Life extension factor klotho prevents mortality and enhances cognition in hAPP transgenic mice

Abstract

Aging is the principal demographic risk factor for Alzheimer disease (AD), the most common neurodegenerative disorder. Klotho is a key modulator of the aging process and, when overexpressed, extends mammalian lifespan, increases synaptic plasticity, and enhances cognition. Whether klotho can counteract deficits related to neurodegenerative diseases, such as AD, is unknown. Here we show that elevating klotho expression decreases premature mortality and network dysfunction in human amyloid precursor protein (hAPP) transgenic mice, which simulate key aspects of AD. Increasing klotho levels prevented depletion of NMDA receptor (NMDAR) subunits in the hippocampus and enhanced spatial learning and memory in hAPP mice. Klotho elevation in hAPP mice increased the abundance of the GluN2B subunit of NMDAR in postsynaptic densities and NMDAR-dependent long-term potentiation, which is critical for learning and memory. Thus, increasing wild-type klotho levels or activities improves synaptic and cognitive functions, and may be of therapeutic benefit in AD and other cognitive disorders.

Keywords: Alzheimer's disease; NMDA receptors; aging; cognition; klotho; mice.

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.

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.

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.

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.

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.

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.

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.

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.