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Cited by 111Genetics and Neurodevelopmental DisordersAlzheimer's disease research and treatmentsNeuroscience and Neuropharmacology Research

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Reversal of Fragile X Phenotypes by Manipulation of AβPP/Aβ Levels in Fmr1KO Mice

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Westmark, Cara J., Westmark, Pamela R., O'Riordan, Kenneth J., Ray, Brian C., Hervey, Crystal M., Salamat, M. Shahriar, Abozeid, Sara H., Stein, Kelsey M., Stodola, Levi A., Tranfaglia, Michael, Burger, Corinna, Berry-Kravis, Elizabeth M., Malter, James S.·2011·PLoS ONE
DOI 10.1371/journal.pone.0026549PMC PMC3202540
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Abstract

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and the leading known genetic cause of autism. Fragile X mental retardation protein (FMRP), which is absent or expressed at substantially reduced levels in FXS, binds to and controls the postsynaptic translation of amyloid β-protein precursor (AβPP) mRNA. Cleavage of AβPP can produce β-amyloid (Aβ), a 39-43 amino acid peptide mis-expressed in Alzheimer's disease (AD) and Down syndrome (DS). Aβ is over-expressed in the brain of Fmr1(KO) mice, suggesting a pathogenic role in FXS. To determine if genetic reduction of AβPP/Aβ rescues characteristic FXS phenotypes, we assessed audiogenic seizures (AGS), anxiety, the ratio of mature versus immature dendritic spines and metabotropic glutamate receptor (mGluR)-mediated long-term depression (LTD) in Fmr1(KO) mice after removal of one App allele. All of these phenotypes were partially or completely reverted to normal. Plasma Aβ(1-42) was significantly reduced in full-mutation FXS males compared to age-matched controls while cortical and hippocampal levels were somewhat increased, suggesting that Aβ is sequestered in the brain. Evolving therapies directed at reducing Aβ in AD may be applicable to FXS and Aβ may serve as a plasma-based biomarker to facilitate disease diagnosis or assess therapeutic efficacy.

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Figures

Figure 1 AGS are rescued by genetic manipulation of App or mGluR5 blockade. (A) western blot analyses of AβPP levels in Fmr1KO, WT, AppHE T and Fmr1KO/AppHET mice (n = 3 male mice per strain, 1 month old). Statistics: one-way ANOVA p<0.0001, F = 53.95. Stars (★) denote statistically different levels by Student T-test analyses and by Bonferroni's multiple comparison test (p<0.05). Error bars represent SEM. (B) Assessment of WR, AGS and death rates in Fmr1KO mice (age P21, n = 58)) after genetic manipulation of AβPP levels (n = 23) or treatment with MPEP (n = 14). Stars (★) denote rates that are statistically different from untreated mice (p<0.5) by Fisher exact tests.
Figure 1 AGS are rescued by genetic manipulation of App or mGluR5 blockade. (A) western blot analyses of AβPP levels in Fmr1KO, WT, AppHE T and Fmr1KO/AppHET mice (n = 3 male mice per strain, 1 month old). Statistics: one-way ANOVA p<0.0001, F = 53.95. Stars (★) denote statistically different levels by Student T-test analyses and by Bonferroni's multiple comparison test (p<0.05). Error bars represent SEM. (B) Assessment of WR, AGS and death rates in Fmr1KO mice (age P21, n = 58)) after genetic manipulation of AβPP levels (n = 23) or treatment with MPEP (n = 14). Stars (★) denote rates that are statistically different from untreated mice (p<0.5) by Fisher exact tests.CC BY · PubMed Central
Figure 2 Rescue of behavioral phenotypes in adult Fmr1KO mice by genetic manipulation of App. (A) male AppKO (n = 8), AppHET (n = 14), WT (n = 7), Fmr-1KO (n = 8), Fmr1KO/AppHET (n = 10) and Fmr1KO/AppKO (n = 4) mice (8–10 weeks old) were assessed for marble burying activity. Statistics: one-way ANOVA p<0.022, F = 2.95. (B, C and D) Mice were assessed for anxiety levels and locomotion in the open field. Both genders were included as the males and females exhibited equivalent locomotion and anxiety in the open field [AppKO (n = 11), AppHET(n = 23), WT (n = 15), Fmr1KO (n = 14), Fmr-1KO/AppHET (n = 18) and Fmr1KO/AppKO (n = 7)]. (B) Total distance (cm) traveled is plotted against mouse strain. Statistics: one-way ANOVA p<0.0005, F = 4.59. (C) Time (sec) spent in the center of the arena is plotted versus mouse strain. Statistics: one-way ANOVA p<0.0001, F = 5.98. (D) Distance (cm) traveled in the center of the arena is plotted versus mouse strain. Statistics: one-way ANOVA p<0.0001, F = 9.17. Stars (★) denote statistically different levels by Student T-test analyses (p<0.5). All error bars represent SEM.
Figure 2 Rescue of behavioral phenotypes in adult Fmr1KO mice by genetic manipulation of App. (A) male AppKO (n = 8), AppHET (n = 14), WT (n = 7), Fmr-1KO (n = 8), Fmr1KO/AppHET (n = 10) and Fmr1KO/AppKO (n = 4) mice (8–10 weeks old) were assessed for marble burying activity. Statistics: one-way ANOVA p<0.022, F = 2.95. (B, C and D) Mice were assessed for anxiety levels and locomotion in the open field. Both genders were included as the males and females exhibited equivalent locomotion and anxiety in the open field [AppKO (n = 11), AppHET(n = 23), WT (n = 15), Fmr1KO (n = 14), Fmr-1KO/AppHET (n = 18) and Fmr1KO/AppKO (n = 7)]. (B) Total distance (cm) traveled is plotted against mouse strain. Statistics: one-way ANOVA p<0.0005, F = 4.59. (C) Time (sec) spent in the center of the arena is plotted versus mouse strain. Statistics: one-way ANOVA p<0.0001, F = 5.98. (D) Distance (cm) traveled in the center of the arena is plotted versus mouse strain. Statistics: one-way ANOVA p<0.0001, F = 9.17. Stars (★) denote statistically different levels by Student T-test analyses (p<0.5). All error bars represent SEM.CC BY · PubMed Central
Figure 3 Dendritic spine morphology is partially rescued in Fmr1KO/AppHET or MPEP treated Fmr1KO mice. (A) Representative fluorescent images of primary cultured neurons prepared from WT (upper left), Fmr1KO (upper right and lower left) and Fmr1KO/AppHET (lower right) embryos stained with DiI and visualized by fluorescent microscopy (100× objective). The arrows denote dendritic spines. (B) The lengths of dendritic protrusions were quantitated with StereoInvestigator software and plotted against mouse strain/treatment. The percentage of filopodia versus spines for each condition is given below the histogram. Statistics: one-way ANOVA comparison of the three genotypes (untreated) p<0.0001, F = 27.18. All genotypes are statistically different from each other by Student T-Test and Bonferroni's multiple comparison tests. Two-way ANOVA comparison of WT versus Fmr1KO ± MPEP: p<0.0001, F = 12.89 (interaction), F = 35.01 (genotype) and F = 27.62 (MPEP). The untreated and 15 min MPEP treated WT spines are statistically different from the corresponding Fmr1KO spines by the Bonferroni multiple comparison test (p<0.5). Stars (★) denote statistically different spine lengths by Student T-test analyses (p<0.5). Error bars indicate SEM [Fmr1KO: untreated (n = 746 spines), 15 min MPEP (n = 263), 1 hr MPEP (n = 300), 4 hr MPEP (n = 293); WT: untreated (n = 994), 15 min MPEP (n = 535), 1 hr MPEP (n = 373), 4 hr MPEP (n = 1221); Fmr1KO/AppHET (n = 2469)].
Figure 3 Dendritic spine morphology is partially rescued in Fmr1KO/AppHET or MPEP treated Fmr1KO mice. (A) Representative fluorescent images of primary cultured neurons prepared from WT (upper left), Fmr1KO (upper right and lower left) and Fmr1KO/AppHET (lower right) embryos stained with DiI and visualized by fluorescent microscopy (100× objective). The arrows denote dendritic spines. (B) The lengths of dendritic protrusions were quantitated with StereoInvestigator software and plotted against mouse strain/treatment. The percentage of filopodia versus spines for each condition is given below the histogram. Statistics: one-way ANOVA comparison of the three genotypes (untreated) p<0.0001, F = 27.18. All genotypes are statistically different from each other by Student T-Test and Bonferroni's multiple comparison tests. Two-way ANOVA comparison of WT versus Fmr1KO ± MPEP: p<0.0001, F = 12.89 (interaction), F = 35.01 (genotype) and F = 27.62 (MPEP). The untreated and 15 min MPEP treated WT spines are statistically different from the corresponding Fmr1KO spines by the Bonferroni multiple comparison test (p<0.5). Stars (★) denote statistically different spine lengths by Student T-test analyses (p<0.5). Error bars indicate SEM [Fmr1KO: untreated (n = 746 spines), 15 min MPEP (n = 263), 1 hr MPEP (n = 300), 4 hr MPEP (n = 293); WT: untreated (n = 994), 15 min MPEP (n = 535), 1 hr MPEP (n = 373), 4 hr MPEP (n = 1221); Fmr1KO/AppHET (n = 2469)].CC BY · PubMed Central
Figure 4 Rescue of mGluR-dependent synaptic depression in the CA1 in Fmr1KO/AppHET mice. (Top) Representative fEPSP taken before the addition of DHPG (solid lines) and at the end of the recordings (dotted lines). (Bottom) Time course of CA1 fEPSP slope after incubation with DHPG (50 µM, 10 min) in hippocampal slices from WT (black circles) (n = 20), Fmr1KO (white circles) (n = 13), AppHET (black triangles) (n = 15) and Fmr1KO/AppHET (white triangles) (n = 10) male mice (3 months old). WT, AppHET and Fmr1KO/AppHET were all statistically different from Fmr1KO by two-way ANOVA/Bonferroni multiple comparison tests. WT versus AppHET, p = 0.92; WT versus Fmr1KO, p<0.006; WT versus Fmr1KO/AppHET, p<0.02; AppHET versus Fmr1KO/AppHET, p<0.04; AppHET versus Fmr1KO, p = 0.02; Fmr1KO versus Fmr1KO/AppHET, p<0.0002. Error bars indicate SEM.
Figure 4 Rescue of mGluR-dependent synaptic depression in the CA1 in Fmr1KO/AppHET mice. (Top) Representative fEPSP taken before the addition of DHPG (solid lines) and at the end of the recordings (dotted lines). (Bottom) Time course of CA1 fEPSP slope after incubation with DHPG (50 µM, 10 min) in hippocampal slices from WT (black circles) (n = 20), Fmr1KO (white circles) (n = 13), AppHET (black triangles) (n = 15) and Fmr1KO/AppHET (white triangles) (n = 10) male mice (3 months old). WT, AppHET and Fmr1KO/AppHET were all statistically different from Fmr1KO by two-way ANOVA/Bonferroni multiple comparison tests. WT versus AppHET, p = 0.92; WT versus Fmr1KO, p<0.006; WT versus Fmr1KO/AppHET, p<0.02; AppHET versus Fmr1KO/AppHET, p<0.04; AppHET versus Fmr1KO, p = 0.02; Fmr1KO versus Fmr1KO/AppHET, p<0.0002. Error bars indicate SEM.CC BY · PubMed Central
Figure 5 Aβ1–42 alters dendritic protein levels. (A) WT neuronal cells treated with vehicle, 20 or 100 nM Aβ1–42 for 1 hr followed by fixation and staining for AβPP, Arc/Arg, Map1B, RhoB and PSD95 and analyses by confocal fluorescent microscopy. (B) WT neuronal cells pre-treated with vehicle, 2.5 µM MPEP or 40 µM anisomycin for 15 min prior to treatment with 20 nM Aβ1–42 for 1 hr and fixation and staining for AβPP. Statistics: one-way ANOVA p<0.0001, F = 7.04. All treatments are statistically different from the 20 nM Aβ1–42 treatment by the Bonferroni multiple comparison test (p<0.05). A minimum of 1793 particles were analyzed per treatment cohort. (C) WT neuronal cells treated with mouse IgG or anti-Aβ antibody in transwells for 3 days prior to fixation and staining for AβPP. Statistics: one-way ANOVA p<0.05, F = 3.03. A minimum of 2,644 particles were analyzed per treatment cohort. Stars (★) denote statistical differences by Student T-test analyses (p<0.5). Error bars indicate SEM.
Figure 5 Aβ1–42 alters dendritic protein levels. (A) WT neuronal cells treated with vehicle, 20 or 100 nM Aβ1–42 for 1 hr followed by fixation and staining for AβPP, Arc/Arg, Map1B, RhoB and PSD95 and analyses by confocal fluorescent microscopy. (B) WT neuronal cells pre-treated with vehicle, 2.5 µM MPEP or 40 µM anisomycin for 15 min prior to treatment with 20 nM Aβ1–42 for 1 hr and fixation and staining for AβPP. Statistics: one-way ANOVA p<0.0001, F = 7.04. All treatments are statistically different from the 20 nM Aβ1–42 treatment by the Bonferroni multiple comparison test (p<0.05). A minimum of 1793 particles were analyzed per treatment cohort. (C) WT neuronal cells treated with mouse IgG or anti-Aβ antibody in transwells for 3 days prior to fixation and staining for AβPP. Statistics: one-way ANOVA p<0.05, F = 3.03. A minimum of 2,644 particles were analyzed per treatment cohort. Stars (★) denote statistical differences by Student T-test analyses (p<0.5). Error bars indicate SEM.CC BY · PubMed Central
Figure 6 AβPP/Aβ are abnormal in blood and brain from FXS patients. (A) AβPPα levels in control (n = 7) and FXS (n = 10) plasma and PBMC culture media (n = 7 controls and 4 FXS) and AβPP in PBMC (n = 7 controls and 5 FXS). For the plasma and PBMC culture media data, AβPPα is expressed as ng/mL and for the PBMC lysate measurements, AβPP is expressed as ng/mL/µg lysate. (B) Aβ1–40 and Aβ1–42 levels in control (n = 7) and FXS (n = 10) plasma. For Aβ1–42, p<0.004 as determined by Student T-Test analyses. (C) AβPP/AβPPα in control (n = 3) and FXS (n = 4) neocortex and hippocampus. Controls were gender- and age-matched to FXS donors by the University of Maryland Brain Bank. All of the donors were Caucasian males and their ages ranged from 21–85 years old. (D) Soluble Aβ1–40 levels in control (n = 3) and FXS (n = 4) neocortex and hippocampus. White bars = control samples and gray bars = FXS. Error bars represent SEM.
Figure 6 AβPP/Aβ are abnormal in blood and brain from FXS patients. (A) AβPPα levels in control (n = 7) and FXS (n = 10) plasma and PBMC culture media (n = 7 controls and 4 FXS) and AβPP in PBMC (n = 7 controls and 5 FXS). For the plasma and PBMC culture media data, AβPPα is expressed as ng/mL and for the PBMC lysate measurements, AβPP is expressed as ng/mL/µg lysate. (B) Aβ1–40 and Aβ1–42 levels in control (n = 7) and FXS (n = 10) plasma. For Aβ1–42, p<0.004 as determined by Student T-Test analyses. (C) AβPP/AβPPα in control (n = 3) and FXS (n = 4) neocortex and hippocampus. Controls were gender- and age-matched to FXS donors by the University of Maryland Brain Bank. All of the donors were Caucasian males and their ages ranged from 21–85 years old. (D) Soluble Aβ1–40 levels in control (n = 3) and FXS (n = 4) neocortex and hippocampus. White bars = control samples and gray bars = FXS. Error bars represent SEM.CC BY · PubMed Central

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Research works printed but not linked: 2

  1. [1]Hagerman RJ Hagerman PJ 2002 Physical and behavioral phenotype Baltimore John Hopkins University Press 3 109
  2. [69]Miller LJ McIntosh DN McGrath J Shyu V Lampe M 1999 Electrodermal responses to sensory stimuli in individuals with fragile X syndrome: A preliminary report. Am J Med Genet 83 4 268 279 10208160

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Metadata

Authors
Westmark, C. J., Westmark, P. R., O'Riordan, K. J., Ray, B. C., Hervey, C. M., Salamat, M. S., Abozeid, S. H., Stein, K. M., Stodola, L. A., Tranfaglia, M., Burger, C., Berry-Kravis, E. M., & Malter, J. S.
Affiliations
  • University of Wisconsin–Madison (US)
  • Rush University Medical Center (US)
  • Fraxa Research Foundation (US)
Corresponding author
Westmark, Cara J.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2011-10-26
Volume
6
Issue
10
Pages
e26549
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0026549
PMC
PMC3202540
OpenAlex
W1966255973
Cited by
110
References
92
Citations by year
2026: 3 · 2025: 3 · 2024: 2 · 2023: 6 · 2022: 5 · 2021: 5 · 2020: 4 · 2019: 5 · 2018: 6 · 2017: 9 · 2016: 13 · 2015: 16 · 2014: 13 · 2013: 11 · 2012: 8
FWCI
6.38
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • FRAXA Research Foundation
  • Waisman Center (P30 HD03352, HD03352)
  • National Institutes of Health (HD033.52)
  • National Institute on Drug Abuse (R01DA026067)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (P30HD03352., P30HD003352)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Keywords
fragile X syndrome, FMRP, amyloid precursor protein, amyloid-beta, Fmr1 knockout mice, audiogenic seizures, anxiety, dendritic spines, metabotropic glutamate receptors, long-term depression, plasma biomarkers, intellectual disability, autism spectrum disorder, Alzheimer's disease
MeSH terms
Animals, Brain Chemistry, Female, Fragile X Syndrome, Male, Neurons, Peptide Fragments, Phenotype, Genetic Therapy, Down-Regulation, Amyloid beta-Peptides, Amyloid beta-Protein Precursor, Receptors, Metabotropic Glutamate, Mice, Knockout, Dendritic Spines, Mice, Fragile X Mental Retardation Protein, Fragile X Messenger Ribonucleoprotein 1

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