The autism- and schizophrenia-associated protein CYFIP1 regulates bilateral brain connectivity and behaviour

Abstract

Copy-number variants of the CYFIP1 gene in humans have been linked to autism spectrum disorders (ASD) and schizophrenia (SCZ), two neuropsychiatric disorders characterized by defects in brain connectivity. Here, we show that CYFIP1 plays an important role in brain functional connectivity and callosal functions. We find that Cyfip1-heterozygous mice have reduced functional connectivity and defects in white matter architecture, similar to phenotypes found in patients with ASD, SCZ and other neuropsychiatric disorders. Cyfip1-deficient mice also present decreased myelination in the callosal axons, altered presynaptic function, and impaired bilateral connectivity. Finally, Cyfip1 deficiency leads to abnormalities in motor coordination, sensorimotor gating and sensory perception, which are also known neuropsychiatric disorder-related symptoms. These results show that Cyfip1 haploinsufficiency compromises brain connectivity and function, which might explain its genetic association to neuropsychiatric disorders.

Fig. 1

Cyfip1^+/− mice show reduced functional connectivity. a Functional connectivity matrices of WT and Cyfip1^+/− mice (lower and upper half of the matrix, respectively) (postnatal day 60, P60), in which functional correlation (z-score) between pairs of regions is represented by a colour scale (abbreviations: Cg = cingulate cortex, Cpu = caudate putamen, HC = hippocampus, MC = motor cortex, OFC = orbitofrontal cortex, PLC = prelimbic cortex, Resp = retrosplenial cortex, SS = somatosensory cortex, T = thalamus). b Based on the matrix in a, average functional connectivity strength of each region with all other regions is plotted for WT and Cyfip1^+/− mice (WT n = 15 and Cyfip1^+/− n = 17 mice; mean ± SEM; Holm-Sidak t-test; Cg, *p = 0.0178 and T, *p = 0.0196). c Seed-based analysis represented by functional connectivity maps for WT and Cyfip1^+/− animals. The strength of connectivity for the seed region, indicated above each image, is mapped by a colour scale representing the T-score. d Average bilateral functional connectivity strength for selected brain regions in WT and Cyfip1^+/− mice (WT n = 15 and Cyfip1^+/− n = 17 mice; mean ± SEM; Holm-Sidak t-test; MC *p = 0.0452, SS *p = 0.0157, HC *p = 0.0148)

Fig. 2

Cyfip1^+/− mice show defects in callosal architecture. a Diffusion Tensor Imaging (DTI) of WT and Cyfip1^+/− mice at postnatal day 60 (P60). Fractional anisotropy (FA) maps in which average FA values for WT and Cyfip1^+/− mice are represented by a colour scale (WT n = 7 and Cyfip1^+/− n = 6 mice). b Upper, FA differential maps between WT and Cyfip1^+/− mice (Δ(WT- Cyfip1^+/−)). Lower, representative anatomical MRI images as reference. c Statistical map of the FA differences between WT and Cyfip1^+/− mice. Shown are the T-values in a colour scale ranging from 2.2 to 3.3 (equivalent to uncorrected p values ranging from 0.05 to 0.005). The red scale indicates reduced FA values in the Cyfip1^+/− mice, the blue scale increased values. d The graph shows mean diffusivity (MD), axial diffusivity (AD), radial diffusivity (RD) and fractional anisotropy (FA) in the corpus callosum (WT n = 7 and Cyfip1^+/− n = 6 mice) (mean ± SEM; two-tailed t-test; FA *p = 0.0165)

Fig. 3

Cyfip1^+/− mice show defects in callosal myelination. a Representative electron micrographs of axons in the genu of the corpus callosum (CC) in WT and Cyfip1^+/− mice at P60. Lower panel, zoom image (scale bar 1 µm). b Schematic of the measured parameters. c Graphs show average axonal diameter, myelin thickness and g-ratio. The histogram on the right shows the g-ratio of axons with different diameters (n > 10,000 axons for each genotype, n = 3 mice for each genotype, mean ± SEM; two-tailed Mann–Whitney test; myelin thickness ***p < 0.0001, g-ratio ***p < 0.0001; Two-way ANOVA F_(1,20585) = 311.8, with Holm-Sidak’s multiple comparison test, g-ratio ***p < 0.0001 for all axon diameters)

Fig. 4

Spontaneous neuronal activity is reduced in Cyfip1^+/− adult mice. a Upper panel, representative image of a P60 brain slice (Image credit: Allen Institute. Coronal Allen Mouse Brain Atlas (https://mouse.brain-map.org/static/atlas), with the recorded area highlighted by a red circle. Lower panel, representative image of the P60 somatosensory cortex slice positioned on the microelectrode array (MEA) system. Dashed black lines delineate the corpus callosum (CC). The field of electrodes is visible above the CC. b Representative traces of single electrodes recording from WT and Cyfip1^+/− slices. c Representative raster plots of all 59 electrodes showing the spike events in WT and Cyfip1^+/− cortical slice recordings. The x-axis corresponds to the recording time and the y-axis to the electrode ID. d Quantification of the spike rate (left) and burst rate (right, both on a logarithmic scale) in WT and Cyfip1^+/− brain slices at P60 (WT n = 12 slices, 6 mice, and Cyfip1^+/− n = 28 slices, 13 mice; mean ± SEM; two-tailed t-test; *p = 0.032 for spike rate and *p = 0.027 for burst rate). Centre, cumulative frequency distribution of the spike rate for WT and Cyfip1^+/− mice (Kolmogorov–Smirnov test, p < 0.001)

Fig. 5

Reduced presynaptic function in Cyfip1^+/− adult mice. a Schematic of the experimental setup. b Representative sample traces induced in layer II/III cortical neurons at P60 by trains of callosal stimulation (5 pulses, 20 Hz). Recordings obtained from WT (black) and Cyfip1^+/− (grey) mice. c Left, graph of the normalised amplitude of EPSCs, and right, histogram and scatter plot of the amplitudes of the fifth EPSC, relative to the first. (WT n = 8 cells, three mice and Cyfip1^+/- n = 12 cells, three mice; mean ± SEM; two-way repeated measures ANOVA, genotype main effect **p = 0.0017, F_(1,18) = 13.53, and interaction p < 0.0001; two-tailed t-test **p = 0.0046)

Fig. 6

Motor coordination is reduced in Cyfip1^+/− mice. a Average latency to fall measured in the accelerating rotarod across the four trials (WT n = 15 and Cyfip1^+/− n = 16 mice; mean ± SEM; two-tailed t-test; *p = 0.024). b Latency to fall per trial measured in the accelerating rotarod (WT n = 15 and Cyfip1^+/− n = 16 mice; mean ± SEM; Two-way repeated measures ANOVA; p = 0.024 for genotype, F_(1,29) = 5.68, trial 1 **p = 0.0015). c Percentage of slips measured in the ladder rung walking test averaged across the three trials (WT n = 14 and Cyfip1^+/− n = 15 mice; mean ± SEM; two-tailed t-test; ***p < 0.0001). d Percentage of slips per trial in the ladder rung walking test (WT n = 14 and Cyfip1^+/– n = 15 mice; mean ± SEM; Two-way repeated measures ANOVA; genotype p < 0.0001, F_(1,27) = 22.04, trial 1 *p = 0.014, trial 2 *p = 0.038, trial three **p = 0.0048 in a post-hoc Sidak test)

Fig. 7

Cyfip1^+/− mice present ASD and SCZ-related phenotypes. a Preference for novel vs. familiar texture (tNORT) was measured for WT and Cyfip1^+/− mice (WT n = 14 and Cyfip1^+/− n = 15 mice; mean ± SEM; two-tailed t-test; ***p < 0.0001). b Average percentage of prepulse inhibition (PPI) for all intensities is shown for WT and Cyfip1^+/− mice (WT n = 11 and Cyfip1^+/− n = 8 mice) (mean ± SEM; two-tailed t-test; **p = 0.0081). c Percentage of PPI for each prepulse intensity in WT and Cyfip1^+/− mice (WT n = 11 and Cyfip1^+/− n = 8 mice) (mean ± SEM; Two-way repeated measures ANOVA; genotype p = 0.0072, F_(1,17) = 9.317, with Sidak’s multiple comparison 68 dB *p = 0.0367, 72 dB *p = 0.0113, 76 dB *p = 0.0134)