Autism gene Ube3a and seizures impair sociability by repressing VTA Cbln1

Abstract

Maternally inherited 15q11-13 chromosomal triplications cause a frequent and highly penetrant type of autism linked to increased gene dosages of UBE3A, which encodes a ubiquitin ligase with transcriptional co-regulatory functions. Here, using in vivo mouse genetics, we show that increasing UBE3A in the nucleus downregulates the glutamatergic synapse organizer Cbln1, which is needed for sociability in mice. Epileptic seizures also repress Cbln1 and are found to expose sociability impairments in mice with asymptomatic increases in UBE3A. This Ube3a–seizure synergy maps to glutamate neurons of the midbrain ventral tegmental area (VTA), where Cbln1 deletions impair sociability and weaken glutamatergic transmission. We provide preclinical evidence that viral-vector-based chemogenetic activation of, or restoration of Cbln1 in, VTA glutamatergic neurons reverses the sociability deficits induced by Ube3a and/or seizures. Our results suggest that gene and seizure interactions in VTA glutamatergic neurons impair sociability by downregulating Cbln1, a key node in the expanding protein interaction network of autism genes.

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Figure 1: Nuclear-targeted increases of UBE3A repress ASD network gene Cbln1 that is required in VGLUT2+ neurons for sociability.
Figure 2: Seizures synergize with increased UBE3A to repress sociability and Cbln1.
Figure 3: UBE3A–seizure synergy localizes to the VTA.
Figure 4: VTA VGLUT2+ neuron-targeted deletions of Cbln1 impair sociability and VTA-NAc glutamatergic synaptic transmission.
Figure 5: Sociability deficits produced by Ube3a-seizure synergy are rescued by chemogenetic activation or increased Cbln1 mRNA in VTA VGLUT2+ neurons.

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Acknowledgements

We thank O. DiStefano, G. Salimando and R. Broadhurst for colony work and the Harvard Medical School Neurobiology Imaging Facility (NINDS P30 Core Center Grant NS07203) and Boston Children’s Hospital IDDRC (1U54HD090255, P30HD18655). Supported by funding to V.K. (1R25NS070682 and an American Academy of Neurology Research Training Fellowship) and to M.P.A. from the NIH (1R01NS08916, 1R21MH100868, 1R21HD079249), Nancy Lurie Marks Family Foundation, Landreth Foundation, Simons Foundation, and Autism Speaks/National Alliance for Autism Research. R.A. was supported by Klarman Family Foundation.

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V.K., D.C.S., Y.N. and M.P.A. designed the study. V.K. and M.P.A wrote the manuscript. V.K., D.C.S. and Y.N. performed all experiments and analyses except for the following: E.O., M.J.S.N. and T.J.C validated microarray-identified gene regulations by qRT–PCR; F.M., R.A. and M.P.A. performed Gene Ontology and cluster visualization; I.N. performed nuclear/cytosolic fractionation studies; S.P. performed olfaction and open-field experiments; M.A.J., B.L.T., M.A.S., E.M.K. and M.J.S.N. developed molecular probes and constructs.

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Correspondence to Matthew P. Anderson.

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Reviewer Information Nature thanks E. Kim and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Protein–protein interactions between genes regulated in Ube3a-2× mice and autism-related genes.

Edges between gene-labelled nodes represent documented protein–protein physical interactions. Clusters shown here are labelled with their major statistically significant Gene Ontology classification(s) together with P values representing significant enrichment. Within each cluster, all the genes (UBE3A-regulated and autism-related) were analysed.

Extended Data Figure 2 Ube3a-NLS mice and mice with increased untagged UBE3A levels display impaired sociability.

a, Total UBE3A protein levels by western blot in wild-type, Ube3a-1×, Ube3a-NLS3 and Ube3a-NLS7 mice (F[3,17] = 12.30, P < 0.001, 5 females per group). b, Flag staining in Ube3a-1× and Ube3a-NLS mice, counterstained with DAPI (somatosensory cortex; 3 females per genotype). Scale bar, 50 μm. c, Quantified nuclear and neuropil Flag immunofluorescence in arbitrary units (a.u.). d, UBE3A protein levels in cytosolic and nuclear fractions obtained from wild-type and Ube3a-NLS7 mice (4 females per group), demonstrating a selective increase in nuclear UBE3A protein levels (see Supplementary Fig. 1). TBP, TATA-box binding protein. e, Chamber times (for Fig. 1d) during the social trial showing absolute times spent in social, middle and opposite chambers. f, Distances moved during acclimation/social trials analysed by two-way ANOVA: WT/Ube3a-1× (trial factor F[1,56] = 18.09, P < 0.001, genotype factor F[1,56] = 0.73, P > 0.1), WT/Ube3a-2× (trial factor F[1,52] = 12.2, P < 0.001, genotype factor F[1,52] = 2.046, P > 0.1), WT/Ube3a-2× untagged (trial factor F[1,50] = 12.21, P < 0.01, genotype factor F[1,50] = 9.67, P < 0.01), WT/Ube3a-NLS3 (trial factor F[1,56] = 14.41, P < 0.001), genotype factor F[1,56] = 0.53, P > 0.1), WT/Ube3a-NLS7 (trial factor F[1,56] = 22.68, P < 0.001, genotype factor F[1,56] = 4.88, P < 0.05), WT/p-Ube3a-mKO (trial factor F[1,62] = 3.67, P > 0.05, genotype factor F[1,62] = 216.7, P < 0.0001), WT/m-Ube3a-mKO (trial factor F[1,48] = 1.75, P > 0.1, genotype factor F[1,48] = 197.9, P < 0.001). g, Total time spent physically interacting during genotype-matched paired female interactions (for Fig. 1e). h, Time spent self-grooming by female mice during a 10-min observation session across Ube3a-1× (n = 10 versus n = 14 WT), Ube3a-2× (n = 11 versus n = 12 WT), Ube3a-NLS3 (n = 14 per group) and Ube3a-NLS7 (n = 14 per group) mice. i, Ube3a-NLS7 (n = 15) and wild-type (n = 12) littermates take similar times to find a food item buried in fresh bedding. j, k, In an open field, Ube3a-NLS7 and wild-type (n = 5 per group) littermates spend equal times in the centre of an open field (j) and ambulate equal distances (k). l, m, In an elevated plus maze test, Ube3a-NLS7 mice (n = 10) spend significantly less time in the open arms (l) and ambulate slightly shorter distances overall than wild-type littermates (n = 7) (m). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Two-tailed unpaired Student’s t-tests are used for all comparisons unless otherwise specified. Source data

Extended Data Figure 3 qPCR-based validation of various genes downregulated in cortical samples from Ube3a-2× mice.

a, b, In separate cohorts of wild-type and transgenic or maternal knockout Ube3a mice, we confirmed the downregulation of the following genes: Fat2, Arhgef33, Crtam, Dao, Smpx, Pcp2, Gsbs (also known as Ppp1r17) (a), A2m, Mab21l1, Zic1, Car8, Lhx1, Calb2, Slc1a6, Chn2 and St18 (b). Sample sizes: Ube3a-1× (ref. 4), Ube3a-NLS, Ube3a-2× (ref. 4) and m-Ube3a-mKO (ref. 21) (n = 3 pooled samples from two mice each); Ube3a-2× (untagged) and p-Ube3a-mKO (ref. 5) (n = 6–12 per group, unpooled). Data are mean ± s.e.m. Transgene constructs and/or breeding schemes are shown on the right. *P < 0.05, **P < 0.01, ***P < 0.001. Unpaired two-tailed Students t-tests. Source data

Extended Data Figure 4 qRT–PCR-based validation of various genes upregulated in cortical samples from Ube3a-2× mice.

a, In separate cohorts of wild-type and Ube3a-2× mice, the upregulation of the following genes was confirmed by qPCR: S100a8, S100a9, Gh, Cdhr1, Nptx2, Blnk, Islr2, Grasp, Agmat, Pcdh8, Rasl10a, Dlx1, Wisp1, Glt8d2, Igfbp4, Ptgs2, Dmkn, Gcnt2, Pcdh15, Efna3, Kalrn, Tnnt2, Kcnh5, Prkg2, Trpc6, Kcnq3 and Homer2. bd, Regulation of these genes in cortical samples from Ube3a-NLS7 (b), Ube3a-1× (c) and Ube3a-mKO (d) mice. n = 3 per genotype, with each biological replicate representing a pooled sample of tissue from two genotype-matched mice. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Unpaired two-tailed Students t-tests. Source data

Extended Data Figure 5 Characterization of VGLUT2-cre;Cbln1fl/fl mice.

a, Representative coronal sections from the Allen Mouse Brain Atlas66, with shaded red regions depicting the approximate geometric centre of punch biopsy samples used for qPCR experiments. Image credit: Allen Institute for Brain Science. b, Chamber times (for Fig. 1g) during the social trial showing absolute time spent in social, middle and opposite chambers. c, Distances moved during acclimation/social trials analysed by two-way ANOVA (trial factor F[1,32] = 27.21, P < 0.0001, genotype factor F[1,32] = 8.186, P < 0.01). d, Total time spent physically interacting during genotype-matched paired female interactions (for Fig. 1h, male–female pairs: Cre n = 14, Cre+ n = 8, genotype-matched female pairs: Cre n = 11, Cre+ n = 16). e, Total time spent self-grooming during a 10-min observation period (males only, n = 8 per group). f, g, In the elevated plus maze, VGLUT2-cre;Cbln1fl/fl (n = 10) mice spent greater time in the open arms than Cbln1fl/fl (WT) mice (n = 8) (f) and ambulated significantly greater distances (g). h, Performance of VGLUT2-cre;Cbln1fl/fl (n = 16) and Cbln1fl/fl (n = 13) mice on buried food testing. i, j, Open-field testing of VGLUT2-cre;Cbln1fl/fl and Cbln1fl/fl mice (n = 10 per group) with time in centre (i) and distances moved (j). k, VGLUT2-cre;Cbln1fl/fl mice (n = 16) display reduced latencies to fall off of an accelerating rotarod compared with Cbln1fl/fl mice (two-way repeated measures ANOVA, n = 13 per group, genotype factor, F[1, 81] = 18.10, P < 0.001). Data are mean ± s.e.m. *P < 0.05, **P < 0.01. Two-tailed unpaired Student’s t-tests are used for all comparisons unless otherwise specified. Source data

Extended Data Figure 6 Recurrent seizures induced by PTZ impair sociability.

a, Ten daily 30 mg kg−1 PTZ injections applied to male wild-type mice resulted in a gradual daily increase in the percentage of animals that displayed a PTZ-induced generalized convulsion (n = 25). b, Weight change for PTZ- (n = 10) or saline- (n = 8) treated mice, with test day factor analysed by two-way repeated measures ANOVA (F[9,160] = 0.28, P > 0.1, treatment factor F[1,160] = 0.34, P > 0.1). c, d, 24 h after the last PTZ (n = 16) or saline (n = 14) injection, open-field testing identified no significant differences in the time spent in the centre of the field (c) or the total distance ambulated in the field (d) (n = 10 per group). e, f, Similarly, on the elevated plus maze, 10 PTZ-induced seizures did not affect the time spent in the open arms (e) or distance moved (f) (n = 10 per group). g, Distances moved during acclimation/social trials for male mice analysed by two-way ANOVA (for Fig. 2b, treatment factor F[1,32] = 0.38, P > 0.1, trial factor F[1,32] = 0.06, P > 0.1). h, i, Female wild-type FVB mice exposed to an identical 10-day PTZ protocol (n = 10) demonstrated impaired sociability 24 h after the last injection compared with mice that received saline alone (n = 9) (h), whereas the distances moved during acclimation/social trials were not affected (treatment factor F[1,34] = 2.47, P > 0.1, trial factor F[1,34] = 1.79, P > 0.1) (i). j, k, Ten PTZ-induced seizures do not affect self-grooming rates (n = 10 females per group) (j) or performance on the buried food test (k) (males: saline n = 10, PTZ n = 7 per group, females: saline n = 7, PTZ n = 9 per group). l, m, 30 days after the last of 10 daily PTZ injections, male wild-type FVB mice continue to display impaired sociability (saline n = 10 per group, PTZ n = 7 per group) (l), as do daily PTZ-treated female mice (saline n = 6 per group, PTZ n = 7 per group) (m). n, Distances moved during acclimation/social trials for Fig. 2f, g, analysed by two-way ANOVA. WT/p-Ube3a-mKO (trial factor F[1,62] = 5.06, P < 0.05, genotype factor F[1,62] = 209.2, P < 0.0001) and WT/m-Ube3a-mKO (trial factor F[1,54] = 0.20, P > 0.1, genotype factor F[1,54] = 56.06, P < 0.001). o, Distances moved for Fig. 2h (trial factor F[1.62] = 3.57, P > 0.05, group factor F[2,62] = 1.57, P > 0.05). p, Distances moved for Fig. 2i (trial factor F[1,62] = 13.29, P < 001, group factor F[2,62] = 2.43, P > 0.05). q, Ube3a mRNA measured in punches from wild-type FVB males treated with 10 daily injections of 30 mg kg−1 PTZ or saline (HPc, n = 8 per group, ECx and VTA: saline n = 10, PTZ n = 8). Data are mean ± s.e.m. ***P < 0.001. Two-tailed unpaired Student’s t-tests are used for all comparisons unless otherwise specified. Source data

Extended Data Figure 7 Validation oflox-TB-Ube3a mice.

a, qPCR analysis of Ube3a mRNA from hippocampal samples (Extended Data Fig. 5a) demonstrating the upregulation of Ube3a in VGLUT2-cre;lox-TB-Ube3a and Emx-cre;lox-TB-Ube3a mice. b, Similar analysis of Ube3a mRNA levels from tissue punches of the VTA of DAT-cre;lox-TB-Ube3a mice (n = 8 per group). ch, Spatial confirmation of Flag staining in various crosses of lox-TB-Ube3a mice, confirming expression of UBE3A in inhibitory forebrain neurons (c), cortical and hippocampal neurons (d), scattered staining in midbrain and pontine nuclei (e), locus coeruleus (f), striatal cholinergic interneurons (g), and raphe regions (h). Images are representative of n = 2–3 mice per group. Data are mean ± s.e.m. *P < 0.05, ***P < 0.001. Two-tailed unpaired Student’s t-tests. Source data

Extended Data Figure 8 Targeted overexpression of Ube3a in VGLUT2+ and DAT+ neurons increases the susceptibility to seizure-induced deficits in sociability.

ah, For each line of mice, we provide baseline sniffing time measures on sociability testing (far left), distances moved during this baseline test (second from left), seizure severity scores during five (alternate day) 30 mg kg−1 PTZ injections (middle), post-seizure sniffing time measures on three-chamber testing (second from right) and associated distances moved (far right). For sociability data, black and white bars represent sniffing times for social and opposite wire mesh cages, respectively. As described in Fig. 1, sniffing time is analysed by two-way repeated-measures ANOVA. F statistics and P values for ‘interaction effects’ between chamber side (social versus opposite) or experimental group (viruses, treatments or genotypes) and ‘main effects’ are shown in Supplementary Table 5. For significant interaction and/or main effects, a Bonferroni post-hoc test P value is reported (adjusted for multiple comparisons). For distance measures, black and white bars represent distances moved during acclimation and social trials, respectively. For VGLUT2-cre and DAT-cre lines, see Fig. 3c–e, g–i. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Source data

Extended Data Figure 9 Stereotaxic manipulations of Ube3a, VGLUT2 and Cbln1 in VTA.

a, qPCR analysis of Cbln1 mRNA in VTA tissue punches of wild-type and Ube3a-1× (ref. 4) mice obtained 24 h after the last of 5 alternate day PTZ injections. b, Distances moved during acclimation/social trials for mice in Fig. 3k (genotype factor F[1,28] = 14.45, P < 0.001, trial factor F[1,28] = 5.16, P < 0.05). c, Distances moved for Fig. 3l (genotype factor F[1,28] = 1.267, P > 0.1, trial factor F[1,28] = 2.72, P > 0.1). df, Distances moved for Fig. 4a (virus factor F[1,34] = 2.81, P > 0.1, trial factor F[1,34] = 2.02, P > 0.1) (d), time spent in the centre (e), or total distance moved (f) in an open field for AAV-Cre-GFP-injected (n = 12) or AAV-GFP-injected (n = 8) Cbln1fl/fl mice. g, Distances moved for Fig. 4b (virus factor F[1,44] = 7.9, P < 0.01, trial factor F[1,44] = 20.49, P < 0.001). h, Distances moved for Fig. 4c (genotype factor F[1,26] = 0.22, P > 0.1, trial factor F[1,26] = 5.86, P < 0.05). i, Cumulative frequency plots of spontaneous EPSC (sEPSC) amplitudes for Fig. 4d, e. j, Representative response of an MSN to injected currents (−20 to +280 pA). k, Average resting membrane potentials in the NAc medial shell in control mice (n = 23 neurons) and mice with VTA-targeted AAV-Cre-GFP-mediated deletions of Cbln1 (n = 22 neurons, P > 0.1). l, Laser intensity needed to induce EPSC currents in medial shell NAc MSNs from ChR2-eYFP mice with AAV-Cre-GFP infused into the VTA (n = 5 neurons). m, Distances moved for Fig. 4i (trial factor F[1,36] = 9.45, P < 0.01, virus factor F[1,36] = 8.49, P < 0.01). n, o, Time spent in the centre (n) and distances moved (o) during the open field test after AAV-Cre-GFP-mediated deletion of VGLUT2 in the VTA (n = 8). p, Distances moved for Fig. 4i (genotype factor F[1,36] = 1.15, P > 0.1, trial factor F[1,36] = 6.18, P < 0.1). Data are mean ± s.e.m. *P < 0.05. Two-tailed unpaired Student’s t-tests are used for all comparisons across two groups, whereas two-way ANOVA is used for all comparisons of distances moved. Kolmogorov–Smirnov test was used in i. Source data

Extended Data Figure 10 Chemogenetic manipulations of VTA VGLUT2+ neurons.

a, Distances moved for Fig. 5a (trial factor F[1,48] = 20.11, P < 0.001, treatment factor F[1,48] = 0.73, P > 0.5). b, Distances moved for Fig. 5b (treatment factor F[2,111] = 1.04, P > 0.5, trial factor F[2,111] = 9.55, P < 0.01). c, Distances moved for Fig. 5c (trial factor F[1,102] = 18.16, P < 0.0001, group factor F[2,102] = 5.7, P < 0.01). d, Distances moved for Fig. 5d (trial factor F[1,90] = 3.64, P > 0.05, group factor F[2,90] = 0.77, P > 0.05). e, Left, distances moved for Fig. 5e, left (baseline) (trial factor F[1,52] = 7.08, P < 0.05, virus factor F[1.52] = 0.022, P > 0.5). Right, distances moved for Fig. 5e, right (after PTZ) (trial factor F[1,76] = 8.48, P < 0.01, virus factor F[1,76] = 1.16, P > 0.05). f, qPCR analysis of Rest (also known as Nrsf) mRNA levels in VTA 24 h after 10 PTZ (n = 8) or saline (n = 10) injections. g, Data presented from the Encode Project Consortium67 showing locations of REST binding within Cbln1 intron of human (top) and mouse (bottom). Data are mean ± s.e.m. *P < 0.05. Two-tailed unpaired Student’s t-tests are used for all comparisons across two groups, whereas two-way ANOVA is used for all comparisons of distances moved. Source data

Supplementary information

Supplementary Figure 1

This file shows the source gel for the representative bands provided in Extended Data Figure 2d. (PDF 140 kb)

Supplementary Table 1

This file lists genes identified as up or downregulated in cortical samples from Ube3a2x mice compared with WT littermates. A +/- 1.4 fold change cutoff was used. Accession numbers, fold change and p values are shown. (XLSX 46 kb)

Supplementary Table 2

This file lists microarray-identified genes by gene ontology classifications together with a calculated p value for enrichment within a specific biological process. Worksheet 1 within this table displays results of annotation through the Kyoto Encyclopedia of Genes and Genomes (KEGG). Worksheets 2-4 display the same results when gene lists were annotated by classifications as defined by the Gene Ontology Consortium (biological process, cellular component and molecular function). (XLSX 3250 kb)

Supplementary Table 3

This file contains a list of selected 276 genes identified as strongly linked to autism from a larger list of 706 genes from the Simons Foundation Autism Research Initiative (SFARI, https://gene.sfari.org/autdb/HG_Home.do - see Basu, S. N., Kollu, R. & Banerjee-Basu, S. AutDB: a gene reference resource for autism research. Nucleic acids research 37, D832-836, doi:10.1093/nar/gkn835 (2009)). (XLSX 34 kb)

Supplementary Table 4

This file contains sequences for forward and reverse primers utilized in quantitative real-time PCR experiments. (XLSX 37 kb)

Supplementary Table 5

This file comprises: Worksheet 1 - A table of chamber times (in seconds) provided for each graph where "sniffing times" are depicted in the main and extended figures. This excludes Fig. 1d and Fig. 1h, where chamber times are depicted graphically in Extended Data Figs. 2e and 5b respectively; Worksheet 2 - Distances moved, chamber times and “sniffing times” (for "social" and "opposite") for a population of 101 wildtype seizure naïve FVB mice (males and females) with column statistics (mean and measures of variance) as well as results of Kolmogorov-Smirnov testing for the presence of a normal distribution; Worksheet 3 - Two-way repeated measures ANOVA analysis results for each graph depicting three chamber sociability data. The results of post-hoc testing are provided comparing "social" versus "opposite" sniffing times for each group separately. (XLSX 66 kb)

Supplementary Table 6

This file contains experimental data. (XLSX 667 kb)

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Krishnan, V., Stoppel, D., Nong, Y. et al. Autism gene Ube3a and seizures impair sociability by repressing VTA Cbln1. Nature 543, 507–512 (2017). https://doi.org/10.1038/nature21678

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