Research ArticleNeurodegeneration

Reelin protects against amyloid β toxicity in vivo

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Science Signaling  07 Jul 2015:
Vol. 8, Issue 384, pp. ra67
DOI: 10.1126/scisignal.aaa6674
  • Fig. 1 Adult loss of Reelin causes increased Dab1 abundance without changing the abundance of downstream effectors or glutamate receptors.

    (A) Relnflox/flox mice were generated by flanking exon 1 of the Reln gene with loxP sites. The mice were bred to homozygosity and crossed with the CAG-CreERT2 line, which ubiquitously expresses a tamoxifen-inducible Cre recombinase. (B) Western blot of whole hippocampal lysates with G10 antibody, demonstrating inducible Reelin knockout. The positions of the180- and 380-kD Reelin forms are indicated. TAM−, tamoxifen-injected Relnflox/flox mouse; VEH+, vehicle-injected CAG-CreERT2:Relnflox/flox mouse; cKO, tamoxifen-injected CAG-CreERT2:Relnflox/flox mouse. (C) Quantification of 180-kD Reelin band Western blotting. Data are presented as means ± SEM (TAM−, VEH+, n = 10 mice; cKO, n = 11 mice; reeler, n = 4 mice). (D to M) Whole hippocampal lysates from mice of the indicated genotypes were evaluated by Western blot. Total Dab1 abundance normalized to actin [same samples as in (B)] (D) [one-way analysis of variance (ANOVA), P < 0.0001; post hoc TAM− compared to cKO, P < 0.001; VEH+ compared to cKO, P < 0.001; n.s., not significant] (TAM−, n = 11 mice; VEH+, n = 12 mice; cKO, n = 12 mice; reeler, n = 7 mice); phosphorylated (p) GSK3β (Ser9) normalized to total GSK3β (E) (one-way ANOVA, P = 0.0012; post hoc cKO compared to KO, P < 0.001); phosphorylated Akt normalized to total (t) Akt (F); phosphorylated ERK normalized to total ERK (G); phosphorylated Src normalized to total Src (H); GluA1 (I); GluA2/3 (J); NR2A (K) (one-way ANOVA, PpAkt/tAkt = 0.5329, PpERK/tERK = 0.9946, PpSrc = 0.2660, PGluR1 = 0.1458, PGluR2/3 = 0.3996, PNR2A = 0.1168); and NR2B (L) (one-way ANOVA, P = 0.0593; post hoc cKO compared to reeler, P < 0.05). For (E) to (G) and (I) to (L): TAM−, n = 8 mice; VEH+, n = 11 mice; cKO, n = 11 mice; reeler, n = 5 mice. For (H): TAM−, n = 6 mice; VEH+, n = 6 mice; cKO, n = 6 mice; reeler, n = 5 mice. Phosphorylated tau normalized to total tau (M) (one-way ANOVA, PAT8 = 0.9610, PThr231 = 0.7995; n = 4 mice per genotype) is also shown. All samples in (D) to (L) were normalized to actin or RAP and then to the control (TAM− and VEH+) mice. Data are presented as means ± SEM.

  • Fig. 2 Adult Reelin cKO mice have normal architecture, no granule cell dispersion, and no alterations in dendritic spine density or morphology.

    (A to P) Sections were compared from TAM− (A, D, G, and K), VEH+ (B, E, H, and L), and cKO (C, F, I, and M) mice. Cortical sections were evaluated for disrupted layering by NeuN immunohistochemistry (A to C). Scale bar, 200 μm. Cerebellar (D to F) (scale bar, 200 μm) and hippocampal (G to I) (scale bar, 200 μm) sections were stained with cresyl violet. The granule cell layer thickness was measured to assess granule cell (GC) dispersion (J) (one-way ANOVA, P = 0.5016). For (A) to (F), n = 3 sections per mice, 3 mice per genotype. For (G) to (J), n = 6 sections per mice, 3 mice per genotype. Golgi-stained CA1 apical dendrites (K to M) were analyzed for changes in spine density (N) and spine morphology (O and P) (scale bar, 5 μm) (one-way ANOVA, PDensity = 0.8681, PHeight = 0.4822, PWidth = 0.7753) (TAM−, n = 38; VEH+, n = 29; cKO, n = 25, where n represents total neurons analyzed from three mice per genotype). Data are presented as means ± SEM.

  • Fig. 3 Reelin cKO mice have mild behavioral changes and increased LTP.

    (A to D) Reelin cKO mice have hypoanxiety and mildly enhanced PPI, but no changes in learning. (A) The percent time mice spent in the center of an open-field apparatus for each 2-min bin is shown. A two-way repeated-measures ANOVA shows a strong effect of time (P < 0.001), a nonsignificant trend of genotype (P = 0.0783), and a significant interaction between the two (P = 0.0129) (TAM−, n = 11 mice; VEH+, n = 10 mice; cKO, n = 14 mice). (B) Mice were tested for PPI with the indicated tones preceding a 120-dB tone. Data shown are reduction of startle at each prepulse relative to the startle when no prepulse was played before the 120-dB tone (two-way ANOVA, PInteraction = 0.876, PPrepulse intensity < 0.0001, PGenotype = 0.0008; post hoc at 78 dB: TAM− compared to cKO, P = 0.0013; VEH+ compared to cKO, P = 0.0306) (TAM−, n = 16 mice; VEH+, n = 18 mice; cKO, n = 17 mice). (C and D) Morris water maze results from cKO mice. The latency of mice to find a hidden platform over a 10-day training period was measured (C) (two-way repeated-measures ANOVA, PInteraction = 0.9993, PTime < 0.0001, PGenotype = 0.5616; n = 10 per group). On the 11th day, the platform was removed, and the time spent in the quadrant (Q) that had previously contained the platform was measured (D) (two-way ANOVA, PGenotype > 0.999, PQuadrant < 0.0001, PInteraction = 0.1874) (TAM−, n = 16 mice; VEH+, n = 17 mice; cKO, n = 17 mice). (E to H) Reelin cKO mice have enhanced late LTP. (E) Field recordings were made from the stratum radiatum of the CA1 region of hippocampal slices from 7-month-old Reelin cKO mice before and after application of θ-burst stimulation–induced LTP. Every 2 min were averaged for analysis. Sample traces are shown before and after θ-burst stimulation (TBS) (right). (F and G) The average LTP at 0 to 20 min was similar between control and cKO mice (F) (unpaired t test, P = 0.9511). At 40 to 60 min, cKO mice had increased LTP (G) (unpaired t test, P = 0.0483). (H) The input-output curves were unaltered between cKO and control mice (unpaired t test, P = 0.9828) (TAM−, n = 15 slices from 6 mice; cKO, n = 11 slices from 4 mice). Data are presented as means ± SEM.

  • Fig. 4 Loss of Reelin does not accelerate amyloid pathology in 7-month-old Tg2576 mice.

    (A and B) Immunohistochemistry with 4G8 antibody was performed on brains of (A) Tg2576 and (B) Tg2576:cKO mice to evaluate plaque deposition (n = 3 mice per genotype). (C) Nine-month-old APP/PS1 mice were used as a positive control because they had no obvious plaques. Scale bar, 200 μm. (D) The abundance of PBS-soluble and PBS-insoluble Aβ was measured with Western blot and quantified. No difference was observed between cKO and control mice (unpaired t test, Psoluble = 0.9945, Pinsoluble = 0.7554) (n = 6 mice per genotype). (E) The abundance of PBS-soluble and PBS-insoluble Aβ40 and Aβ42 was measured by ELISA. No difference was observed between cKO and control mice (unpaired t test: Aβ40, Psoluble = 0.6016, Pinsoluble = 0.3398; Aβ42, Psoluble = 0.6116, Pinsoluble = 0.4789) (n = 9 Tg2576 mice, n = 4 Tg2576:cKO mice). Data are presented as means ± SEM.

  • Fig. 5 Low amounts of endogenously produced Aβ induce severe memory impairment and reduce hyperexcitability in Tg2576:Reelin cKO mice.

    (A to C) Morris water maze testing of 7-month-old Tg2576:Reelin cKO mice showed that they had impaired acquisition of the task (A) [two-way ANOVA: F18,306 (Interaction) = 3.370, P <0.0001, F9,306 (Time) = 10.38, P < 0.0001, F2,34 (Genotype) = 0.564, P = 0.5742] and poor performance on the probe trial (B). (C) Swim speeds between genotypes were similar (one-way ANOVA, P = 0.9866) (Tg2576:TAM−, n = 9 mice; Tg2576:VEH+, n = 12 mice; Tg2576:cKO, n = 16 mice). Data are presented as means ± SEM. (D to F) θ-Burst LTP was performed in Tg2576:Reelin cKO mice (D). No difference was observed at 0 to 20 min (E). However, at 40 to 60 min, the cKO mice had increased LTP, whereas the Tg2576:cKO mice showed similar LTP to control mice (F). One-way ANOVA revealed a significant difference at 40 to 60 min, with post hoc analyses indicating a significant difference between the cKO mice and all other genotypes (P < 0.05, compared to TAM−; P < 0.01, compared to Tg2576; P < 0.05, compared to Tg2576:cKO) but no difference between the other genotypes, including the Tg2576:Reelin cKO mice (TAM−, n = 15 slices from 6 mice; cKO, n = 11 slices from 4 mice; Tg2576:TAM−, n = 11 slices from 4 mice; Tg2576:cKO, n = 10 slices from 4 mice). Data are presented as means ± SEM.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/384/ra67/DC1

    Fig. S1. Brain-wide Reelin knockout in Reelin cKO mice.

    Fig. S2. Meox-Cre–driven Reelin knockout results in a reeler phenotype.

    Fig. S3. Behavioral findings in Reelin cKO mice.

    Fig. S4. Morris water maze in 7-month-old cKO mice.

  • Supplementary Materials for:

    Reelin protects against amyloid β toxicity in vivo

    Courtney Lane-Donovan,* Gary T. Philips, Catherine R. Wasser, Murat S. Durakoglugil, Irene Masiulis, Ajeet Upadhaya, Theresa Pohlkamp, Cagil Coskun, Tiina Kotti, Laura Steller, Robert E. Hammer, Michael Frotscher, Hans H. Bock, Joachim Herz*

    *Corresponding author. E-mail: joachim.herz{at}utsouthwestern.edu (J.H.); courtney.lane{at}utsouthwestern.edu (C.L.-D.)

    This PDF file includes:

    • Fig. S1. Brain-wide Reelin knockout in Reelin cKO mice.
    • Fig. S2. Meox-Cre–driven Reelin knockout results in a reeler phenotype.
    • Fig. S3. Behavioral findings in Reelin cKO mice.
    • Fig. S4. Morris water maze in 7-month-old cKO mice.

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    Citation: C. Lane-Donovan, G. T. Philips, C. R. Wasser, M. S. Durakoglugil, I. Masiulis, A. Upadhaya, T. Pohlkamp, C. Coskun, T. Kotti, L. Steller, R. E. Hammer, M. Frotscher, H. H. Bock, J. Herz, Reelin protects against amyloid β toxicity in vivo. Sci. Signal. 8, ra67 (2015).

    © 2015 American Association for the Advancement of Science

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