The NONO protein has been characterized as an important transcriptional regulator in diverse cellular contexts. Here we show that loss of NONO function is a likely cause of human intellectual disability and that NONO-deficient mice have cognitive and affective deficits. Correspondingly, we find specific defects at inhibitory synapses, where NONO regulates synaptic transcription and gephyrin scaffold structure. Our data identify NONO as a possible neurodevelopmental disease gene and highlight the key role of the DBHS protein family in functional organization of GABAergic synapses.
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We are grateful to the patients and their family members for their participation in our study and to the Functional Genomics Center Zürich (FGCZ) for transcriptomic services. This program has received a state subsidy managed by the National Research Agency under the Investments for the Future program bearing the reference ANR-10-IAHU-01.This study was also supported by the Centre National de la Recherche Scientifique (CNRS), the Fondation pour la Recherche Médicale (DEQ20120323702) and the Ministère de la Recherche et de l'Enseignement Supérieur, as well as by the Swiss National Science Foundation, the Zurich Clinical Research Priority Program “Sleep and Health,” the Zurich Fonds zur Förderung des akademischen Nachwuchses and the Zurich Neurozentrum (ZNZ). D.M., S.A.B. and S.K.T. are affiliates of the ZNZ Life Sciences Zurich graduate program, and S.A.B. is a member of the Zürich Center for Interdisciplinary Sleep Research (ZIS). The DDD study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the UK Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). The views expressed in this publication are those of the author(s) and not necessarily those of the Wellcome Trust or the Department of Health. The research team acknowledges the support of the UK National Institute for Health Research, through the Comprehensive Clinical Research Network.
The authors declare no competing financial interests.
A full list of consortium members appears in the Supplementary Note.
Integrated supplementary information
(a) Sanger sequencing chromatograms showing the NONO mutations in the probands and their parents. (b) Schematic representation of the NONO transcript showing exon structure. (c) Schematic overview of the NONO protein showing the different functional domains.
Western blot of NONO protein in patients and controls, using a different polyclonal antibody from the one used in Figure 1.
(a) Graph of average circadian oscillations of bioluminescence from Bmal1-luciferase reporter-infected fibroblasts from patients (grey) or siblings (black). Y-axis, background-subtracted bioluminescence in photons per minute; X-axis, time in days. Depicted curve is the average of three independent experiments in technical quadruplicate. (b) Circadian period measured in cells from controls (black bars) and patients (open bars). (c) Circadian amplitude measured in the same cells (arbitrary units). Student t-test, p<0.001. Values for both panels are the average of three independent experiments with fibroblasts from both patients and their siblings in technical quadruplicate.
(a) Photograph of representative brains from WT (left) and Nonogt (right) mice. (b) Quantification of weight of whole brain, cortex, and cerebellum. N=5-9 mice per genotype. Student t-test. White bars, WT. grey bars, Nonogt. In this and subsequent figures, ***p<0.001, **p<0.01, *p<0.05. Quantification of all brain parameters from different morphological tests is shown in detail in Table S4.
(a) Percentage of time spent in open arms of elevated plus-maze. N=13-14 mice per genotype. Student t-test, p<0.001 (b) Post-conditioning startle response in prepulse inhibition test. Y-axis, whole-body startle response in volts; X-axis, stimulus in decibels. N=13-14 mice per genotype. Student t-test, p<0.01 or 0.001 as indicated. (c) Open-field exploration. Y-axis, percentage of area surface tiles visited. X-axis, subsequent 5-minute intervals after commencement of test. N=18 mice per genotype, repeated ANOVA, gene p<.0002, time p<.0001, time x gene n.s. (d) Light-dark transition test. Y-axis, percentage of time spent in zones indicated on X-axis. N=18 mice per genotype, repeated ANOVA, gene p<.0001, zone p<.0001, zone x gene p<0.0001. In all panels, Nonogt mice are represented by grey bars/circles, compared to WT littermates (open).
(a) Staining of hippocampal cell nuclei by DAPI (blue), anti-NONO (green), and neuron-specific anti-NeuN or astrocyte-specific anti-GFAP (red, left or right column respectively). (b) Identical staining of cortex.
(a) Volcano plot of deregulated genes. Red dots, p<0.01 and log2>0.5. (b) Reduced GABRA2 mRNA levels in hippocampi of Nonogt mice. White bars, WT. grey bars, Nonogt. Student t-test, N=4. (c) List of most severely deregulated transcripts, showing upregulation of sister DBHS family members Sfpq and Pspc1.
(a) Western blots of hippocampal protein extracts from widtype and Nonogt mice hippocampi, probed with anti-NONO, anti-PSPC1, anti-SFPQ, and anti-β-actin. N=3 mice per genotype. (b) Quantification of (a). White bars, WT. Black bars, Nonogt. Student t-test, p<0.01.
(a) Profile of the mouse forebrain synaptic transcriptome. Y-axis, ratio of transcript reads from synaptome RNA sequencing compared to total. Selected transcripts previously characterized to be transported to synapses (Kif5a, Shank3, CamK2A, Arc, Gabra2), present throughout the cell (Map2, Actb), or retained in the nucleus (Neat1) are indicated to demonstrate the quality of synaptosomal transcript enrichment. Blue shading, transcripts more than 1.5x enriched. (b) Quantification of Neat1 compared to Gapdh in whole-cell homogenate, purified nuclei, supernatant, or gradient-purified synaptosomes from WT (white bars) or Nonogt mice (solid bars). (c) Quantification of CamkII compared to Gapdh. (d) Quantification of Gabra2 compared to Gapdh.
(a) Western blot showing fractionation of mouse forebrain into different neuronal compartments by density gradient centrifugation. Immunohistochemistry using antibodies against NONO, gephyrin, and GABA is pictured. As a control, β-actin and dendrite-enriched PSD-95 are also shown. (b) Quantification of the reduction in GABRA2 levels in each compartment.
(a) Single-transcript-resolution RNA in-situ hybridization to detect abundance of collybistin (CB, blue), Gabra1 (blue), Gabra2 (red), and CamKII (red) in WT or Nonogt neurons. Two transcripts were tested in different colors in a single plate of cells, and depicted in a single column. (b) Quantification of collybistin transcript distribution in puncta number per cell for 44-56 cells from 2 experiments. (C) Similar quantification of Gabra2. Solid circles, Nonogt neurons; open circles, WT.
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Mircsof, D., Langouët, M., Rio, M. et al. Mutations in NONO lead to syndromic intellectual disability and inhibitory synaptic defects. Nat Neurosci 18, 1731–1736 (2015). https://doi.org/10.1038/nn.4169
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