually neuron death.

    “Pro-inflammatory cytokines and chemokines secreted into the immediate neurological environment accelerate neuronal injury and eventually neuron death (Aktas et al., 2007; Heckmann et al., 2018; Morales et al.,2014).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Aktas et al., 2007 source

      Not checked in the free run.

    • cites Heckmann et al., 2018

      Not checked in the free run.

    • cites Morales et al.,2014 source

      Not checked in the free run.

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

    Premisecitation

    Rubicon is an inhibitor of canonical autophagy and a key component of a non-canonical function of autophagy proteins that modulates inflammatory immune activation.

    “Rubicon has been shown to be an inhibitor of canonical autophagy (Matsunaga et al., 2009) and a key component of a non-canonical function of autophagy proteins that modulates inflammatory immune activation (Cunha et al., 2018; Martinez et al., 2016).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Matsunaga et al., 2009 source

      Not checked in the free run.

    • cites Cunha et al., 2018 source

      Not checked in the free run.

    • cites Martinez et al., 2016 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Microglia are the principal mediators of inflammation in response to Aβ accumulation, and their contribution to neuroinflammation is highly correlated with progression of neurodegeneration and synaptic dysfunction, particularly in Alzheimer's disease.

    “Microglia are the principal mediators of inflammation in response to Aβ accumulation (Machado et al., 2016; Perry and Holmes, 2014; Wang et al., 2015a), and their contribution to neuroinflammation is highly correlated to the progression of neurodegeneration and synaptic dysfunction, particularly with respect to Alzheimer’s disease (AD).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Machado et al., 2016 source

      Not checked in the free run.

    • cites Perry and Holmes, 2014

      Not checked in the free run.

    • cites Wang et al., 2015a

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    LANDO may contribute to not only the regulation of aberrant Aβ deposition but also the immune activation of microglial cells in response to Aβ exposure.

    “Taken as a whole, these data suggest LANDO may contribute to not only the regulation of aberrant Aβ deposition but also the immune activation of microglial cells in response to Aβ exposure.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Hyperphosphorylation of tau is a marker of progressive Alzheimer's disease in both humans and mice; its incidence increases as disease progresses and leads to microtubule de-stabilization and failure of the microtubule architecture, especially within neuronal axons.

    “A marker of progressive AD in both humans and mice is the hyperphosphorylation of the microtubule-stabilizing protein tau. The incidence of tau phosphorylation increases as disease progresses and leads to microtubule de-stabilization and failure of the microtubule architecture, especially within neuronal axons (Frost et al., 2015; Gong and Iqbal, 2008; Noble et al., 2013).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Frost et al., 2015 source

      Not checked in the free run.

    • cites Gong and Iqbal, 2008

      Not checked in the free run.

    • cites Noble et al., 2013 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    ATG5 and Rubicon are necessary for recruitment of LC3 to Aβ-containing endosomes, whereas FIP200 is dispensable.

    “Taken together, these data suggest that both ATG5 and Rubicon are necessary for recruitment of LC3 to Aβ-containing endosomes, whereas FIP200 is dispensable.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The requirements for recycling of TLR4, TREM2, and CD36 to the plasma membrane appear to be identical to those for LAP and distinct from those of autophagy.

    “Therefore, the requirements for recycling of these receptors to the plasma membrane appear to be identical to those for LAP and distinct from those of autophagy.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    LAP has a vital role in regulating inflammatory immune responses.

    “Since we have previously shown a vital role for LAP in regulating inflammatory immune responses (Martinez et al., 2016), we hypothesized that defective LANDO may have a similar effect.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Martinez et al., 2016 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Uptake of Aβ in BV2 cells occurs primarily through endocytosis (receptor-mediated endocytosis), as latrunculin A prevents zymosan internalization but has no effect on dextran or Aβ.

    “Inhibition of actin polymerization and phagocytosis using latrunculin A (de Oliveira and Mantovani, 1988; Oliveira et al., 1996), prevented the internalization of the phagocytic substrate zymosan but had no effect on either the endocytic substrate dextran or Aβ (Fig. 2C), suggesting that uptake of Aβ in our model occurs primarily through endocytosis (RME).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites de Oliveira and Mantovani, 1988

      Not checked in the free run.

    • cites Oliveira et al., 1996 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    LANDO facilitates the recycling of internalized Aβ receptors to the plasma membrane, supporting continued rounds of endocytosis.

    “Therefore, LANDO facilitates the recycling of internalized Aβ receptors to the plasma membrane, supporting continued rounds of endocytosis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    LANDO is distinct from LAP and may have a unique role in Aβ clearance in vivo.

    “Based on the association of LC3 to Aβ-containing endosomes, we propose the term LC3-Associated eNDOcytosis (LANDO) to describe this effect and suggest that LANDO is distinct from LAP and may have a unique role in Aβ clearance in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The results strongly suggest a role for microglial Rubicon and ATG5 in the clearance of Aβ and mitigation of microglial activation that is distinct from their functions in canonical autophagy.

    “Therefore, our results strongly suggest a role for microglial Rubicon and ATG5 in the clearance of Aβ and mitigation of microglial activation that is distinct from their functions in canonical autophagy.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Anhedonia can be analyzed in mice using the sucrose preference test (SPT).

    “which can be analyzed in mice using a sucrose preference test (SPT) (Briones et al., 2012; Liu et al., 2018).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Briones et al., 2012 source

      Not checked in the free run.

    • cites Liu et al., 2018 source

      Not checked in the free run.

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

    Premisecitation

    The CA3 field of the hippocampus has been implicated as one of the first sites of neuronal dysfunction and neuronal death in Alzheimer's disease.

    “This region has been implicated as one of the first sites of neuronal dysfunction and neuronal death in AD (Belvindrah et al., 2014; Padurariu et al., 2012; Zhang et al., 2013).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Belvindrah et al., 2014 source

      Not checked in the free run.

    • cites Padurariu et al., 2012

      Not checked in the free run.

    • cites Zhang et al., 2013 source

      Not checked in the free run.

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

    Premisenone given

    Tau phosphorylation is likely to promote axonal degeneration and eventual neuronal death.

    “Because tau phosphorylation is likely to promote axonal degeneration and eventual neuronal death, we analyzed cell death within the brains of our 5xFAD models.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Frost et al., 2015 source

      Not checked in the free run.

    • cites Gong and Iqbal, 2008

      Not checked in the free run.

    • cites Noble et al., 2013 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    These analyses illustrate the importance of molecular regulation of microglial function by LANDO in maintaining CNS integrity and immune function upon Aβ deposition, allowing for homeostasis in memory and behavior.

    “These analyses illustrate the importance for the molecular regulation of microglial function by LANDO in maintaining CNS integrity and immune function upon Aβ deposition, allowing for homeostasis in memory and behavior.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The paper's data support a physiological role for LANDO in microglia in mitigating microglial activation and immune-mediated aggregate Aβ removal.

    “Our data support a physiological role for LANDO in microglia in the mitigation and protection against microglial activation and immune-mediated aggregate Aβ) removal.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    In advanced AD, patients often complain of anhedonia.

    “In advanced AD, patients often complain of anhedonia, or the inability to sense or experience pleasure (Naudin et al., 2015; Reichman and Coyne, 1995)”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Naudin et al., 2015 source

      Not checked in the free run.

    • cites Reichman and Coyne, 1995

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    All animals were generated on the C57BL/6J background, confirmed by microsatellite analysis, so the differences observed do not reflect a background effect between strains.

    “Moreover, all animals were generated on the C57BL/6J background which was confirmed by microsatellite analysis, therefore the differences observed do not reflect a background effect between strains.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Increased cytokines, such as interleukin-1, can influence Aβ uptake.

    “In turn, increased cytokines, such as interleukin-1, can influence Aβ uptake (Mrak and Griffin, 2000; Shaftel et al., 2007).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Mrak and Griffin, 2000

      Not checked in the free run.

    • cites Shaftel et al., 2007 source

      Not checked in the free run.

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

    Premisecitation

    Similar effects were observed in response to dying cells in macrophages lacking Rac1 or the efferocytosis receptors merTK and TIM4.

    “Similar effects were observed in response to dying cells in macrophages lacking Rac1 or the efferocytosis receptors merTK and TIM4 (Cai et al., 2018; Camenisch et al., 1999; Martinez et al., 2011; Scott et al., 2001; Thorp et al., 2008), consistent with this idea.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Cai et al., 2018 source

      Not checked in the free run.

    • cites Camenisch et al., 1999 source

      Not checked in the free run.

    • cites Martinez et al., 2011 source

      Not checked in the free run.

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

    Premisecitation

    A previous study showed that recycling of CD36 and TREM2 is dependent on Beclin1 and VPS34.

    “A previous study had shown that this recycling of CD36 and TREM2 is dependent on Beclin1 and VPS34 (Lucin et al., 2013), which we confirmed in primary BMDM (Fig. 3).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Lucin et al., 2013 source

      Not checked in the free run.

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

    Premisecitation

    Whole body acute deletion of ATG7 causes lethal neuronal degeneration.

    “Whole body acute deletion of ATG7 causes lethal neuronal degeneration (Karsli-Uzunbas et al., 2014).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Karsli-Uzunbas et al., 2014 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    LANDO functions in microglia not only to promote Aβ clearance but also to promote an anti-inflammatory immune response.

    “Based on our findings using myeloid ATG5-deficiency and conditional ablation of Rubicon in microglia, we suggest that LANDO functions in microglia not only to promote Aβ clearance but also to promote an anti-inflammatory immune response.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Autophagy proteins have been shown to impact the clearance of protein aggregates in neurons, most likely by canonical aggrephagy.

    “While autophagy proteins have been shown to impact the clearance of protein aggregates in neurons, most likely by the canonical autophagic process of aggrephagy (Frake et al., 2015; Menzies et al., 2017; Qaisiya et al., 2017; Sarkar et al., 2007; Sarkar et al., 2009; Webb et al., 2003; Yamamoto and Simonsen, 2011), effects in microglia of the CNS are more likely due to LANDO.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Frake et al., 2015 source

      Not checked in the free run.

    • cites Menzies et al., 2017 source

      Not checked in the free run.

    • cites Qaisiya et al., 2017 source

      Not checked in the free run.

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

    Study design and conductnone given

    The assay used does not evaluate the role of LANDO in receptor degradation or other potential perturbations in endosome trafficking.

    “The assay used herein does not evaluate the role of LANDO in receptor degradation, or other potential perturbations in endosome trafficking, and it remains possible that such alterations alter receptor signaling to promote cytokine expression in response to Aβ.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    LANDO is distinct from canonical autophagy and plays a requisite role in the recycling of putative Aβ receptors.

    “We conclude that LANDO is distinct from canonical autophagy and plays a requisite role in the recycling of putative Aβ receptors.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    BV2 microglia deficient in FIP200, ATG5, and Rubicon were generated using CRISPR/Cas9, and transduction efficiency was confirmed by immunoblot analysis.

    “BV2 microglia deficient in FIP200, ATG5, and Rubicon were generated using CRISPR/Cas9 technology by lentiviral transduction and puromycin selection.”
    • Also stated in: § METHOD DETAILS · plain
      “BV2 cells were subsequently transduced and transduction efficiency was confirmed by immunoblot analysis following two weeks of puromycin selection.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All experiments were designed and powered to a minimum of 0.8 as calculated using G*Power.

    “All experiments were designed and are powered to a minimum of 0.8 as calculated using G*Power.”
    Quote in the paperNot a substantial claim

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

    Study design and conductcitation

    The membrane-associated LC3 analysis assay has been well-established and verified previously.

    “This assay has been well-established and verified previously (Eng et al., 2010; He et al., 2009; Martinez et al., 2015).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Eng et al., 2010 source

      Not checked in the free run.

    • cites He et al., 2009

      Not checked in the free run.

    • cites Martinez et al., 2015 source

      Not checked in the free run.

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

    Study design and conductnone given

    Following the receptor recycling protocol, fluorescent signal from recycled receptors is intracellular and readily determined by fluorescent microscopy.

    “Following the above protocol, fluorescent signal from recycled receptors is intracellular and readily determined by fluorescent microscopy as described above.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    MG-cre− animals were exposed to tamoxifen and allowed the same reconstitution period as MG-cre+ mice to ensure no off-target effects from tamoxifen administration.

    “MG-cre− animals used in the present study have been exposed to tamoxifen and allowed the same reconstitution period as the MG-cre+ mice to ensure no off-target effects from tamoxifen administration.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Specificity of the antibody targeting TREM2 was confirmed by staining wild-type and TREM2−/− BV2 microglial cells (data not shown).

    “Specificity of the antibody targeting the primary β-amyloid receptor TREM2 was confirmed by staining wild-type and TREM2 −/− BV2 microglial cells (data not shown).”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    All strains used had an overall background identity of greater than 97%.

    “All strains used had an overall background identity of greater than 97%.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    All cell lines used were confirmed as mycoplasma negative.

    “All the cell lines used were confirmed as mycoplasma negative using MycoAlert Mycoplasma Detection kit (Lonza #LT07).”
    Quote in the paperNot a substantial claim

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

    Study design and conductcitation

    The receptor recycling method is well-established and was used as previously reported by Lucin et al. (2013).

    “This method is well-established and used as previously reported (Lucin et al., 2013).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Lucin et al., 2013 source

      Not checked in the free run.

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

    Study design and conductnone given

    Mice with fewer than 5 arm entries during the 5-minute Y-maze trial were excluded from the analysis.

    “Mice with less than 5 arm entries during the 5 min. trial were excluded from the analysis.”
    Quote in the paperNot a substantial claim
68 of 68

  • The paper relies on this without support.

    BV2 murine microglial cells are a valid model of primary microglia for studying LANDO and Aβ responses.

    “we engineered BV2 murine microglial cells expressing GFP-LC3 that are deficient in FIP200, ATG5, or Rubicon using CRISPR/Cas9 (Fig. S2A).”

  • The paper relies on this without support.

    The 5xFAD mouse model is a well-characterized model of Aβ aggregate deposition and Alzheimer's disease.

    “We employed a well-characterized model of Aβ aggregate deposition, the 5xFAD AD mouse model with genetic manipulation of our target autophagy-related genes.”

  • The paper relies on this without support.

    The behavioral tests used are well-characterized and known to be affected at late stages in the 5xFAD model.

    “We therefore subjected mice to a variety of well-characterized behavioral tests known to be affected at late stages in the 5xFAD model.”

  • The paper relies on this without support.

    The commercially available antibodies for CD36, TLR4, and TREM2 used in the recycling experiments are validated and specific.

    “Furthermore, validated commercially available antibodies for CD36, TLR4, and TREM2 were used for all recycling experiments.”

  • The paper relies on this without support.

    Latrunculin A inhibits phagocytosis but not endocytosis, allowing delineation between the two processes.

    “To delineate between phagocytosis and endocytosis, cells were treated as indicated with the phagocytic inhibitor latrunculin A.”

  • The paper relies on this without support.

    Zymosan is a specific control substrate for phagocytosis and dextran is a specific control substrate for endocytosis.

    “The following control substrates were used, zymosan (phagocytosis) and dextran (endocytosis)”
6 of 6

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.

  • Conflicting sources for BV2 cells

    Minor

    The key resources table lists Washington University as the source of BV2 microglia, but the methods state that the BV2 cells were obtained from ATCC.

1 of 1

Cites

3 retracted

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1 of the 90 references couldn’t be matched to a known paper.

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Authors
Heckmann, B. L., Teubner, B. J. W., Tummers, B., Boada-Romero, E., Harris, L., Yang, M., Guy, C. S., Zakharenko, S. S., & Green, D. R.
Affiliations
St. Jude Children's Research Hospital (US)
Corresponding author
Green, Douglas R.
ORCID
Venue
Cell
Publisher
Elsevier BV
Published
2019-07
Volume
178
Issue
3
Pages
536-551.e14
ISSN
0092-8674 (ISSN-L)
DOI
10.1016/j.cell.2019.05.056
PMC
PMC6689199
OpenAlex
W2953948138
Semantic Scholar
f14bdb656ef7b729cf199d36d42394767974c4eb
Cited by
503
References
90
Citations by year
2026: 42 · 2025: 77 · 2024: 73 · 2023: 71 · 2022: 82 · 2021: 76 · 2020: 65 · 2019: 16 · 2018: 1
FWCI
40.96
Type
journal article
Language
en
Update notices
Open access
Licence
Locations
  • Cell (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • Lupus Research Alliance
  • American Lebanese Syrian Associated Charities
  • National Institutes of Health (AI138492, AI40646, CA231423, CA231620)
Topics
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
  • Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
Lando, Alzheimer's disease, amyloid-beta clearance, microglia, autophagy, neuroinflammation, reactive microgliosis, tau hyperphosphorylation, neurodegeneration, memory impairment, inflammatory cytokines
MeSH terms
RAW 264.7 Cells, Autophagy-Related Proteins, Autophagy-Related Protein 5, Alzheimer Disease, Animals, Disease Models, Animal, Endocytosis, Hippocampus, Membrane Glycoproteins, Mice, Transgenic, Microtubule-Associated Proteins, Receptors, Immunologic, Cytokines, Amyloid beta-Peptides, Microglia, CD36 Antigens, Intracellular Signaling Peptides and Proteins, Toll-Like Receptor 4, Mice

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 2026-10-10.

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

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