Autism spectrum disorder (ASD) is genetically heterogeneous with convergent symptomatology, suggesting common dysregulated pathways. In this study, we analyzed brain transcriptional changes in five mouse models of Pitt–Hopkins syndrome (PTHS), a syndromic form of ASD caused by mutations in the TCF4 gene, but not the TCF7L2 gene. Analyses of differentially expressed genes (DEGs) highlighted oligodendrocyte (OL) dysregulation, which we confirmed in two additional mouse models of syndromic ASD (Ptenm3m4/m3m4 and Mecp2tm1.1Bird). The PTHS mouse models showed cell-autonomous reductions in OL numbers and myelination, functionally confirming OL transcriptional signatures. We also integrated PTHS mouse model DEGs with human idiopathic ASD postmortem brain RNA-sequencing data and found significant enrichment of overlapping DEGs and common myelination-associated pathways. Notably, DEGs from syndromic ASD mouse models and reduced deconvoluted OL numbers distinguished human idiopathic ASD cases from controls across three postmortem brain data sets. These results implicate disruptions in OL biology as a cellular mechanism in ASD pathology.
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Impaired macroglial development and axonal conductivity contributes to the neuropathology of DYRK1A-related intellectual disability syndrome
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RNA-seq data from the cortex and whole brain of Tcf4-mutant mice are available via Globus: http://research.libd.org/globus/NatNeuro_TCF4_Data/ and at BioProject under accession number PRJNA601252.
R code used to analyze data in this study and analyzed data are available at https://github.com/LieberInstitute/PTHS_mouse.
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We are grateful for the vision and generosity of the Lieber and Maltz families, who made this work possible. We thank D.R. Weinberger for his helpful comments and feedback. We thank the Johns Hopkins School of Medicine Microscope Core Facility and specifically L. Roker for generating TEM images of CC used in this study. This work was supported by the Lieber Institute for Brain Development, the Pitt–Hopkins Research Foundation Awards (to B.J.M., B.D.P., C.T., D.S. and A.J.K.), National Institute of Mental Health (NIMH) grant R56MH104593 (to B.J.M.), NIMH grant R01MH110487 (to B.J.M.), a Johns Hopkins PURA grant (to B.N.P.), UPenn Orphan Disease Center Million Dollar Bike Ride grant MDBR-15-108-PH (to B.D.P. and C.T.), NARSAD Young Investigator grant 20653 from the Brain Behavior Research Foundation (to C.T.), National Institute of Neurological Disorders and Stroke grant P30NS045892 (to J.M.S.), National Institute of Child Health and Human Development grant P30HD03110 (to J.M.S.), NIMH grant R01MH104158 (to D.S. and A.J.K.), National Institute of General Medical Sciences training grant T32GM008208 (to B.N.P.) and NIMH training grant T32MH015330 (to B.A.D.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The authors declare no competing interests.
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Comparison of lifespan expression patterns of TCF4 in heterozygous (Tcf4tr/+) mice and wild-type (Tcf4+/+) littermates in qRT-PCR and RNA-sequencing analyses. mRNA and protein were extracted from frontal cortex of mice across developmental ages. (A) qRT-PCR analysis of full-length Tcf4 transcripts from mouse frontal cortex. Tcf4tr/+ mice show overall reduced expression compared to Tcf4+/+ mice (n = 66 mice, ANOVA p = 0.02) with the greatest decrease in Tcf4 expression around postnatal days 1–4 (P1–4, n = 18 mice, Posthoc p<0.05). (B) RNA-seq analysis also shows Tcf4 expression decreased in the Tcf4tr/+ mouse in the exon after the truncation (n = 35 mice). Tcf4tr/+ mice had significant decrease of Tcf4 exons (differentially expressed exon by genotype FDR = 3.35 × 10−35). The boxplot shows the quartile breaks of residualized variance stablilized count of a Tcf4 exon after the truncation (see methods on residualization for visual interpretation). (C) Western blot of endogenous mouse TCF4 at three ages (E12, P1, and P42). A single full-length (TCF4 fl; 80kDa) protein is observed in lysates from Tcf4+/+mouse brain and Tcf4tr/+ mouse brain expresses a truncated (TCF4 tr) and full-length TCF4 protein. These representative gel images are compiled across several different gel images and stitched together. (D) Full-length TCF4 protein is decreased in the Tcf4tr/+ mouse brain (n = 3 mice per genotype per timepoint, pAnova = 0.0009) with the largest effect occuring at P1 in the TCF4tr/+ mice (n = 3 mice per condition, two-sided unpaired t-test, p<0.01). Center values indicate mean and errors bars are the S.E.M., *<0.05, **<0.01, ***< 0.001.
a, Table of DEGs (NP1 = 28, NAdult = 69, using the two-sided differential expression cutoff of FDR<0.05) and percent of differential expression replication across different forms of Tcf4 mutation P1 and adult mice. Most DEGs and replication occur in adult mice. The replication rate was defined as the proportion/percentage of DE genes that were p < 0.01 in at least one other mouse model of the same age group divided by those DEGs in the reference mouse model. b, Differential expression log2 fold-change heatmap comparing replicated DEGs across various models of Tcf4 mutations in P1 (replication defined the same gene having differential expression with two-sided p<0.05).
Bulls-eye plots from CSEA analysis of DEGs in (a) P1 and (b) adult Tcf4tr/+ mice. The bulls-eye plot size is scaled to the number of genes specific to a cell type at increasing levels of specificity as published by Xu et al., 201461. The FDR-adjusted hypergeometric test p-value is plot for each level of specificity, with unenriched groups colored gray. Cell type bulls-eye plots are arranged by hierarchical distance of their specific gene expression levels. (a) P1 DEGs (N = 36 DEG at Padj < 0.05) enriched for D1+, D2+, and cholinergic neurons (Padj<0.05). (b) Adult DEGs (N = 1832 DEG at Padj < 0.05) strongly enrich for OLs among other neuronal cell types.
a, Plot of gRatio and corresponding radius for all axons assessed. Axon radius is significantly correlated with gRatio (p = 2.86e-34), and this correlation is different by genotype (p = 0.03). (b–f) No significant differences were observed between genotypes for gRatio (p = 0.796), axon area (p = 0.844), myelin area (p = 0.852), myelin + axon radius (p = 0.615), or axon radius (p = 0.873).
The peak time of N1 and N2 waveform (y-axis) is the amount of time between stimulation artifact and the amplitude peak of the compound action potential. The peak time is plotted against distance (x-axis) which is the distance between the stimulating electrode and recording electrode. The slope of the line generated from both N1(a) and N2 (b) does not differ between genotypes (N1 slope p = 0.96, N2 slope p = 0.36, N = 30 slices from 4 Tcf4+/+ and 5 Tcf4tr/+ mice) Center values indicate mean and errors bars are the S.E.M.
a, Example images of fluorescent in situ hybridization showing Tcf4 transcript co-localizes with both Pdgfrα and Mbp. b, Summary plots of single-cell RNA-seq data across oligodendrocyte development showing expression levels for Pdgfrα, Tcf4, Olig2, and Mbp. This data was adapted from Marques et al.24.
a, Primary neuronal culture stained with CNP and GFAP as a positive control for antibody staining. b1, Primary OL culture stained with CNP and GFAP. b2, Cell counts showing primary OL cultures have very few neurons (Tuj1+) or astrocytes (GFAP+). Numbers indicate number of cells counted for that condition. c, Primary neuronal cultures stained with OLIG2, NeuN, and GFAP as a positive control for antibody staining. d, Primary OL culture stained with OLIG2, NeuN, and GFAP. d1, Cell counts showing primary OL cultures have very few neurons (NeuN+) or astrocytes (GFAP+).
Extended Data Fig. 8 OPCs derived from Tcf4tr/+ mice show inefficient maturation into oligodendrocytes.
a, Representative images of OPCs (PDGFRα) and mature OLs (MBP, CNP) derived from Tcf4+/+ and Tcf4tr/+ mice. To control for cell numbers all cell counts are normalized by the pan-OL marker Olig2 that labels both OPC and mature OLs. Tcf4tr/+ produce significantly more OPCs (n = 23 mice, two-tailed unpaired t-test, p<0.0001) and fewer MBP positive OLs (Tcf4+/+ 0.19 ± 0.02 vs. Tcf4tr/+ 0.05 ± 0.01, n = 23 mice, two-tailed unpaired t-test, p<0.0001). b, Representative images of OPCs (PDGFRα) and mature OLs (CNP). Tcf4tr/+ produce significantly more OPCs (two-tailed unpaired t-test, n = 17 mice, two-tailed unpaired t-test, p<0.0001) and fewer CNP positive OLs (Tcf4+/+ 0.56 ± 0.04 vs. Tcf4tr/+ 0.22 ± 0.02, n = 17 mice, two-tailed unpaired t-test, p<0.0001). All scale bars equal 100µm. For all bar graphs, center values represent the mean and error bars are S.E.M.,***p<0.001, ****p<0.0001.
Comparison of differential expression in adult PTHS mice with human ASD and 15q duplication (15q Dup) in postmortem frontal, temporal, and cerebellum. (NTemp = 68, NFrontal = 73, NVermis = 63, Human two-sided differential expression p<0.05, mouse DEGs, FDR<0.01). a, Log2 fold-change comparison of adult PTHS mouse DEGs replicated in human ASD and 15q Dup in each tissue region (p<0.05). Gene regulation in PTHS mice cluster closest with ASD differential expression in cortex. b, More than 50% of replicated PTHS DEGs had concordant fold-change directionality. Null permutation for empirical p-value significance of human-mouse gene fold-change concordance from 1000 permutations are reported (Two-sided Fisher’s exact test, *, padj<0.05; **, padj<0.01; *** padj<0.001). c, Replicated DEGs in ASD and 15q Dup are significantly enriched in all tissues, mostly in the cortex (FDR-adjusted Fisher Exact test for overlap of Tcf4 mouse DEG with ASD DEG, padj<0.05). d, Venn diagram showing overlap of PTHS mouse DEGs with human ASD or 15q Dup in cortical tissues. e, Gene ontology analysis shows tissue-specific biological processes and cellular components between overlap of PTHS mouse and human ASD or 15q Dup (NASD = 10896 and N15qDup = 13149 DEGs at p < 0.01,s q-adjusted two-sided hypergeometric test). The gene sets are largely brain region specific and concordant between human ASD and 15q Dup. The color of the dot plots shows the q-adjusted hypergeometric test p-value for gene set enrichment of the DEG of each diagnosis group.
Extended Data Fig. 10 Mouse concordant ASD genes (CAGs) are not convergent with Schizophrenia or Down Syndrome.
a, The eigengene of the CAGs found across the three models of syndromic ASD explains 65.8% of the gene expression variance and is not associated with Schizophrenia diagnosis (linear regression two-sided p-value = 0.538). b, The eigengene of the CAGs found across the three models of syndromic ASD explains 53.2% of the gene expression variance and is not associated with Down Syndrome diagnosis (linear regression two-sided p-value = 0.34). c, Estimated cellular composition differences between individuals with schizophrenia and controls using reference-based deconvolution. There were significant increases of astrocytes (p = 0.0002) and endothelial cells (p = 0.0118) and decreases in microglia (p = 0.0076) in individuals with schizophrenia compared to controls using linear regression analysis.
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Phan, B.N., Bohlen, J.F., Davis, B.A. et al. A myelin-related transcriptomic profile is shared by Pitt–Hopkins syndrome models and human autism spectrum disorder. Nat Neurosci 23, 375–385 (2020). https://doi.org/10.1038/s41593-019-0578-x
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