Research ArticleRett Syndrome

mGlu7 potentiation rescues cognitive, social, and respiratory phenotypes in a mouse model of Rett syndrome

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Science Translational Medicine  16 Aug 2017:
Vol. 9, Issue 403, eaai7459
DOI: 10.1126/scitranslmed.aai7459
  • Fig. 1. mGlu7 expression is reduced in RTT autopsy samples.

    (A) Diagram of luciferase reporter constructs. (B) Bar graph showing the effect of transfection of increasing amounts of human MECP2 plasmid on luciferase expression from the pGL4 vector with and without the GRM7 promoter [#P < 0.0001 and *P < 0.0001, one-way analysis of variance (ANOVA) with Bonferroni’s post hoc; n = 3]. (C) GRM7 messenger RNA (mRNA) expression in the motor cortex of control and RTT autopsy samples (P = 0.07, two-tailed Student’s t test; n = 7 to 8 biological replicates, df = 13). (D) mGlu7 (***P < 0.0001, two-tailed Student’s t test; n = 7 to 8, df = 13) and (E) vGlut2 (P = 0.39, two-tailed Student’s t test; n = 7 to 8, df = 13) protein expression in control and RTT autopsy samples. (F) Representative Western blots showing mGlu7 and vGlut2 expression in control and RTT motor cortex samples. MECP2 mutations are listed below the representative images.

  • Fig. 2. Grm7 and mGlu7 expression is decreased in Mecp2−/y mice.

    (A) Grm7 mRNA expression in total cortex (*P < 0.05), hippocampus (P > 0.05), and striatum (***P < 0.001) tissue samples from symptomatic Mecp2−/y mice relative to Mecp2+/y mice (P < 0.0001, two-way ANOVA, genotype × brain region, with Bonferroni’s post hoc; n = 3 to 5, df = 3, 21). (B) Representative Western blots for mGlu7 total protein expression from cortex, hippocampus, and striatum in Mecp2+/y and Mecp2−/y mice. (C) Quantification of mGlu7 total protein expression in the cortex (*P < 0.05), hippocampus (P > 0.05), and striatum (P > 0.05) of Mecp2−/y mice (two-way ANOVA, genotype × brain region, with Bonferroni’s post hoc; n = 7 control and 8 RTT, df = 3, 13). (D) Representative synaptosome Western blots for mGlu7 and vGlut2. (E) Quantification of mGlu7 expression in synaptosomes prepared from cortex (P > 0.05) and hippocampus (***P < 0.001) tissue samples from Mecp2−/y mice. Two-way ANOVA, genotype × brain region with Bonferroni’s post hoc, P < 0.003, n = 5 samples from individual mice, df = 1, 16.

  • Fig. 3. mGlu7 function is impaired in Mecp2−/y slices and can be restored by application of VU0422288.

    Recordings performed at SC-CA1 synapses. (A) Effect of bath application of LSP4-2022 (blue line) on fEPSP slope in Mecp2+/y and Mecp2−/y slices. Mecp2+/y versus Mecp2−/y [***P < 0.0001, two-tailed Student’s t test; n = 4, 4 (slices, mice), df = 6]. (B) Sample traces of fEPSPs before (1) and during (2) LSP4-2022 treatment in slices from Mecp2+/y and Mecp2−/y mice. (C) PPR after application of LSP4-2022 in Mecp2+/y and Mecp2−/y slices (**P = 0.003, two-tailed paired t test; n = 4, 4, df = 3). n.s., not significant. (D) Effects of pretreatment with VU0422288 alone and in combination with LSP4-2022 in Mecp2−/y slices. Mecp2−/y baseline is from (A). ***P < 0.0001, one-way repeated-measures ANOVA with Bonferonni’s post hoc; n = 4, 4, df = 4, 8. (E) Sample traces of fEPSPs during baseline (1) and after application of VU0422288 and LSP4-2022 (2) in slices from Mecp2−/y mice. (F) PPR after application of VU0422288 and LSP4-2022 in Mecp2−/y slices. P < 0.0001, one-way repeated-measures ANOVA with Bonferonni’s post hoc; n = 5, 5, df = 4, 8. ***P < 0.001 VU0422288 alone versus VU0422288 + LSP4-2022 and ***P < 0.001 VU0422288 + LSP4-2022 versus baseline. (G) Post hoc analysis of PPR comparing baseline Mecp2+/y levels relative to Mecp2−/y baseline (***P < 0.001), VU0422288 alone (***P < 0.001), and VU0422288 + LSP4-2022 (#P < 0.05 relative to Mecp2−/y baseline). Two-tailed Student’s t test. Scale bars, 0.2 mV by 2 ms (B and E).

  • Fig. 4. VU0422288 rescues synaptic plasticity defects and learning and memory phenotypes in Mecp2+/− mice.

    (A) mGlu7 expression in synaptosome preparations from the cortex (P > 0.05) and hippocampus (*P < 0.05) of Mecp2+/+ and Mecp2+/− mice. n = 7 per genotype, two-tailed Student’s t test. (B) LTP in Mecp2+/+ (filled black circles), Mecp2+/− (*P < 0.05 relative to Mecp2+/+, filled red circles), and Mecp2+/− slices pretreated with VU0422288 (#P < 0.05 relative to Mecp2+/− untreated, empty red circles). The gray bar represents the area from which LTP was measured in the accompanying bar graph. One-way ANOVA with Student’s t test post hoc; n = 4 to 5, 4 (slices, mice). (C) Contextual fear conditioning in Mecp2+/+ and Mecp2+/− mice treated with vehicle (n = 22 and 16), VU0422288 (n = 17 and 15), and ADX88178 (n = 10 and 15). ***P < 0.001, ##P < 0.01, ****P < 0.0001, two-way ANOVA with Student’s t test post hoc analysis; df = 2, 89. # denotes within-genotype comparison. (D) NOR in Mecp2+/+ and Mecp2+/− mice treated with vehicle, VU0422288 and ADX88178. *P < 0.05, #P < 0.05, two-tailed Student’s t test; n = 10 per genotype per treatment. (E) EPM in Mecp2+/+ and Mecp2+/− mice treated with vehicle, VU0422288, and ADX88178. **P < 0.01, *P < 0.05, ##P < 0.01, #P < 0.05, two-tailed Student’s t test; n = 10 per genotype per treatment. * denotes within-treatment comparison, # denotes within-genotype comparison. (F) Social preference assay in Mecp2+/+ and Mecp2+/− mice treated with vehicle, VU0422288, and ADX88178. ***P < 0.001, *P < 0.05, two-tailed Student’s t test, n = 9 to 12 per treatment.

  • Fig. 5. VU0422288 reduces the number of apneas in Mecp2+/− mice.

    WBP. (A) Average breath rate over 30 min in Mecp2+/+ (black) and Mecp2+/− (red) mice in response to vehicle (n = 14 and 11), VU0422288 (n = 11 and 11), and ADX88178 (n = 11 and 14) treatment. ###P < 0.001, #P < 0.05, two-way ANOVA with Student’s t test post hoc. # denotes within-genotype comparison. (B and C) Effects of vehicle, VU0422288, and ADX88178 treatment in Mecp2+/+ and Mecp2+/− mice on (B) inspiration and (C) expiration time. ###P < 0.001, #P < 0.05, two-way ANOVA with Student’s t test post hoc. (D) Quantification of apneas per 10,000 breaths in Mecp2+/+ and Mecp2+/− treated with vehicle, VU0422288, and ADX88178. *P < 0.001, #P < 0.05, two-way ANOVA with Student’s t test post hoc. (E) Sample plethysmography traces.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/403/eaai7459/DC1

    Materials and Methods

    Fig. S1. Antibody #07-239 is selective for mGlu7.

    Fig. S2. Gad65, but not mGlu4, expression is reduced in Mecp2−/y mice.

    Fig. S3. Mecp2−/y hippocampal slices display enhanced excitatory transmission at the SC-CA1 synapse.

    Fig. S4. VU0155094, a PAM that is structurally distinct from VU0422288, also rescues the efficacy of LSP4-2022 on fEPSPs at SC-CA1 in Mecp2−/y slices.

    Fig. S5. VU0422288 rescues attenuated LTP at the SC-CA1 synapse in Mecp2−/y mice.

    Fig. S6. ADX88178 is active at mGlu4 and mGlu8, but not at mGlu7.

    Fig. S7. Startle response threshold is not affected by VU0422288 and ADX88178 treatment in Mecp2+/+ or Mecp2+/− mice.

    Fig. S8. Coadministration of VU0422288 and ADX71743 confirm mGlu7’s role in cognition but evokes sedative effects.

    Fig. S9. Efficacy is conserved with repeated VU0422288 administration.

    Table S1. Human motor cortex sample data.

    Table S2. Pharmacokinetic analysis of VU0422288.

  • Supplementary Material for:

    mGlu7 potentiation rescues cognitive, social, and respiratory phenotypes in a mouse model of Rett syndrome

    Rocco G. Gogliotti, Rebecca K. Senter, Nicole M. Fisher, Jeffrey Adams, Rocio Zamorano, Adam G. Walker, Anna L. Blobaum, Darren W. Engers, Corey R. Hopkins, J. Scott Daniels, Carrie K. Jones, Craig W. Lindsley, Zixiu Xiang, P. Jeffrey Conn, Colleen M. Niswender*

    *Corresponding author. Email: colleen.niswender{at}vanderbilt.edu

    Published 16 August 2017, Sci. Transl. Med. 9, eaai7459 (2017)
    DOI: 10.1126/scitranslmed.aai7459

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Antibody #07-239 is selective for mGlu7.
    • Fig. S2. Gad65, but not mGlu4, expression is reduced in Mecp2−/y mice.
    • Fig. S3. Mecp2−/y hippocampal slices display enhanced excitatory transmission at the SC-CA1 synapse.
    • Fig. S4. VU0155094, a PAM that is structurally distinct from VU0422288, also rescues the efficacy of LSP4-2022 on fEPSPs at SC-CA1 in Mecp2−/y slices.
    • Fig. S5. VU0422288 rescues attenuated LTP at the SC-CA1 synapse in Mecp2−/y mice.
    • Fig. S6. ADX88178 is active at mGlu4 and mGlu8, but not at mGlu7.
    • Fig. S7. Startle response threshold is not affected by VU0422288 and ADX88178 treatment in Mecp2+/+ or Mecp2+/− mice.
    • Fig. S8. Coadministration of VU0422288 and ADX71743 confirm mGlu7’s role in cognition but evokes sedative effects.
    • Fig. S9. Efficacy is conserved with repeated VU0422288 administration.
    • Table S1. Human motor cortex sample data.
    • Table S2. Pharmacokinetic analysis of VU0422288.

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