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Articles, Neurobiology of Disease

Life Extension Factor Klotho Prevents Mortality and Enhances Cognition in hAPP Transgenic Mice

Dena B. Dubal, Lei Zhu, Pascal E. Sanchez, Kurtresha Worden, Lauren Broestl, Erik Johnson, Kaitlyn Ho, Gui-Qiu Yu, Daniel Kim, Alexander Betourne, Makoto Kuro-o, Eliezer Masliah, Carmela R. Abraham and Lennart Mucke
Journal of Neuroscience 11 February 2015, 35 (6) 2358-2371; DOI: https://doi.org/10.1523/JNEUROSCI.5791-12.2015
Dena B. Dubal
1Gladstone Institute of Neurological Disease, San Francisco, California 94158,
2Department of Neurology, University of California, San Francisco, California 94158,
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Lei Zhu
1Gladstone Institute of Neurological Disease, San Francisco, California 94158,
2Department of Neurology, University of California, San Francisco, California 94158,
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Pascal E. Sanchez
1Gladstone Institute of Neurological Disease, San Francisco, California 94158,
2Department of Neurology, University of California, San Francisco, California 94158,
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Kurtresha Worden
1Gladstone Institute of Neurological Disease, San Francisco, California 94158,
2Department of Neurology, University of California, San Francisco, California 94158,
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  • ORCID record for Kurtresha Worden
Lauren Broestl
2Department of Neurology, University of California, San Francisco, California 94158,
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Erik Johnson
1Gladstone Institute of Neurological Disease, San Francisco, California 94158,
2Department of Neurology, University of California, San Francisco, California 94158,
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Kaitlyn Ho
1Gladstone Institute of Neurological Disease, San Francisco, California 94158,
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Gui-Qiu Yu
1Gladstone Institute of Neurological Disease, San Francisco, California 94158,
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Daniel Kim
1Gladstone Institute of Neurological Disease, San Francisco, California 94158,
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Alexander Betourne
2Department of Neurology, University of California, San Francisco, California 94158,
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Makoto Kuro-o
3Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75390,
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Eliezer Masliah
4Departments of Neurosciences and Pathology, University of California, San Diego, San Diego, California 92093, and
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Carmela R. Abraham
5Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
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Lennart Mucke
1Gladstone Institute of Neurological Disease, San Francisco, California 94158,
2Department of Neurology, University of California, San Francisco, California 94158,
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    Figure 1.

    Klotho is decreased in the DG of hAPP mice. Klotho protein levels in dentate gyrus homogenates from NTG and hAPP mice (n = 10–13 mice per genotype, age 5–7 months) were determined by Western blot analysis. Actin was used as a loading control. Representative Western blots (top) and relative levels of mouse klotho determined by densitometric quantitations of Western blot signals (bottom). Mean levels in controls were arbitrarily defined as 1.0; *p < 0.05 (t test). Bar graphs represent mean ± SEM.

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    Figure 2.

    Elevating klotho reduces mortality and improves network dysfunction in hAPP mice. A, Hippocampal klotho levels in KL and hAPP/KL mice determined by Western blot analysis and expressed relative to mean levels in NTG controls (n = 7–14 mice per genotype, age 3 months). KL effect p < 0.0001 by two-way ANOVA. B, Kaplan–Meier curves showing differences in survival between weaning and 12.5 months of age among the four genotypes indicated (n = 366 mice: 111 NTG, 67 hAPP, 110 KL, and 78 hAPP/KL; p < 0.0001 by log rank test; hAPP vs all other groups p < 0.05). Proportional hazard testing revealed the KL effect in hAPP mice to be independent of age (p = 0.5). C, Representative EEG traces recorded over the parietal cortex depicted with a compressed time scale. Gray arrowheads indicate abnormal spikes. D, Frequency of spikes recorded during 24 h measured from uncompressed, raw EEG traces (n = 3–7 mice per genotype, age 2–3.5 months). Two-way ANOVA: hAPP effect p < 0.01; *p < 0.05, ***p < 0.001 by Bonferroni-Holm test (A) or Welch's t test (D). Bar graphs represent mean ± SEM.

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    Figure 3.

    Klotho elevation improves cognitive and behavioral abnormalities in hAPP mice. A–C, NTG, KL, hAPP, and hAPP/KL mice (n = 14–18 per genotype) were tested in the Morris water maze at 4–7 months. A, Spatial learning curves (platform hidden). Data points represent the daily average of total distance traveled to reach the platform in four trials. Mixed-model ANOVA: hAPP effect p < 0.001, KL effect p < 0.0001, hAPP by KL interaction p < 0.05; hAPP versus all other groups p < 0.001, NTG versus KL p = 0.06 (Bonferroni-Holm test). B, C, Probe trial (platform removed) results obtained 24 h after completion of hidden-platform training. B, Latency to reach the original platform location. Two-way ANOVA: hAPP effect p < 0.05, KL effect p < 0.001, hAPP by KL interaction p = 0.05; *p < 0.05 versus NTG, **p < 0.01 versus hAPP (Bonferroni-Holm test). C, Percentage of time mice spent in the target quadrant (black) versus the average percentage of time they spent in the other three quadrants (white); **p < 0.01, *p < 0.05 versus 25% (two-tailed, one-sample t tests). Dashed gray line indicates performance expected based on chance. Water maze data from some of the NTG and KL mice were reported previously (Dubal et al., 2014). D, Object recognition memory as reflected by the percentage of time mice spent exploring a novel object (n = 11–12 mice per genotype, age 5–8 months); *p < 0.05, **p < 0.01 versus 50% (two-tailed, one-sample t tests). Dashed gray line is average time spent with object during training, which was equivalent in all groups (data not shown). E, Passive avoidance memory, as reflected by latency to enter the dark chamber during the test session (n = 13–23 mice per genotype, age 5–8 months). Two-way ANOVA: KL effect p < 0.05, hAPP by KL interaction p < 0.05; *p < 0.05 versus all other groups (Bonferroni-Holm test). Dashed gray line is average latency to enter chamber during training, which was similar in all groups (data not shown). F, Total number of movements during exploration of an open field (n = 16–24 mice per genotype, age 4–7 months). Two-way ANOVA: hAPP effect p < 0.01; KL effect p < 0.05; *p < 0.05 versus NTG (Bonferroni-Holm test), op < 0.05 (Newman–Keuls test). G, Percentage of time mice spent exploring the open arms of an elevated plus maze (n = 16–24 mice per genotype, age 4–7 months). Two-way ANOVA: hAPP effect p < 0.001, hAPP by KL interaction p = 0.06; ***p < 0.001 versus NTG (Bonferroni-Holm test). Data represent mean ± SEM.

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    Figure 4.

    Elevation of klotho in hAPP mice does not alter levels of hAPP, Aβ, β-CTFs, Aβ oligomers, total tau, or phospho-tau and does not prevent neuritic dystrophy. A, Representative Western blots showing hippocampal levels of hAPP, total mouse tau, phosphorylated (p) mouse tau (PHF-1 antibody, Ser 396/404), and β-actin. Images were captured from the same gel. B, Relative hAPP levels determined by quantitation of Western blot signals (n = 8–9 mice per genotype, age 3 months). GAPDH was used as a loading control. C, Representative Western blots showing hippocampal levels of hAPP, β-CTFs and α-tubulin. D, Relative β-CTF levels determined by quantitation of Western blot signals (n = 4 mice per genotype, age 3–4.5 months). E, Hippocampal Aβ1-x and Aβ1-42 levels determined by ELISA (n = 7–9 mice per genotype, age 3 months). F, Hippocampal Aβ oligomer levels determined by ECL assay (n = 4 mice per genotype, age 3–4.5 months). G, Immunostaining of hippocampal Aβ deposits in coronal brain sections from an hAPP (top) and an hAPP/KL mouse (bottom). Scale bar, 200 μm. H, Relative levels of hippocampal Aβ deposition (n = 12–14 mice per genotype, age 10–12.5 months). The percentage area covered by Aβ deposits in hAPP mice was arbitrarily defined as 1.0. I, Representative double-labeling of hippocampus for dystrophic neurites (antibody 8E5, red) and amyloid plaques (thioflavin-S, green) in hAPP and hAPP/KL mice (age 10–12.5 months; n = 10–12 per genotype). Scale bar, 50 μm. J, Quantification of dystrophic neurites expressed as a percentage of thioflavin-S-labeled plaques with surrounding neuritic dystrophy. K, L, Quantitation of total (K) and phosphorylated (L) mouse tau levels relative to levels found in hAPP mice (n = 8–9 mice per genotype, age 3 months). Actin was used as a loading control. Bar graphs represent mean ± SEM.

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    Figure 5.

    Elevation of klotho prevents loss of spinophilin in the hippocampus of hAPP mice. A, Representative immunostaining of spinophillin (spino, red) and MAP-2 (green) in the hippocampus of NTG, KL, hAPP, and hAPP/KL mice. Scale bars: Spino, MAP-2, and merge, 5 μm; zoom, 10 μm. B, C, Quantification of spinophilin-immunoreactive spines in the DG (n = 10–27 mice per group, age 10–12.5 months) expressed as percentage area of MAP-2-positive structures covered (B) and number of punctae per 10 μm of MAP-2-positive dendrite (C). Two-way ANOVA: hAPP effect p < 0.05 (B) and p < 0.01 (C), KL effect p < 0.05 (B), and p < 0.0001 (C), hAPP by KL interaction p < 0.01 (B) and p < 0.0001 (C); **p < 0.01, ***p < 0.001 versus hAPP/KL or as indicated by brackets (Bonferroni-Holm test). Bar graphs represent mean ± SEM.

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    Figure 6.

    Klotho elevation prevents depletion of NMDAR subunits GluN1 and GluN2A, and increases hippocampal levels of GluN2B in hAPP mice. A, Representative Western blots showing hippocampal levels of NMDAR subunits GluN1, GluN2A, and GluN2B, and AMPAR subunits GluA1 and GluA2. Actin served as a loading control. B–F, Quantitation of Western blot signals relative to mean levels found in NTG mice (n = 7–15 mice per genotype, age 3 months). B, GluN1 levels. Two-way ANOVA: hAPP effect p < 0.05. C, GluN2A levels. Two-way ANOVA: KL effect p < 0.05; hAPP by KL interaction p < 0.05. D, GluN2B levels. Two-way ANOVA: KL effect p < 0.001. Dashed gray line is level of GluN2B in NTG mice. E, GluA1 levels. F, GluA2 levels; *p < 0.05, **p < 0.01 versus NTG or as indicated by brackets (Bonferroni-Holm test). Levels of NMDAR and AMPAR subunits in the NTG and KL mice were reported previously (Dubal et al., 2014). Bar graphs represent mean ± SEM.

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    Figure 7.

    Klotho elevation increases GluN2B levels in hippocampal PSD fractions of hAPP mice. Non-PSD (1) and PSD (2) synaptic membrane fractions were isolated from hippocampal homogenates from mice of the indicated genotypes (n = 6–14 per genotype, age 3 months). A, Representative Western blots. B, Quantitation of GluN2B Western blot signals in the non-PSD fraction relative to NTG levels. Two-way ANOVA: hAPP effect p < 0.01; *p < 0.05 versus NTG, #p = 0.06 versus KL (Bonferroni-Holm test). C, Quantitation of GluN2B signals in PSD fraction relative to NTG levels. Two-way ANOVA: KL effect p < 0.05; *p < 0.05 versus NTG or as indicated by bracket (Bonferroni-Holm test). D, Ratio of PSD/non-PSD GluN2B levels in hAPP and hAPP/KL mice relative to ratio in hAPP mice; *p < 0.05 (t test). Bar graphs represent mean ± SEM.

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    Figure 8.

    Klotho elevation ameliorates synaptic dysfunction in the hippocampus of hAPP mice. Field EPSPs were recorded from acute hippocampal slices obtained from 3.5- to 4.5-month-old mice. A, LTP induction and decay in the DG were monitored for 45–50 min following theta burst stimulation of the medial perforant pathway. Mixed-model ANOVA: hAPP effect, KL effect, and hAPP by KL interaction p < 0.001; NTG versus KL p < 0.05; hAPP versus all other groups p < 0.001 (Bonferroni-Holm test). B, Mean of the last 10 min of LTP recordings. Mixed-model ANOVA: hAPP effect p < 0.001, KL effect p < 0.001; **p < 0.01 versus all other groups or as indicated by brackets (Bonferroni-Holm test). The fEPSP results in NTG and KL slices were reported previously (Dubal et al., 2014). C, Input/output curves reflecting AMPAR-mediated synaptic transmission at Schaffer collateral to CA1 pyramidal cell synapses. Mixed-model ANOVA: hAPP effect p < 0.001, KL effect <0.05, hAPP by KL interaction p < 0.001; hAPP versus all other groups p < 0.001 (Bonferroni-Holm test). Number of slices/number of mice: NTG 3/3, KL 6/5, hAPP 7/5, hAPP/KL 7/3 (A, B), and NTG 9/3, KL 10/5, hAPP 9/5, hAPP/KL 11/3 (C). Data represent mean ± SEM.

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    Table 1.

    Mice analyzed in different experiments

    FigureMice per groupTotal no.From cohort(s)Age at analysis
    Longevity
    2BNTG, 111366 miceA–H3 weeks to 12.5 months
    hAPP, 67
    KL, 110
    hAPP/KL, 78
    EEG
    2DNTG, 321 miceB2–3.5 months
    KL, 4
    hAPP, 7
    hAPP/KL, 7
    Behavior
        Water maze3A–CNTG, 1463 miceB, C4–7 months
    KL, 14
    hAPP, 18
    hAPP/KL, 17
        Novel object3DNTG, 1045 miceC5–8 months
    KL, 11
    hAPP, 12
    hAPP/KL, 12
        Passive avoidance3ENTG, 2372 miceC5–8 months
    KL, 21
    hAPP, 15
    hAPP/KL, 13
        Open field, elevated plus maze3F, GNTG, 2481 miceC4–7 months
    KL, 21
    hAPP, 20
    hAPP/KL, 16
    Biochemistry/histology
        Western1NTG, 1023 miceD5–7 months
    hAPP, 13
        Western2A, 6B–F, 7B, CNTG, 14–1543–45 miceE3 months
    KL, 13
    hAPP, 7–8
    hAPP/KL, 9
        Westerns, Aβ ELISA4B, E, K, LhAPP, 7–815–17 miceE3 months
    hAPP/KL, 8–9
        Aβ oligomer ECL assay4FhAPP, 48 miceF3–4.5 months
    hAPP/KL, 4
        Western4DhAPP, 48 miceF3–4.5 months
    hAPP/KL, 4
        Immunohistochemistry4H, JhAPP, 12–1321 miceC10–12.5 months
    hAPP/KL, 8–9
        Immunohistochemistry5B, CNTG, 2575 miceC10–12.5 months
    KL, 27
    hAPP, 14
    hAPP/KL, 9
    Electrophysiology
        EPSP (LTP)8A, BNTG 3 slices/3 mice23 slices, 16 miceG3.5–4.5 months
    KL 6 slices/5 mice
    hAPP 7 slices/5 mice
    hAPP/KL 7 slices/3 mice
        fEPSP (I/O curves)8CNTG 9 slices/3 mice39 slices, 16 miceH3.5–4.5 months
    KL 10 slices/5 mice
    hAPP 9 slices/5 mice
    hAPP/KL 11 slices/3 mice
    • Cohorts are independent groups of mice, some or all of which were analyzed at one or more time points/ages in one or more paradigms.

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Journal of Neuroscience
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11 Feb 2015
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Life Extension Factor Klotho Prevents Mortality and Enhances Cognition in hAPP Transgenic Mice
Dena B. Dubal, Lei Zhu, Pascal E. Sanchez, Kurtresha Worden, Lauren Broestl, Erik Johnson, Kaitlyn Ho, Gui-Qiu Yu, Daniel Kim, Alexander Betourne, Makoto Kuro-o, Eliezer Masliah, Carmela R. Abraham, Lennart Mucke
Journal of Neuroscience 11 February 2015, 35 (6) 2358-2371; DOI: 10.1523/JNEUROSCI.5791-12.2015

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Life Extension Factor Klotho Prevents Mortality and Enhances Cognition in hAPP Transgenic Mice
Dena B. Dubal, Lei Zhu, Pascal E. Sanchez, Kurtresha Worden, Lauren Broestl, Erik Johnson, Kaitlyn Ho, Gui-Qiu Yu, Daniel Kim, Alexander Betourne, Makoto Kuro-o, Eliezer Masliah, Carmela R. Abraham, Lennart Mucke
Journal of Neuroscience 11 February 2015, 35 (6) 2358-2371; DOI: 10.1523/JNEUROSCI.5791-12.2015
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Keywords

  • aging
  • Alzheimer's disease
  • cognition
  • klotho
  • mice
  • NMDA receptors

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