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Cited by 83Alzheimer's disease research and treatmentsAutophagy in Disease and TherapyNeuroinflammation and Neurodegeneration Mechanisms

NRBF2 is involved in the autophagic degradation process of APP-CTFs in Alzheimer disease models

Open access
Yang, Chuanbin, Cai, Cui-Zan, Song, Ju-Xian, Tan, Jie-Qiong, Durairajan, Siva Sundara Kumar, Iyaswamy, Ashok, Wu, Ming-Yue, Chen, Lei-Lei +3 more·2017·Autophagy
DOI 10.1080/15548627.2017.1379633PMC PMC5788544
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Abstract

Alzheimer disease (AD) is the most common neurodegenerative disease characterized by the deposition of amyloid plaque in the brain. The autophagy-associated PIK3C3-containing phosphatidylinositol 3-kinase (PtdIns3K) complex has been shown to interfere with APP metabolism and amyloid beta peptide (Aβ) homeostasis via poorly understood mechanisms. Here we report that NRBF2 (nuclear receptor binding factor 2), a key component and regulator of the PtdIns3K, is involved in APP-CTFs homeostasis in AD cell models. We found that NRBF2 interacts with APP in vivo and its expression levels are reduced in hippocampus of 5XFAD AD mice; we further demonstrated that NRBF2 overexpression promotes degradation of APP C-terminal fragments (APP-CTFs), and reduces Aβ1–40 and Aβ1-42 levels in human mutant APP-overexpressing cells. Conversely, APP-CTFs, Aβ1–40 and Aβ1-42 levels were increased in Nrbf2 knockdown or nrbf2 knockout cells. Furthermore, NRBF2 positively regulates autophagy in neuronal cells and NRBF2-mediated reduction of APP-CTFs levels is autophagy dependent. Importantly, nrbf2 knockout attenuates the recruitment of APP and APP-CTFs into phagophores and the sorting of APP and APP-CTFs into endosomal intralumenal vesicles, which is accompanied by the accumulation of the APP and APP-CTFs into RAB5-positive early endosomes. Collectively, our results reveal the potential connection between NRBF2 and the AD-associated protein APP by showing that NRBF2 plays an important role in regulating degradation of APP-CTFs through modulating autophagy.

AI snapshot

Written from the paper’s full text.

TL;DR

NRBF2, a PtdIns3K component and positive autophagy regulator, is reduced in the hippocampus of 5XFAD Alzheimer disease mice and regulates APP-CTF homeostasis in AD cell models. NRBF2 overexpression lowers APP-CTFs and Aβ1-40/Aβ1-42, while Nrbf2 knockdown/knockout increases them, with effects dependent on autophagy and on APP/APP-CTF trafficking into autophagic and endosomal degradative compartments.

Key findings

  • NRBF2 protein levels were significantly reduced in the hippocampus of 12-mo-old 5XFAD mice (n = 6) versus wild-type C57BL/6 mice; BECN1 was also reduced, ATG12–ATG5 was unchanged, and LC3B-II and SQSTM1/p62 were increased.
  • Nrbf2 overexpression reduced APP-CTFα and APP-CTFβ and intracellular/extracellular Aβ1-40 and Aβ1-42 in N2S and 7PA2 cells, without affecting FL-APP, sAPPα, sAPPβ, or PSEN1 levels. Nrbf2 knockdown produced about 60% NRBF2 reduction and about 50% increases in APP-CTFα/β; nrbf2 knockout increased APP-CTFs and intracellular/secreted Aβ1-40/Aβ1-42, without affecting FL-APP or γ-secretase activity.
  • NRBF2 positively regulated autophagy in neuronal N2S cells: Nrbf2 knockdown/knockout increased SQSTM1 and attenuated starvation-induced LC3B-II increase/SQSTM1 reduction, while Nrbf2 overexpression lowered SQSTM1 and increased LC3 puncta.
  • NRBF2-mediated APP-CTF reduction was autophagy dependent: chloroquine and Atg5 knockdown reversed it; nrbf2 knockout increased APP-CTF half-life and the APP-CTFs:FL-APP ratio. NRBF2 interacted with APP in 5XFAD brains and HEK293 cells, and nrbf2 knockout reduced APP/APP-CTF recruitment into phagophores and sorting into endosomal ILVs, with accumulation in RAB5-positive early endosomes and reduced distribution in RAB7- and LAMP1-positive late endosomes/lysosomes.

Methods

  • Cell models: mouse neuroblastoma N2a cells stably expressing human Swedish mutant APP695 (N2S), and CHO cells stably expressing human APP751 with V717F (7PA2); Nrbf2 knockdown by siRNA and knockout by lentivirus-mediated CRISPR/Cas9.
  • Animal model: 5XFAD AD mice and C57BL/6J controls; hippocampus from 12-mo-old mice was analyzed (n = 6 per group).
  • Main assays: western blotting, co-immunoprecipitation, ELISA for Aβ1-40/Aβ1-42, γ-secretase luciferase assay, LysoTracker Red flow cytometry, immunocytochemistry for colocalization, cycloheximide half-life, chloroquine treatment, and Atg5 knockdown; statistics used one-way ANOVA followed by Student-Newman-Keuls test, with n ≥ 3 independent experiments and mean ± SEM.

Limitations

  • Stated: whether NRBF2 levels are reduced in AD patient brains remains to be examined; mechanisms by which NRBF2 specifically affects APP-CTFα/β rather than FL-APP need further elucidation; other methods are required to confirm NRBF2’s roles in APP/APP-CTF redistribution and whether it specifically affects APP/APP-CTFs versus other endocytic proteins; how NRBF2 mediates redistribution into different organelles is largely unclear; the NRBF2–AD connection needs confirmation using nrbf2 KO AD animal models.
  • Stated: NRBF2–APP-CTF interaction could not be detected by co-immunoprecipitation, possibly due to fast APP-CTF turnover.
  • Evident: the study relies mainly on cell models and 5XFAD mouse hippocampus, without human AD tissue validation; the authors note about 5% changes in FL-APP with NRBF2 overexpression or knockout were not statistically significant, so they do not fully exclude an effect on FL-APP degradation.

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

Integrity signals

Registry

Crossref · 10.1080/15548627.2017.1379633

Cites

59 of 59 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 16 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Nrbf2 overexpression does not affect FL-APP levels in N2S cells.

    “but did not affect the levels of FL-APP (Fig. 2A andB).”
    • Also stated in: § Statistical analysis · plain
      “Quantitative data in Fig. 2A showed that NRBF2 overexpression significantly reduced APP-CTFs levels but not FL-APP levels.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: N2S cells stably expressing human Swedish mutant APP695 are a valid model for studying APP processing relevant to Alzheimer disease.
  • Findingown data

    Nrbf2 overexpression significantly lowers cellular SQSTM1.

    “Nrbf2 overexpression significantly lowered cellular SQSTM1 (Fig. 4G andH).”
    Quote in the paperSubstantial claim
  • Findingown data

    nrbf2 KO does not affect lysosomal numbers as reflected by LysoTracker Red staining.

    “nrbf2 KO did not affect lysosomal numbers as reflected by LysoTracker Red staining (Fig. 5C)”
    Quote in the paperSubstantial claim
  • Findingown data

    Cellular APP-CTFa and APP-CTFb levels are increased about 50% in Nrbf2 KD cells.

    “the cellular APP-CTFa and APP-CTFb levels were increased about 50% in Nrbf2 KD cells (Fig. 3C andD).”
    Quote in the paperSubstantial claim
  • Findingown data

    Nrbf2 overexpression reduces Ab 1-40 and Ab 1-42 levels in human mutant APP-overexpressing cells.

    “reduces Ab 1-40 and Ab 1-42 levels in human mutant APP-overexpressing cells.”
    Quote in the paperSubstantial claim
  • May combine two claims. 11
  • Classified as probably not a substantial claim. 10

  • May combine two claims.

    Findingown data

    Nrbf2 overexpression lowers both intracellular and extracellular Ab40 levels.

    “overexpression of Nrbf2 lowered both the intracellular and extracellular Ab40 levels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    nrbf2 KO dramatically increases APP-CTFa and APP-CTFb levels but not FL-APP levels.

    “KO of nrbf2 dramatically increased APP-CTFa and APP-CTFb levels but not FL-APP levels (Fig. 3F andG).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    nrbf2 KO dramatically increases SQSTM1 protein levels rather than mRNA levels.

    “nrbf2 KO also dramatically increased SQSTM1 protein levels (Fig. 4E andF) rather than mRNA levels (Fig. S3)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Nrbf2 overexpression decreases APP-CTFa and APP-CTFb, as well as intracellular and extracellular Ab 1-40 and Ab 1-42 levels in different cells.

    “These results suggest that Nrbf2 overexpression decreases APP-CTFa and APP-CTFb, as well as intracellular and extracellular Ab 1-40 and Ab 1-42 levels in different cells.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisereasoning

    Autophagy dysfunction has been implicated in the pathogenesis of AD.

    “Autophagy dysfunction has been implicated in the pathogenesis of AD.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    5XFAD mice co-express 5 mutations of familial AD and recapitulate major features of AD amyloid pathology.

    “5XFAD mice co-express 5 mutations of familial AD, which recapitulate major features of AD amyloid pathology. 31”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [31] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    nrbf2 KO results in the accumulation of APP and APP-CTFs in early endosomes and attenuates the sorting of APP and APP-CTFs into late endosomes/lysosomes for degradation.

    “these results suggest that nrbf2 KO results in the accumulation of APP and APP-CTFs in early endosomes and attenuates the sorting of APP and APP-CTFs into late endosomes/lysosomes for degradation.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The lumen of these endosomes contains the ILVs.

    “The lumen of these endosomes contains the ILVs. 46”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [46] source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisereasoning

    APP-CTFs levels and their subcellular locations are essential for Ab production.

    “Thus, APP-CTFs (hereafter referred to as APP-CTFa and APP-CTFb) levels and their subcellular locations are essential for Ab production.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [4][5][6][7]

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductown data

    nrbf2 knockout in the expanded colonies was confirmed by western blotting.

    “nrbf2 knockout in the expanded colonies was confirmed by western blotting.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All animal experiments were approved by the Committee on the Use of Human & Animal Subjects in Teaching and Research (HASC) of Hong Kong Baptist University.

    “All animal experiments were approved by the Committee on the Use of Human & Animal Subjects in Teaching and Research (HASC) of Hong Kong Baptist University.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    NRBF2 is a novel component of the PtdIns3K complex and associates with ATG14 in both mammalian cells and yeast.

    “NRBF2 is a novel component of the PtdIns3K complex, and associates with ATG14 in both mammalian cells [25][26][27][28] and in yeast (where it is named Atg38).29”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [25][26][27][28]

      Not checked in the free run.

    • cites [29] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    nrbf2 KO attenuates the degradation of APP-CTFs via reducing the recruitment of APP and APP-CTFs into both phagophores and ILVs, and increasing the distribution of APP and APP-CTFs into RAB5-positive early endosomes, which may further affect the degradation of APP-CTFs by lysosomes and the processing of APP in different organelles.

    “Collectively, nrbf2 KO attenuates the degradation of APP-CTFs via reducing the recruitment of APP and APP-CTFs into both phagophores and ILVs, and increasing the distribution of APP and APP-CTFs into RAB5-positive early endosomes, which may further affect the degradation of APP-CTFs by lysosomes and the processing of APP in different organelles.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Ab 1-40 accounts for about 90% of all Ab fragments generated from APP-CTFb in amyloid plaques.

    “Ab 1-40 accounts for about 90% of all Ab fragments generated from APP-CTFb in amyloid plaques. 36”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] source

      Not checked in the free run.

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

    Premisecitation

    N2S cells are commonly used as an AD cell model.

    “N2a mouse neuroblastoma cells stably expressing the human Swedish mutant APP695 (N2S cells), which is commonly used as an AD cell model. 35”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    NRBF2 interacts with APP in both 5XFAD mice and neuronal cells, and the interaction may contribute to the redistribution of APP and APP-CTFs in nrbf2 KO cells.

    “We found that NRBF2 interacts with APP in both 5XFAD mice and in neuronal cells, and the interaction may contribute to the redistribution of APP and APP-CTFs in nrbf2 KO cells.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Cells expressing the active GTP-bound form of RAB5, RAB5 Q79L, show an enlargement of endosomes that is commonly used to facilitate discrimination between the limiting membrane and the lumen of these organelles.

    “Cells expressing the active (GTP-bound) form of RAB5, RAB5 Q79L , show an enlargement of endosomes, which is commonly used to facilitate the discrimination between the limiting membrane and the lumen of these organelles. 44,45”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [44] source

      Not checked in the free run.

    • cites [45] source

      Not checked in the free run.

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

    Premisecitation

    APP is a type I transmembrane protein that can be processed by either non-amyloidogenic processing or amyloidogenic processing.

    “APP is a type I transmembrane protein that can be processed by either non-amyloidogenic processing or amyloidogenic processing.[4][5][6]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4][5][6]

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    By regulating autophagy and interaction with APP, nrbf2 KO attenuates the degradation of APP-CTFs via reducing the recruitment of APP and APP-CTFs into phagophores and sorting into ILVs, and increasing the distribution of APP and APP-CTFs into RAB5-positive early endosomes.

    “By regulating autophagy and interaction with APP, nrbf2 KO attenuates the degradation of APP-CTFs via reducing the recruitment of APP and APP-CTFs into phagophores and sorting into ILVs, and increasing the distribution of APP and APP-CTFs into RAB5-positive early endosomes.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: APP is processed by a-secretase and BACE1/b-secretase to generate sAPPa/APP-CTFa and sAPPb/APP-CTFb, and APP-CTFb is cleaved by g-secretase to generate Ab.
19 of 19

  • The paper relies on this without support.

    The roles and mechanisms of the PtdIns3K complex components involved in autophagy initiation and regulating APP metabolism are unclear.

    “In addition, though autophagy has been implicated in Ab metabolism, the roles and mechanisms of the PtdIns3K complex components involved in autophagy initiation and regulating APP metabolism are unclear.”

  • The paper relies on this without support.

    Whether NRBF2 is a positive or negative regulator in neuronal cells remains unclear.

    “Whether NRBF2 is a positive or negative regulator in neuronal cells remains unclear.”

  • The paper relies on this without support.

    PtdIns3P levels in the brains of AD patients and animal models are reduced.

    “PtdIns3P levels in the brains of AD patients and animal models are reduced.”

  • The paper relies on this without support.

    The 5XFAD mouse model is a valid model of Alzheimer disease for studying NRBF2 and APP-CTF metabolism.

    “NRBF2 protein levels are reduced in the hippocampus region of 5XFAD AD mice.”

  • The paper relies on this without support.

    N2S cells stably expressing human Swedish mutant APP695 are a valid model for studying APP processing relevant to Alzheimer disease.

    “After N2a mouse neuroblastoma cells stably expressing human Swedish mutant APP695 (N2S cells) were transfected with Flag-NRBF2 plasmid or vehicle plasmid, the expression of NRBF2, full-length APP (FL-APP), APP-CTFa, and APP-CTFb were examined by western blotting; ACTB was used as an internal control.”

  • The paper relies on this without support.

    7PA2 cells are a valid model for studying APP processing relevant to Alzheimer disease.

    “Overexpression of Nrbf2 in Chinese hamster ovary cells stably transfected with a plasmid encoding human APP751 bearing the V717F mutation (7PA2 cells) reduced the expression of APP-CTFs.”

  • The paper relies on this without support.

    APP is processed by a-secretase and BACE1/b-secretase to generate sAPPa/APP-CTFa and sAPPb/APP-CTFb, and APP-CTFb is cleaved by g-secretase to generate Ab.

    “Full-length amyloid precursor protein (FL-APP) can be processed by either a-secretase (non-amyloidogenic processing) or BACE1/b-secretase (amyloidogenic processing) to generate sAPPa/APP-CTFa and sAPPb/APP-CTFb, respectively.APP-CTFb can be further cleaved to generate Ab by g-secretase.”
7 of 7

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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.

  • Figure 5E cites the wrong source panel

    Minor

    The caption says Figure 5E quantifies panel C, which measures lysosomal numbers. Panel D, not C, examines CQ's effect on Nrbf2-mediated APP-CTF degradation.

  • Figure 2G is assigned to different cell models

    Minor

    The Results describe Figures 2F and 2G as confirming the findings in 7PA2 cells, but the Figure 2 caption says panel G reports ELISA results in N2S cells.

  • Figure 3I-J describes overexpression instead of knockout

    Minor

    The Results attribute the measurements in Figures 3I and 3J to nrbf2 knockout, whereas the figure caption attributes them to Nrbf2 overexpression.

3 of 3

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. Pickford F, 2008, J Clin Invest, 118, 2190
  2. 10.1523/JNEUROSCI.0800-08.2008. · DOI · Paper page

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Related

Authors
Yang, C., Cai, C.-Z., Song, J.-X., Tan, J.-Q., Durairajan, S. S. K., Iyaswamy, A., Wu, M.-Y., Chen, L.-L., Yue, Z., Li, M., & Lu, J.-H.
Affiliations
  • Hong Kong Baptist University (HK)
  • University of Macau (MO)
  • State Key Laboratory of Medical Genetics, Xiangya Medical School, Central South University, Changsha, Hunan, China
  • Mr. and Mrs. Ko Chi Ming Centre for Parkinson's Disease Research, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China
  • Icahn School of Medicine at Mount Sinai (US)
  • State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau SAR, China
Corresponding author
Li, Min
ORCID
Venue
Autophagy
Publisher
Informa UK Limited
Published
2017-12-02
Volume
13
Issue
12
Pages
2028-2040
ISSN
  • 1554-8627 (ISSN-L)
  • 1554-8635
DOI
10.1080/15548627.2017.1379633
PMC
PMC5788544
OpenAlex
W2764155369
Semantic Scholar
fbaf978f14e3e5eb6191f7b56e639a53141027fa
Cited by
83
References
59
Citations by year
2026: 3 · 2025: 8 · 2024: 7 · 2023: 3 · 2022: 16 · 2021: 21 · 2020: 12 · 2019: 8 · 2018: 5
FWCI
5.06
Type
journal article
Language
en
Open access
Locations
  • Autophagy (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • HKBU Institutional Repository (Hong Kong Baptist University) (acceptedVersion)
Funders
  • Hong Kong Baptist University (EF001/ICMS-LJH/2015/HKBU)
  • National Natural Science Foundation of China (NSFC-31500831, 31500831, 2014-2016)
  • Fundo para o Desenvolvimento das Ciências e da Tecnologia (092/2015/A3, NSFC-31500831, 022/2015/A1, FDCT-092-2015-A3, FDCT-022/2015/A1)
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
Alzheimer's disease, APP C-terminal fragments, amyloid-beta, early endosomes, phagophore, 5xFAD mice, hippocampus, APP overexpression
MeSH terms
Autophagy-Related Proteins, Alzheimer Disease, Animals, Autophagy, Disease Models, Animal, Hippocampus, Humans, Mice, Transgenic, Models, Biological, Neurons, Peptide Fragments, Protein Binding, Endosomes, Transcription Factors, Trans-Activators, Amyloid beta-Peptides, Proteolysis

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