Contribution of mGluR5 to pathophysiology in a mouse model of human chromosome 16p11.2 microdeletion

Journal name:
Nature Neuroscience
Volume:
18,
Pages:
182–184
Year published:
DOI:
doi:10.1038/nn.3911
Received
Accepted
Published online

Human chromosome 16p11.2 microdeletion is the most common gene copy number variation in autism, but the synaptic pathophysiology caused by this mutation is largely unknown. Using a mouse with the same genetic deficiency, we found that metabotropic glutamate receptor 5 (mGluR5)-dependent synaptic plasticity and protein synthesis was altered in the hippocampus and that hippocampus-dependent memory was impaired. Notably, chronic treatment with a negative allosteric modulator of mGluR5 reversed the cognitive deficit.

At a glance

Figures

  1. mGluR-LTD is protein synthesis independent in 16p11.2 df/+ mice.
    Figure 1: mGluR-LTD is protein synthesis independent in 16p11.2 df/+ mice.

    (a) TBS-LTP was unchanged in mutant (n = 9 animals, 16 slices) compared with WT (n = 7 animals, 15 slices) mice. (b) LFS-LTD was unchanged in mutant (n = 6 animals, 10 slices) compared with WT (n = 6 animals, 10 slices) mice. (c,d) The magnitude of DHPG-LTD was comparable in hippocampal slices from the WT (n = 17 animals, 18 slices) and mutant (Mut, n = 16 animals, 20 slices) mice in the absence of CHX. However, CHX blocked DHPG-LTD in WT slices (n = 17 animals, 21 slices), but not mutant slices (n = 16 animals, 20 slices) (two-way ANOVA, genotype × CHX, P = 0.0074). (e,f) The magnitude of PP-LFS-LTD was comparable in hippocampal slices from WT (n = 12 animals, 14 slices) and mutant (n = 8 animals, 10 slices) mice in the absence of CHX. CHX significantly attenuated PP-LFS-LTD in the WT slices (n = 12 animals, 14 slices), but not mutant slices (n = 8 animals, 9 slices) (two-way ANOVA, genotype × CHX, P = 0.013). Representative field excitatory postsynaptic potential (fEPSP) traces (average of ten sweeps) were taken at the times indicated by numerals. All data are plotted as mean ± s.e.m.

  2. 16p11.2 df/+ mice exhibit deficits in hippocampal-associated CFC and IA.
    Figure 2: 16p11.2 df/+ mice exhibit deficits in hippocampal-associated CFC and IA.

    (a) CFC experimental design. (b) Mutant mice showed significantly less freezing in the familiar context compared with WT (unpaired t test, P = 0.0018). Although WT mice were able to distinguish a novel from familiar context (unpaired t test, P < 0.0001), the mutant mice were impaired (unpaired t test, P = 0.2840). Two-way ANOVA, genotype × context, P = 0.0166. (c) Mutant and WT mice had the same running response to foot shock during the training session (unpaired t test, P = 0.6234). (d) Mutant mice were impaired in IA acquisition (WT versus Mut, 0 h versus 6 h, repeated measures two-way ANOVA, P = 0.0108; WT versus Mut at 6 h, post hoc unpaired t test, P = 0.0033). Unlike WT (6 h versus 48 h, post hoc paired t test, P = 0.0101), mutant mice showed no extinction of fear memory (WT versus Mut, 6 h versus 48 h, repeated measures two-way ANOVA, P = 0.0197; Mut, 6 h versus 48 h, post hoc paired t test, P = 0.6278). (e) CTEP treatment ameliorated behavioral deficits in mutant mice in IA. In mutant mice, CTEP treatment enhanced acquisition (Mut + Veh versus Mut + CTEP, 0 h versus 6 h, repeated measures two-way ANOVA, P = 0.0016; Mut + Veh versus Mut + CTEP at 6 h, post hoc unpaired t test, P = 0.0013) and extinction of fear memory (Mut + Veh versus Mut + CTEP, 6 h versus 48 h, repeated measures two-way ANOVA, P = 0.0039; Mut + Veh, 6 h versus 48 h, post hoc paired t test, P = 0.4281; Mut + CTEP, 6 h versus 48 h, post hoc paired t test, P = 0.0140). There was no statistically significant difference between WT + Veh and Mut + CTEP at 6 h (unpaired t test, P = 0.3471). In WT mice, CTEP had no effect on either acquisition (WT + Veh versus WT + CTEP, 0 h versus 6 h, repeated measures two-way ANOVA, P = 0.6564) or extinction of fear memory (WT + Veh versus WT + CTEP, 6 h versus 48 h, repeated measures two-way ANOVA, P = 0.9882). All data are plotted as mean ± s.e.m. with individual values superimposed.

  3. 16p11.2 df/+ mice exhibit a decrease in basal protein synthesis which is accompanied by an increase in Arc protein levels.
    Figure 3: 16p11.2 df/+ mice exhibit a decrease in basal protein synthesis which is accompanied by an increase in Arc protein levels.

    (a) Metabolic labeling of hippocampal slices revealed a significant reduction of basal protein synthesis in 2 df/+ compared to WT mice (unpaired t test, P = 0.0210). (b) MVP, ERK1 and pERK1 were decreased in 2 df/+ mice relative to WT mice (unpaired t test, P = 0.0036, P = 0.0001 and P = 0.0013 respectively), whereas ERK2 and pERK2 levels were comparable between 2 df/+ and WT mice (unpaired t test, P = 0.9091 and P = 0.8568 respectively). Arc protein levels were significantly increased in 2 df/+ mice as compared with WT mice (unpaired t test, P = 0.0191). All data are plotted as mean ± s.e.m. with individual values superimposed; n indicates number of animals.

  4. Steady decline of transmission rate of the 16p11.2 df/+ allele.
    Supplementary Fig. 1: Steady decline of transmission rate of the 16p11.2 df/+ allele.

    The percentage of the heterozygous 16p11.2 df/+ mutant mice, including both male and female, gradually declines across multiple generations during backcrossing to C57BL/6J. The number of 16p11.2 df/+ and total mice for each generation are: N5: 21/53, N6: 26/118, N7: 41/250, N8: 57/434, N9: 16/202, and N10: 8/102.

  5. Basal synaptic transmission is normal in the 16p11.2 df/+ mice.
    Supplementary Fig. 2: Basal synaptic transmission is normal in the 16p11.2 df/+ mice.

    (a) Input-output functions, plotted as fEPSP slope versus stimulus intensity, are not different between the WT (n = 14 animals) and mutant mice (n = 14 animals). Repeated measures one-way ANOVA, p = 0.92.

    (b) Paired-pulse facilitation is comparable between the WT (n = 16 animals) and mutant (n = 17 animals) mice across multiple stimulus intervals (10, 20, 50, 100, 200, 300, 500 ms). Repeated measures one-way ANOVA, p = 0.76. All data are plotted as mean ± SEM.

  6. Presynaptic LTD is independent of genotype and is not affected by cycloheximide treatment.
    Supplementary Fig. 3: Presynaptic LTD is independent of genotype and is not affected by cycloheximide treatment.

    DHPG increases paired-pulse facilitation in both the WT and 16p11.2 df/+ mutant slices and this effect is not affected by cycloheximide. PPF is calculated at a 50 ms inter-stimulus interval: WT baseline: 1.44 ± 0.019, n = 17 animals, 37 slices, WT DHPG: 1.55 ± 0.035, n = 17 animals, 18 slices, WT DHPG+CHX: 1.55 ± 0.027, n = 17 animals, 19 slices. Mut baseline: 1.45 ± 0.023, n = 16 animals, 42 slices, Mut DHPG:1.56 ± 0.053, n = 16 animals, 21 slices, Mut DHPG+CHX: 1.53 ± 0.038, n = 16 animals, 21 slices. Statistical analyses are performed using unpaired t-tests. All data are plotted as mean ± SEM.

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Author information

Affiliations

  1. Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Di Tian,
    • Laura J Stoppel,
    • Arnold J Heynen &
    • Mark F Bear
  2. The Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Di Tian,
    • Laura J Stoppel,
    • Arnold J Heynen &
    • Mark F Bear
  3. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Di Tian,
    • Laura J Stoppel,
    • Arnold J Heynen &
    • Mark F Bear
  4. Department of Pathology and Laboratory Medicine, Developmental Neuroscience Program, The Saban Research Institute, Children's Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.

    • Di Tian
  5. Pharmaceuticals Division, F. Hoffmann-La Roche, Basel, Switzerland.

    • Lothar Lindemann &
    • Georg Jaeschke
  6. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.

    • Alea A Mills

Contributions

M.F.B. and D.T. conceived and designed the study. M.F.B. and A.J.H. supervised the study. D.T. performed hippocampal electrophysiology and contextual fear conditioning. D.T. and A.J.H. performed inhibitory avoidance test. L.J.S. performed hippocampal protein synthesis and immunoblot experiments. D.T., A.J.H., L.J.S. and M.F.B. wrote the manuscript. L.L. and G.J. provided CTEP. A.A.M. provided the 16p11.2 df/– mice before publication and edited the manuscript.

Competing financial interests

M.F.B. holds patents on the use of mGluR5 inhibitors for treatment of fragile X and autism. M.F.B. and D.T. have patents pending on use of mGluR5 inhibitors for treatment of 16p11.2 microdeletion. L.L. and G.J. are employees of Roche Pharmaceuticals.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Steady decline of transmission rate of the 16p11.2 df/+ allele. (47 KB)

    The percentage of the heterozygous 16p11.2 df/+ mutant mice, including both male and female, gradually declines across multiple generations during backcrossing to C57BL/6J. The number of 16p11.2 df/+ and total mice for each generation are: N5: 21/53, N6: 26/118, N7: 41/250, N8: 57/434, N9: 16/202, and N10: 8/102.

  2. Supplementary Figure 2: Basal synaptic transmission is normal in the 16p11.2 df/+ mice. (28 KB)

    (a) Input-output functions, plotted as fEPSP slope versus stimulus intensity, are not different between the WT (n = 14 animals) and mutant mice (n = 14 animals). Repeated measures one-way ANOVA, p = 0.92.

    (b) Paired-pulse facilitation is comparable between the WT (n = 16 animals) and mutant (n = 17 animals) mice across multiple stimulus intervals (10, 20, 50, 100, 200, 300, 500 ms). Repeated measures one-way ANOVA, p = 0.76. All data are plotted as mean ± SEM.

  3. Supplementary Figure 3: Presynaptic LTD is independent of genotype and is not affected by cycloheximide treatment. (29 KB)

    DHPG increases paired-pulse facilitation in both the WT and 16p11.2 df/+ mutant slices and this effect is not affected by cycloheximide. PPF is calculated at a 50 ms inter-stimulus interval: WT baseline: 1.44 ± 0.019, n = 17 animals, 37 slices, WT DHPG: 1.55 ± 0.035, n = 17 animals, 18 slices, WT DHPG+CHX: 1.55 ± 0.027, n = 17 animals, 19 slices. Mut baseline: 1.45 ± 0.023, n = 16 animals, 42 slices, Mut DHPG:1.56 ± 0.053, n = 16 animals, 21 slices, Mut DHPG+CHX: 1.53 ± 0.038, n = 16 animals, 21 slices. Statistical analyses are performed using unpaired t-tests. All data are plotted as mean ± SEM.

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