CoChat
Sign Up
Cited by 290Alzheimer's disease research and treatmentsDementia and Cognitive Impairment ResearchNeuroinflammation and Neurodegeneration Mechanisms

The DOI matches its Crossref record; 35 of the 36 references it cites were found in a registry or an index.

Detection of a Novel Intraneuronal Pool of Insoluble Amyloid β Protein that Accumulates with Time in Culture

Open access
Skovronsky, Daniel M., Doms, Robert W., Lee, Virginia M.-Y.·1998·The Journal of Cell Biology
DOI 10.1083/jcb.141.4.1031PMC PMC2132781
Open this paper in CoChat — free account

Abstract

The amyloid-β peptide (Aβ) is produced at several sites within cultured human NT2N neurons with Aβ1-42 specifically generated in the endoplasmic reticulum/intermediate compartment. Since Aβ is found as insoluble deposits in senile plaques of the AD brain, and the Aβ peptide can polymerize into insoluble fibrils in vitro, we examined the possibility that Aβ1-40, and particularly the more highly amyloidogenic Aβ1-42, accumulate in an insoluble pool within NT2N neurons. Remarkably, we found that formic acid extraction of the NT2N cells solubilized a pool of previously undetectable Aβ that accounted for over half of the total intracellular Aβ. Aβ1-42 was more abundant than Aβ1-40 in this pool, and most of the insoluble Aβ1-42 was generated in the endoplasmic reticulum/intermediate compartment pathway. High levels of insoluble Aβ were also detected in several nonneuronal cell lines engineered to overexpress the amyloid-β precursor protein. This insoluble intracellular pool of Aβ was exceptionally stable, and accumulated in NT2N neurons in a time-dependent manner, increasing 12-fold over a 7-wk period in culture. These novel findings suggest that Aβ amyloidogenesis may be initiated within living neurons rather than in the extracellular space. Thus, the data presented here require a reexamination of the prevailing view about the pathogenesis of Aβ deposition in the AD brain.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1083/jcb.141.4.1031

Cites

35 of 35 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1 FA extraction of NT2N neurons reveals a large pool of insoluble intracellular Aβ. (A) 10-cm dishes of NT2N cells (replate 2, 4 wk old) were either sequentially extracted in RIPA followed by FA, or lysed directly into FA. Aβ1-40 and Aβ1-42 levels in the RIPA and FA samples were quantified by sandwich ELISA. Mean results and standard errors are shown (six separate experiments, each done with duplicate samples). (B) 1-42/1-40 ratios were calculated for the RIPA soluble pool of Aβ, the RIPA insoluble (FA soluble) pool of Aβ, and the total intracellular pool of Aβ for these NT2N neurons, demonstrating that the insoluble intracellular pool of Aβ consists mainly of Aβ1-42, while the soluble pool contains similar amounts of Aβ1-40 and Aβ1-42.
Figure 1 FA extraction of NT2N neurons reveals a large pool of insoluble intracellular Aβ. (A) 10-cm dishes of NT2N cells (replate 2, 4 wk old) were either sequentially extracted in RIPA followed by FA, or lysed directly into FA. Aβ1-40 and Aβ1-42 levels in the RIPA and FA samples were quantified by sandwich ELISA. Mean results and standard errors are shown (six separate experiments, each done with duplicate samples). (B) 1-42/1-40 ratios were calculated for the RIPA soluble pool of Aβ, the RIPA insoluble (FA soluble) pool of Aβ, and the total intracellular pool of Aβ for these NT2N neurons, demonstrating that the insoluble intracellular pool of Aβ consists mainly of Aβ1-42, while the soluble pool contains similar amounts of Aβ1-40 and Aβ1-42.CC BY-NC-SA · PubMed Central
Figure 2 FA extraction of a variety of cell lines reveals the presence of varying levels of insoluble intracellular Aβ. (A) Uninfected and SFV-APPwt-infected NT2 cells, NT2N cells (replate 2, 4 wk old), CHO Pro5 cells, CHO-695 cells (uninfected only), and BHK-21 cells were sequentially extracted in RIPA followed by FA. Aβ 1-40 and 1-42 levels in the RIPA and FA samples were quantified by sandwich ELISA. Means and standard errors (four separate experiments done in triplicate) of Aβ levels are shown. (B) Samples from RIPA cell lysates of each of these cell lines (both uninfected and SFV-APPwt infected) were resolved on a 7.5% Tris-glycine acrylamide gel, and immunoblotted with Karen antibody; bands were detected by PhosphorImager after using an I125-labeled secondary antibody.
Figure 2 FA extraction of a variety of cell lines reveals the presence of varying levels of insoluble intracellular Aβ. (A) Uninfected and SFV-APPwt-infected NT2 cells, NT2N cells (replate 2, 4 wk old), CHO Pro5 cells, CHO-695 cells (uninfected only), and BHK-21 cells were sequentially extracted in RIPA followed by FA. Aβ 1-40 and 1-42 levels in the RIPA and FA samples were quantified by sandwich ELISA. Means and standard errors (four separate experiments done in triplicate) of Aβ levels are shown. (B) Samples from RIPA cell lysates of each of these cell lines (both uninfected and SFV-APPwt infected) were resolved on a 7.5% Tris-glycine acrylamide gel, and immunoblotted with Karen antibody; bands were detected by PhosphorImager after using an I125-labeled secondary antibody.CC BY-NC-SA · PubMed Central
Figure 3 Aβ can be recovered from RIPA-soluble and RIPA- insoluble (FA-solubilized) fractions of cell lysate. SFV-APPwt– infected NT2N cells and CHO Pro5 cells were metabolically labeled for 12 h before being lysed in either FA or RIPA, followed by extraction of insoluble material by FA. RIPA and FA samples were subjected to immunoprecipitation with 6E10, and were resolved on a 10/16.5% step gradient Tris-tricine gel. Molecular weight standards and Aβ bands are labeled.
Figure 3 Aβ can be recovered from RIPA-soluble and RIPA- insoluble (FA-solubilized) fractions of cell lysate. SFV-APPwt– infected NT2N cells and CHO Pro5 cells were metabolically labeled for 12 h before being lysed in either FA or RIPA, followed by extraction of insoluble material by FA. RIPA and FA samples were subjected to immunoprecipitation with 6E10, and were resolved on a 10/16.5% step gradient Tris-tricine gel. Molecular weight standards and Aβ bands are labeled.CC BY-NC-SA · PubMed Central
Figure 4 Insoluble Aβ1-42 can be produced by APPΔKK expressing NT2N and CHO cells. (A) N2TN cells and (B) CHO Pro5 cells were infected with SFV-APPwt or SFV-APPΔKK for 12 h before sequential extraction in RIPA, followed by FA. Aβ in each sample was quantified by sandwich-ELISA. Means and standard errors (two separate experiments each done in triplicate) are shown.
Figure 4 Insoluble Aβ1-42 can be produced by APPΔKK expressing NT2N and CHO cells. (A) N2TN cells and (B) CHO Pro5 cells were infected with SFV-APPwt or SFV-APPΔKK for 12 h before sequential extraction in RIPA, followed by FA. Aβ in each sample was quantified by sandwich-ELISA. Means and standard errors (two separate experiments each done in triplicate) are shown.CC BY-NC-SA · PubMed Central
Figure 5 Insoluble intracellular Aβ accumulates over time within NT2N cells. 10-cm dishes of Replate 2 NT2N cells were harvested each week 3–7 wk after replating. Cells were lysed in RIPA buffer, and insoluble material was resuspended in FA. Aβ1-40 (A) and 1-42 (B) in the soluble and insoluble intracellular pools were quantified by sandwich-ELISA. Data were normalized for total protein present (in mg). The experiment was repeated three times, and means and standard errors (three to five samples per time point) are shown for a representative experiment.
Figure 5 Insoluble intracellular Aβ accumulates over time within NT2N cells. 10-cm dishes of Replate 2 NT2N cells were harvested each week 3–7 wk after replating. Cells were lysed in RIPA buffer, and insoluble material was resuspended in FA. Aβ1-40 (A) and 1-42 (B) in the soluble and insoluble intracellular pools were quantified by sandwich-ELISA. Data were normalized for total protein present (in mg). The experiment was repeated three times, and means and standard errors (three to five samples per time point) are shown for a representative experiment.CC BY-NC-SA · PubMed Central
Figure 6 The insoluble pool of intracellular Aβ is stable over 24 h, while the soluble pool of cellular Aβ turns over more rapidly. 10-cm dishes of Replate 2 NT2N cells were treated with 150 μg/ml cyclohexamide for the times indicated before being sequentially extracted in RIPA and FA. Aβ1-40 and 1-42 in the soluble (A) and insoluble intracellular pools (B) were quantified by sandwich-ELISA. The experiment was repeated three times, and means and standard errors (three samples per time point) are shown for a representative experiment.
Figure 6 The insoluble pool of intracellular Aβ is stable over 24 h, while the soluble pool of cellular Aβ turns over more rapidly. 10-cm dishes of Replate 2 NT2N cells were treated with 150 μg/ml cyclohexamide for the times indicated before being sequentially extracted in RIPA and FA. Aβ1-40 and 1-42 in the soluble (A) and insoluble intracellular pools (B) were quantified by sandwich-ELISA. The experiment was repeated three times, and means and standard errors (three samples per time point) are shown for a representative experiment.CC BY-NC-SA · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

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.

The self-check needs the paper’s full text.

Citations

Cites

Research works printed but not linked: 1

  1. Kim, 1988, Production and characterization of monoclonal antibodies reactive to synthetic cerebrovascular amyloid peptide, Neurosci Res Commun, 2, 121

Cited by

Sorted by citations

See all 290

Related

See all

Metadata

Authors
Skovronsky, D. M., Doms, R. W., & Lee, V. M.-Y.
Affiliations
University of Pennsylvania (US)
ORCID
Lee, Virginia M.-Y.
Venue
The Journal of Cell Biology
Publisher
Rockefeller University Press
Published
1998-05-18
Volume
141
Issue
4
Pages
1031-1039
ISSN
  • 0021-9525 (ISSN-L)
  • 1540-8140
DOI
10.1083/jcb.141.4.1031
PMC
PMC2132781
OpenAlex
W2130792655
Cited by
290
References
36
Citations by year
2026: 2 · 2025: 4 · 2024: 4 · 2023: 4 · 2022: 6 · 2021: 3 · 2019: 8 · 2018: 2 · 2017: 4 · 2016: 1 · 2015: 6 · 2014: 3 · 2013: 8 · 2012: 8
FWCI
15.16
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Cell Biology (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Health Sciences › Medicine › Psychiatry and Mental health › Dementia and Cognitive Impairment Research
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
amyloid-beta, amyloid-beta 42, Aβ40, endoplasmic reticulum, amyloidogenesis, Alzheimer's disease, amyloid plaques, amyloid precursor protein overexpression, formic acid extraction
MeSH terms
Alzheimer Disease, Animals, Brain, Cell Line, Endoplasmic Reticulum, Genetic Vectors, Cricetinae, Humans, Neurons, Peptide Fragments, Recombinant Proteins, Semliki forest virus, Solubility, Subcellular Fractions, Transfection, Tumor Cells, Cultured, Amyloid beta-Peptides, CHO Cells, Carcinoma, Embryonal

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.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review