Cited by 142Alzheimer's disease research and treatmentsDementia and Cognitive Impairment ResearchCholinesterase and Neurodegenerative Diseases

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Phospho-eIF2α Level Is Important for Determining Abilities of BACE1 Reduction to Rescue Cholinergic Neurodegeneration and Memory Defects in 5XFAD Mice

Open access
Devi, Latha, Ohno, Masuo·2010·PLoS ONE

Abstract

β-Site APP-cleaving enzyme 1 (BACE1) initiates amyloid-β (Aβ) generation and thus represents a prime therapeutic target in treating Alzheimer's disease (AD). Notably, increasing evidence indicates that BACE1 levels become elevated in AD brains as disease progresses; however, it remains unclear how the BACE1 upregulation may affect efficacies of therapeutic interventions including BACE1-inhibiting approaches. Here, we crossed heterozygous BACE1 knockout mice with AD transgenic mice (5XFAD model) and compared the abilities of partial BACE1 reduction to rescue AD-like phenotypes at earlier (6-month-old) and advanced (15-18-month-old) stages of disease, which expressed normal (∼100%) and elevated (∼200%) levels of BACE1, respectively. BACE1(+/-) deletion rescued memory deficits as tested by the spontaneous alternation Y-maze task in 5XFAD mice at the earlier stage and prevented their septohippocampal cholinergic deficits associated with significant neuronal loss. Importantly, BACE1(+/-) deletion was no longer able to rescue memory deficits or cholinergic neurodegeneration in 5XFAD mice at the advanced stage. Moreover, BACE1(+/-) deletion significantly reduced levels of Aβ42 and the β-secretase-cleaved C-terminal fragment (C99) in 6-month-old 5XFAD mouse brains, while these neurotoxic β-cleavage products dramatically elevated with age and were not affected by BACE1(+/-) deletion in 15-18-month-old 5XFAD brains. Interestingly, although BACE1(+/-) deletion lowered BACE1 expression by ∼50% in 5XFAD mice irrespective of age in concordance with the reduction in gene copy number, BACE1 equivalent to wild-type controls remained in BACE1(+/-)·5XFAD mice at the advanced age. In accord, phosphorylation of the translation initiation factor eIF2α, an important mediator of BACE1 elevation, was dramatically increased (∼9-fold) in 15-18-month-old 5XFAD mice and remained highly upregulated (∼6-fold) in age-matched BACE1(+/-)·5XFAD mice. Together, our results indicate that partial reduction of BACE1 is not sufficient to block the phospho-eIF2α-dependent BACE1 elevation during the progression of AD, thus limiting its abilities to reduce cerebral Aβ/C99 levels and rescue memory deficits and cholinergic neurodegeneration.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

Partial (heterozygous) BACE1 reduction rescued memory deficits and septohippocampal cholinergic neurodegeneration in 6-month-old 5XFAD mice but failed to do so at 15–18 months, when BACE1 and phospho-eIF2α were elevated. The results suggest that phospho-eIF2α-driven BACE1 elevation limits the efficacy of partial BACE1 suppression as Alzheimer’s-like disease progresses.

Key findings

  • BACE1+/− deletion rescued spontaneous alternation Y-maze deficits in 6-month-old 5XFAD mice, but not in 15–18-month-old 5XFAD mice (no significant difference between 5XFAD and BACE1+/−·5XFAD, p = 0.24).
  • BACE1+/− deletion restored hippocampal ChAT levels and ChAT-positive Ch1/2 neuron numbers in 6-month-old 5XFAD mice, but not at 15–18 months (ChAT trend p = 0.10; neuron number p = 0.32).
  • BACE1+/− deletion reduced C99 by ∼53% and Aβ42 by ∼67% in 6-month-old 5XFAD brains; at 15–18 months, reductions were only ∼18% for C99 and ∼20% for Aβ42 and not significant (p = 0.14 and p = 0.19).
  • BACE1 levels were ∼200% of wild-type in 15–18-month-old 5XFAD mice; BACE1+/− reduced BACE1 by ∼50% but residual BACE1 remained equivalent to wild-type. Phospho-eIF2α was ∼9-fold elevated in old 5XFAD and remained ∼6-fold elevated in BACE1+/−·5XFAD; Sal 003-induced phospho-eIF2α elevation increased BACE1 in 6-month-old 5XFAD slices.

Methods

  • Crossbred heterozygous BACE1 knockout mice with 5XFAD transgenic mice to generate wild-type, BACE1+/−, 5XFAD, and BACE1+/−·5XFAD genotypes; tested at 6 months and 15–18 months of age.
  • Behavioral testing used the spontaneous alternation Y-maze; biochemical measures included immunoblot for BACE1, APP, C99, phospho-eIF2α, ChAT, and β-actin; ChAT immunohistochemistry in medial septum/vertical limb of diagonal band; and Aβ42 sandwich ELISA.
  • Brain slices from 6-month-old 5XFAD mice were treated with Sal 003 (30 µM) to inhibit phospho-eIF2α phosphatase; statistical analysis used one-way ANOVA with post-hoc Fisher’s PLSD, p < 0.05.

Limitations

  • The paper does not state a dedicated limitations section.
  • Evident from the text: findings are restricted to the 5XFAD mouse model and two age points (6 months and 15–18 months), with small group sizes (n = 4–12 per group in reported experiments).
  • Evident from the text: BACE1 reduction was achieved by heterozygous genetic deletion, not pharmacological BACE1 inhibition, so direct clinical dosing implications remain untested.

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Integrity signals

Registry

Crossref · 10.1371/journal.pone.0012974

Cites

61 of 61 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1 Age-related effects of BACE1+/− deletion on memory deficits in 5XFAD mice. Memory function of mice at 6 months (A, C) and 15–18 months (B, D) of age was tested using the spontaneous alternation Y-maze task. (A, B) Spatial working memory, as assessed by the spontaneous alternation performance, is significantly impaired (around 50% chance levels) in 5XFAD mice irrespective of age compared to wild-type controls (* p<0.05). Note that BACE1+/−·5XFAD are rescued completely back to wild-type levels of alternation performance at 6 months but not at 15–18 months of age (# p<0.05 versus age-matched 5XFAD). n = 5–12 mice per group. (C, D) Total number of arm entries reflecting exploratory activities of mice in the Y-maze does not differ between the four groups irrespective of age. n = 5–12 mice per group. All data are presented as mean ± SEM.
Figure 1 Age-related effects of BACE1+/− deletion on memory deficits in 5XFAD mice. Memory function of mice at 6 months (A, C) and 15–18 months (B, D) of age was tested using the spontaneous alternation Y-maze task. (A, B) Spatial working memory, as assessed by the spontaneous alternation performance, is significantly impaired (around 50% chance levels) in 5XFAD mice irrespective of age compared to wild-type controls (* p<0.05). Note that BACE1+/−·5XFAD are rescued completely back to wild-type levels of alternation performance at 6 months but not at 15–18 months of age (# p<0.05 versus age-matched 5XFAD). n = 5–12 mice per group. (C, D) Total number of arm entries reflecting exploratory activities of mice in the Y-maze does not differ between the four groups irrespective of age. n = 5–12 mice per group. All data are presented as mean ± SEM.CC BY · PubMed Central
Figure 2 Age-related effects of BACE1+/− deletion on cholinergic neurodegeneration in 5XFAD mice. (A, B) Immunoblot analysis of ChAT in hippocampal lysates from wild-type mice and 5XFAD mice with BACE1+/+ or BACE1+/− genotype. Intensities of ChAT-immunoreactive bands of mice at 6 months (A) and 15–18 months (B) of age were quantified by phosphorimaging and expressed as percentage of wild-type levels (n = 6–9 mice per group). (C, D) Brain sections from wild-type mice and 5XFAD mice with BACE1+/+ or BACE1+/− genotype were immunostained for ChAT. Shown are representative photomicrographs of ChAT-immunoreactive neurons in the medial septum of mice at 6 months (C) and 15–18 months (D) of age. Scale bar = 200 µm. The number of ChAT-positive neurons in the medial septum and the vertical limb of the diagonal band (Ch1/2), which provide the cholinergic innervation to the hippocampus, was counted for quantification (n = 4–7 mice per group). Both ChAT measures are significantly reduced in 5XFAD mice irrespective of age as compared with wild-type controls (* p<0.05). Note that ChAT is restored completely back to wild-type levels in BACE1+/−·5XFAD mice at 6 months but not at 15–18 months of age (# p<0.05 versus age-matched 5XFAD). All data are presented as mean ± SEM.
Figure 2 Age-related effects of BACE1+/− deletion on cholinergic neurodegeneration in 5XFAD mice. (A, B) Immunoblot analysis of ChAT in hippocampal lysates from wild-type mice and 5XFAD mice with BACE1+/+ or BACE1+/− genotype. Intensities of ChAT-immunoreactive bands of mice at 6 months (A) and 15–18 months (B) of age were quantified by phosphorimaging and expressed as percentage of wild-type levels (n = 6–9 mice per group). (C, D) Brain sections from wild-type mice and 5XFAD mice with BACE1+/+ or BACE1+/− genotype were immunostained for ChAT. Shown are representative photomicrographs of ChAT-immunoreactive neurons in the medial septum of mice at 6 months (C) and 15–18 months (D) of age. Scale bar = 200 µm. The number of ChAT-positive neurons in the medial septum and the vertical limb of the diagonal band (Ch1/2), which provide the cholinergic innervation to the hippocampus, was counted for quantification (n = 4–7 mice per group). Both ChAT measures are significantly reduced in 5XFAD mice irrespective of age as compared with wild-type controls (* p<0.05). Note that ChAT is restored completely back to wild-type levels in BACE1+/−·5XFAD mice at 6 months but not at 15–18 months of age (# p<0.05 versus age-matched 5XFAD). All data are presented as mean ± SEM.CC BY · PubMed Central
Figure 3 Age-related effects of BACE1+/− deletion on APP processing and Aβ levels in 5XFAD mice. (A) Immunoblot analysis of hemibrain lysates from 5XFAD mice with BACE1+/+ or BACE1+/− genotype at 6 months and 15–18 months of age. (B, C) Intensities of immunoreactive bands for full-length APP (B) and C99 (C) were quantified by phosphorimaging and expressed as percentage of 6-month-old 5XFAD mouse levels (n = 5–7 mice per group). (D) Levels of total Aβ42 were quantified by sandwich ELISA of guanidine extracts of hemibrain samples and expressed in nanograms per milligram of total protein (n = 4 mice per group). APP overexpression levels are not changed in BACE1+/−·5XFAD mice irrespective of age as compared to 5XFAD mice. Levels of C99 and Aβ42 are significantly lower in BACE1+/−·5XFAD mice at 6 months of age as compared with 5XFAD wild-type controls (# p<0.05). Note that C99 and Aβ42 levels are significantly elevated in 15–18-month-old 5XFAD mouse brains compared to 6-month-old 5XFAD brains (* p<0.05), while BACE1+/− deletion is no longer able to reduce levels of the β-cleavage products in 5XFAD mice at the advanced age. All data are presented as mean ± SEM.
Figure 3 Age-related effects of BACE1+/− deletion on APP processing and Aβ levels in 5XFAD mice. (A) Immunoblot analysis of hemibrain lysates from 5XFAD mice with BACE1+/+ or BACE1+/− genotype at 6 months and 15–18 months of age. (B, C) Intensities of immunoreactive bands for full-length APP (B) and C99 (C) were quantified by phosphorimaging and expressed as percentage of 6-month-old 5XFAD mouse levels (n = 5–7 mice per group). (D) Levels of total Aβ42 were quantified by sandwich ELISA of guanidine extracts of hemibrain samples and expressed in nanograms per milligram of total protein (n = 4 mice per group). APP overexpression levels are not changed in BACE1+/−·5XFAD mice irrespective of age as compared to 5XFAD mice. Levels of C99 and Aβ42 are significantly lower in BACE1+/−·5XFAD mice at 6 months of age as compared with 5XFAD wild-type controls (# p<0.05). Note that C99 and Aβ42 levels are significantly elevated in 15–18-month-old 5XFAD mouse brains compared to 6-month-old 5XFAD brains (* p<0.05), while BACE1+/− deletion is no longer able to reduce levels of the β-cleavage products in 5XFAD mice at the advanced age. All data are presented as mean ± SEM.CC BY · PubMed Central
Figure 4 Age-related changes in BACE1 levels and the modification by BACE1+/− deletion in 5XFAD mice. (A, B) Immunoblot analysis of BACE1 in hemibrain lysates from wild-type mice and 5XFAD mice with BACE1+/+ or BACE1+/− genotype. Intensities of BACE1-immunoreactive bands of mice at 6 months (A) and 15–18 months (B) of age were quantified by phosphorimaging and expressed as percentage of wild-type levels (n = 4–8 mice per group). BACE1+/− deletion reduced BACE1 expression by ∼50% in 5XFAD mice irrespective age as compared with age-matched 5XFAD mice (# p<0.05). However, since BACE1 expression is significantly elevated up to ∼200% in 15–18-month-old 5XFAD mouse brains (* p<0.05 versus wild-type), levels of BACE1 equivalent to wild-type controls remain in BACE1+/−·5XFAD brains at the advanced age. All data are presented as mean ± SEM.
Figure 4 Age-related changes in BACE1 levels and the modification by BACE1+/− deletion in 5XFAD mice. (A, B) Immunoblot analysis of BACE1 in hemibrain lysates from wild-type mice and 5XFAD mice with BACE1+/+ or BACE1+/− genotype. Intensities of BACE1-immunoreactive bands of mice at 6 months (A) and 15–18 months (B) of age were quantified by phosphorimaging and expressed as percentage of wild-type levels (n = 4–8 mice per group). BACE1+/− deletion reduced BACE1 expression by ∼50% in 5XFAD mice irrespective age as compared with age-matched 5XFAD mice (# p<0.05). However, since BACE1 expression is significantly elevated up to ∼200% in 15–18-month-old 5XFAD mouse brains (* p<0.05 versus wild-type), levels of BACE1 equivalent to wild-type controls remain in BACE1+/−·5XFAD brains at the advanced age. All data are presented as mean ± SEM.CC BY · PubMed Central
Figure 5 Age-related changes in phospho-eIF2α levels and the modification by BACE1+/− deletion in 5XFAD mice. (A, B) Immunoblot analysis of phospho-eIF2α (p-eIF2α) in hemibrain lysates from wild-type mice and 5XFAD mice with BACE1+/+ or BACE1+/− genotype. Intensities of p-eIF2α-immunoreactive bands of mice at 6 months (A) and 15–18 months (B) of age were quantified by phosphorimaging and expressed as percentage of wild-type levels (n = 4–9 mice per group). While changes in p-eIF2α levels are marginal at 6 months of age, p-eIF2α levels are dramatically elevated in 15–18-month-old 5XFAD (∼9-fold relative to wild-type controls) and BACE1+/−·5XFAD (∼6-fold) mouse brains. * p<0.05 versus wild-type, # p<0.05 versus 5XFAD. All data are presented as mean ± SEM. (C) After brain slices from 6-month-old 5XFAD mice were exposed to normal aCSF or Sal 003-containing aCSF (30 µM), they were lysed for immunoblot analysis. The eIF2α phosphatase inhibitor Sal 003-induced increase in p-eIF2α level caused the elevation of BACE1 in 5XFAD mouse brains.
Figure 5 Age-related changes in phospho-eIF2α levels and the modification by BACE1+/− deletion in 5XFAD mice. (A, B) Immunoblot analysis of phospho-eIF2α (p-eIF2α) in hemibrain lysates from wild-type mice and 5XFAD mice with BACE1+/+ or BACE1+/− genotype. Intensities of p-eIF2α-immunoreactive bands of mice at 6 months (A) and 15–18 months (B) of age were quantified by phosphorimaging and expressed as percentage of wild-type levels (n = 4–9 mice per group). While changes in p-eIF2α levels are marginal at 6 months of age, p-eIF2α levels are dramatically elevated in 15–18-month-old 5XFAD (∼9-fold relative to wild-type controls) and BACE1+/−·5XFAD (∼6-fold) mouse brains. * p<0.05 versus wild-type, # p<0.05 versus 5XFAD. All data are presented as mean ± SEM. (C) After brain slices from 6-month-old 5XFAD mice were exposed to normal aCSF or Sal 003-containing aCSF (30 µM), they were lysed for immunoblot analysis. The eIF2α phosphatase inhibitor Sal 003-induced increase in p-eIF2α level caused the elevation of BACE1 in 5XFAD mouse brains.CC BY · PubMed Central

Verification

Claims the paper makes

40 Own result · 22 Own interpretation · 20 Not checked yet · 1 Own method · 1 No basis given

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Show all 56 claims

  • It may state two separate claims as one. 18
  • Probably not a substantial claim. 12
28 of 28
  • The paper relies on this without support.

    Approximately 50% BACE1 suppression is likely efficacious.

    “Given ∼50% BACE1 suppression generally accepted as it is likely efficacious, we used BACE1+/− mice as a model system to evaluate the efficacies of partial and therapeutic inhibition of BACE1.”
  • The paper relies on this without support.

    C99 is a direct β-cleavage metabolite of APP.

    “Since C99 is a direct β-cleavage metabolite of APP, the high residual levels of C99 may reflect some accumulating species of C99 (similar to Aβ peptides) that could be less sensitive to reduction by BACE1+/− deletion in 15–18-month-old 5XFAD mice.”
  • The paper relies on this without support.

    These phenotypes (liabilities of complete BACE1 deletion) are not observed in BACE1+/− knockout mice.

    “However, these phenotypes are not observed in BACE1+/− knockout mice, suggesting that potential mechanism-based side effects might occur only in completely abolishing BACE1 activity but not in partially inhibiting it.”
  • The paper relies on this without support.

    The mechanisms for cholinergic neuron loss in AD are largely unknown.

    “however, the mechanisms for cholinergic neuron loss is yet largely unknown.”
4 of 4

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Statistics recomputed

Every reported test result, recomputed from its test statistic and degrees of freedom. A result shows as not adding up only once an independent check has confirmed it.

  • ConsistentF(3, 32) = 4.78reported p < .05 · recomputed p = .007

    “F (3,32) = 4.78, p < .05”

  • ConsistentF(3, 33) = 11.38reported p < .05 · recomputed p < .001

    “F (3,33) = 11.38, p < .05”

  • ConsistentF(2, 18) = 8.89reported p < .05 · recomputed p = .002

    “F (2,18) = 8.89, p < .05”

  • ConsistentF(2, 24) = 10.51reported p < .05 · recomputed p < .001

    “F (2,24) = 10.51, p < .05”

  • ConsistentF(2, 13) = 9.8reported p < .05 · recomputed p = .003

    “F (2,13) = 9.80, p < .05”

  • ConsistentF(2, 13) = 8.49reported p < .05 · recomputed p = .004

    “F (2,13) = 8.49, p < .05”

  • ConsistentF(1, 10) = 7.37reported p < .05 · recomputed p = .022

    “F (1,10) = 7.37, p < .05”

  • ConsistentF(1, 10) = 10.71reported p < .05 · recomputed p = .008

    “F (1,10) = 10.71, p < .05”

  • ConsistentF(1, 9) = 2.57reported p = .14 · recomputed p = .143

    “F (1,9) = 2.57, p = .14”

  • ConsistentF(1, 6) = 48.41reported p < .05 · recomputed p < .001

    “F (1,6) = 48.41, p < .05”

  • ConsistentF(1, 6) = 23.53reported p < .05 · recomputed p = .003

    “F (1,6) = 23.53, p < .05”

  • ConsistentF(1, 6) = 2.23reported p = .19 · recomputed p = .186

    “F (1,6) = 2.23, p = .19”

  • ConsistentF(2, 18) = 11.25reported p < .05 · recomputed p < .001

    “F (2,18) = 11.25, p < .05”

  • ConsistentF(2, 11) = 31.91reported p < .05 · recomputed p < .001

    “F (2,11) = 31.91, p < .05”

  • ConsistentF(2, 11) = 25.76reported p < .05 · recomputed p < .001

    “F (2,11) = 25.76, p < .05”

  • ConsistentF(2, 21) = 11.72reported p < .05 · recomputed p < .001

    “F (2,21) = 11.72, p < .05”

16 of 16

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Metadata

Authors
Devi, L., & Ohno, M.
Affiliations
New York University (US)
Corresponding author
Ohno, Masuo
Venue
PLoS ONE
Published
2010-09-23
Volume
5
Issue
9
Pages
e12974
ISSN
1932-6203 (ISSN-L)
OpenAlex
W2012590437
Cited by
140
References
61
Citations by year
2026: 8 · 2025: 8 · 2024: 14 · 2023: 9 · 2022: 16 · 2021: 11 · 2020: 8 · 2019: 8 · 2018: 5 · 2017: 9 · 2016: 6 · 2015: 12 · 2014: 10 · 2013: 6 · 2012: 4
FWCI
3.25
Type
journal article
Language
en
Open access
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Alzheimer's Association (IIRG-08-91231)
  • National Institutes of Health
  • National Institute of Mental Health (R01MH067251)
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
  • Health Sciences › Medicine › Pharmacology (medical) › Cholinesterase and Neurodegenerative Diseases
Keywords
eIF2α phosphorylation, Alzheimer's disease, 5xFAD mice, amyloid-beta, cholinergic neurodegeneration, memory impairment, C99, amyloid-beta 42, translation initiation, gene dosage, BACE1 inhibition
MeSH terms
Alzheimer Disease, Animals, Cholinergic Fibers, Disease Models, Animal, Female, Humans, Male, Memory, Mice, Inbred C57BL, Mice, Transgenic, Nerve Degeneration, Phosphorylation, Eukaryotic Initiation Factor-2, Aspartic Acid Endopeptidases, Mice, Knockout, Mice, Amyloid Precursor Protein Secretases

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