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. 2012;7(8):e42422.
doi: 10.1371/journal.pone.0042422. Epub 2012 Aug 10.

Haploinsufficiency of Cyfip1 produces fragile X-like phenotypes in mice

Ozlem Bozdagi  1 , Takeshi Sakurai, Nathan Dorr, Marion Pilorge, Nagahide Takahashi, Joseph D Buxbaum
Affiliations

Haploinsufficiency of Cyfip1 produces fragile X-like phenotypes in mice

Ozlem Bozdagi et al. PLoS One. 2012.

Abstract

Background: Copy number variation (CNV) at the 15q11.2 region, which includes a gene that codes for CYFIP1 (cytoplasmic FMR1 interacting protein 1), has been implicated in autism, intellectual disability and additional neuropsychiatric phenotypes. In the current study we studied the function of Cyfip1 in synaptic physiology and behavior, using mice with a disruption of the Cyfip1 gene.

Methodology/principal findings: We observed that in Cyfip1 heterozygous mice metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) induced by paired-pulse low frequency stimulation (PP-LFS) was significantly increased in comparison to wildtype mice. In addition, mGluR-LTD was not affected in the presence of protein synthesis inhibitor in the Cyfip1 heterozygous mice, while the same treatment inhibited LTD in wildtype littermate controls. mGluR-agonist (RS)-3,5-dihydroxyphenylglycine (DHPG)-induced LTD was also significantly increased in hippocampal slices from Cyfip1 heterozygous mice and again showed independence from protein synthesis only in the heterozygous animals. Furthermore, we observed that the mammalian Target of Rapamycin (mTOR) inhibitor rapamycin was only effective at reducing mGluR-LTD in wildtype animals. Behaviorally, Cyfip1 heterozygous mice showed enhanced extinction of inhibitory avoidance. Application of both mGluR5 and mGluR1 antagonist to slices from Cyfip1 heterozygous mice reversed the increase in DHPG-induced LTD in these mice.

Conclusions/significance: These results demonstrate that haploinsufficiency of Cyfip1 mimics key aspects of the phenotype of Fmr1 knockout mice and are consistent with the hypothesis that these effects are mediated by interaction of Cyfip1 and Fmrp in regulating activity-dependent translation. The data provide support for a model where CYFIP1 haploinsufficiency in patients results in intermediate phenotypes increasing risk for neuropsychiatric disorders.

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Conflict of interest statement

Competing Interests: OB, TS, and JDB have submitted a patent on this work (“Methods of Treating Psychiatric or Neurological Disorders with MGLUR Antagonists”; No. '61/255,340; File date: 10/27/2009). This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Generation and characterization of a mouse with disruption of the Cyfip1 gene.
(A) The genomic structure of Cyfip1 is shown to scale with larger horizontal boxes representing exons, and the first (ATG) and last (Stop) coding exons indicated. The diagram shows the site of the gene-trap insertion (identified as an LTR-flanked Trapping casette) in intron 1 (5′ to the first coding exon), in order to generate mice with a disruption of the Cyfip1 gene. (B) Synaptoneurosome preparations from wildtype (Wt) and Cyfip1 heterozygous (Het) mice were subjected to quantitative immunoblotting with an antibody to Cyfip1, with actin as a reference protein. The migration of molecular weight markers is shown on the left (in kDa). (C) Brain mRNA from wildtype (black bars) and Cyfip1 heterozygous (white bars) mice were subjected to qPCR for the indicated genes. (D) Quantification of Cyfip1 and Fmrp by immunoblotting of extracts from wildtype (black bars) and heterozygous (white bars) mice. *, P<0.05; **, P = 0.004.
Figure 2
Figure 2. Basal synaptic properties and long-term potentiation are normal but long-term depression is enhanced in Cyfip1 heterozygotes.
(A) Hippocampal slices from 4–6 weeks old wildtype (WT) or Cyfip1 heterozygous (Het) mice were analyzed for baseline synaptic properties, determined by input/output function, representing the relationship between stimulus intensity and the size of the field EPSP slope. (B) Paired-pulse facilitation in the Schaffer collateral-commissural pathway is not different between genotypes over the test interpulse interval of 50 ms. (C) HFS-induced LTP was not significantly different between wildtype (WT) or Cyfip1 heterozygous (Het) mice. (D) PP-LFS-induced LTD in Cyfip1 heterozygous mice was significantly increased. Inset: Representative EPSP traces recorded before stimulation (arrow) or 60 min after stimulation in wildtype and heterozygous animals (scale: 10 ms and 0.5 mV).
Figure 3
Figure 3. Long-term depression but not long-term potentiation is independent of protein synthesis in Cyfip1 heterozygotes.
(A, B) LTD induced by PP-LFS in wild type (A) or Cyfip1 heterozygous (B) mice, in the absence (o) or presence (•) of the protein synthesis inhibitor cycloheximide (60 µM). The effect of cycloheximide is significantly different across genotypes. (C, D) LTP induced by HFS in wildtype (C) or Cyfip1 heterozygous (D) mice, in the absence (o) or presence (•) of cycloheximide. In all panels, the large arrow indicates onset of stimulation. (E, F) Rapamycin blocks LTP induced by HFS in wildtype (E) and Cyfip1 heterozygous (F) mice, as shown by incubating slices in the absence (o) or presence (•) of the mTOR inhibitor rapamycin (20 nM). Onset of stimulation is indicated by an arrow.
Figure 4
Figure 4. DHPG-induced long-term depression is not dependent on protein synthesis or mammalian Target of Rapamycin in Cyfip1 heterozygotes.
(A) LTD was induced by DHPG (50 µM for 5 minutes, indicated by the short horizontal bar) in hippocampal slices from wildtype (Wt) and Cyfip1 heterozygous (Het) mice. LTD is significantly enhanced in the heterozygotes as compared to wildtype. Inset: Representative EPSP traces recorded before (arrow) or 40 min after DHPG in wildtype and heterozygous animals (scale: 10 ms and 0.5 mV). (B, C) LTD was induced with DHPG in wildtype (B) or Cyfip1 heterozygous (C) mice, in the absence (o) or presence (•) of cycloheximide (Cyclohex, 60 µM, indicated by the long horizontal bar). Cycloheximide significantly inhibited LTD in slices from wildtype but not heterozygous animals. (D, E) LTD was induced by DHPG (50 µM, indicated by the short horizontal bar) in hippocampal slices from wildtype (D) or Cyfip1 heterozygous (E) mice, in the absence (o) or presence (•) of rapamycin (20 nM, indicated by the long horizontal bar). (F) LTD was induced by DHPG (50 µM, indicated by the short horizontal bar) in hippocampal slices from wildtype (o) or Cyfip1 heterozygous (•) mice, the latter in the absence (•) or presence (▪) of both MPEP (10 µM) and LY367385 (indicated by the long horizontal bar). Application of the mGluR1 and mGluR5 antagonists decreased the magnitude of DHPG-induced-LTD in heterozygotes.
Figure 5
Figure 5. Normal learning and memory in Morris Water Maze and in fear conditioning but enhanced extinction of inhibitory avoidance in Cyfip1 heterozygous mice.
(A) Mice were tested using the Morris Water Maze. Time (s) to travel to the target platform was not significantly different between genotypes. (B) Mice were tested for fear conditioning, with mice receiving shocks at 120 and 180 seconds during training. Testing was performed 24 hours later, in the same test chamber, without footshock. (C) Inhibitory avoidance was measured by latency to enter the dark side of the box associated with prior shock. Extinction of inhibitory avoidance is enhanced in the heterozygotes. The lower panel shows the experimental design. WT, wildtype mice; Het, heterozygous mice; acq, acquisition; ext, extinction; IA, inhibitory avoidance. *, P = 0.027.
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