Abstract
The molecular mechanisms and evolutionary changes accompanying synapse development are still poorly understood1,2. Here we generate a cross-species proteomic map of synapse development in the human, macaque and mouse neocortex. By tracking the changes of more than 1,000 postsynaptic density (PSD) proteins from midgestation to young adulthood, we find that PSD maturation in humans separates into three major phases that are dominated by distinct pathways. Cross-species comparisons reveal that human PSDs mature about two to three times slower than those of other species and contain higher levels of Rho guanine nucleotide exchange factors (RhoGEFs) in the perinatal period. Enhancement of RhoGEF signalling in human neurons delays morphological maturation of dendritic spines and functional maturation of synapses, potentially contributing to the neotenic traits of human brain development. In addition, PSD proteins can be divided into four modules that exert stage- and cell-type-specific functions, possibly explaining their differential associations with cognitive functions and diseases. Our proteomic map of synapse development provides a blueprint for studying the molecular basis and evolutionary changes of synapse maturation.
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Data availability
All raw proteomic data were deposited to ProteomeXchange through MassIVE (human PFC dataset: MSV000091887 and PXD042067; human V1 dataset: MSV000091888 and PXD042068; macaque dataset: MSV000091889 and PXD042069; mouse dataset: MSV000091890 and PXD042071). All processed data are available in Supplementary Tables 1–13 and at an online interactive portal (https://liwang.shinyapps.io/PSD_development_explorer). The reference proteomes (Homo sapiens UP000005640_9606, Macaca mulatta UP000006718_9544 and Mus musculus UP000000589_10090) can be downloaded from UniProt (https://www.uniprot.org/proteomes). The PPI data can be downloaded from the BioGRID database (https://thebiogrid.org/). Bulk-level RNA-seq data of developing human brain can be downloaded from the BrainSpan database (http://www.brainspan.org) or from the PsychENCODE website (http://evolution.psychencode.org/). Protein domain information can be downloaded from the SMART database (https://smart.embl.de/). Single-cell and single-nucleus RNA-seq data from developing and adult human neocortex can be downloaded from the UCSC Cell Browser (https://cells.ucsc.edu). GWAS summary statistics for human cognitive function can be downloaded from the GWAS ATLAS resource (https://atlas.ctglab.nl). LOEUF scores, missense z-scores and synonymous z-scores for all human genes can be downloaded from the gnomAD database (https://gnomad.broadinstitute.org). De novo variants in neurodevelopmental disorders can be obtained from denovo-db (https://denovo-db.gs.washington.edu/denovo-db). GWAS summary statistics for psychiatric disorders can be downloaded from The Psychiatric Genomics Consortium database (https://www.med.unc.edu/pgc).
Code availability
Code used for data analysis in this manuscript is available at GitHub (https://github.com/alexwang1001/PSD_development).
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Acknowledgements
We thank NIH NeuroBioBank, the University of Maryland School of Medicine Brain and Tissue Bank for providing postmortem brain tissue samples; L. Kohlstaedt for help in mass spectrometry data analysis; and S. Sanders, R. Nicoll, N. Parikshak, T. Mukhtar, M. Song and H. Zoghbi for comments on the manuscript. This work used the Vincent J. Proteomics/Mass Spectrometry Laboratory at UC Berkeley, supported in part by NIH S10 Instrumentation grant S10RR025622. This study was supported by National Institute of Neurological Disorders and Stroke grant R35NS097305 to A.R.K., National Institute of Mental Health grant K99MH131832 to L.W., National Human Genome Research Institute grant HG010898 to N.S. and National Institute of Mental Health grant MH124619 to N.S.
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Conceptualization: L.W.; data curation: L.W.; formal analysis: L.W.; funding acquisition: L.W., N.S., A.R.K.; investigation: L.W., L.Z., A.C.-S., S.G.-G., S.W., M.L.W., B.H., T.L.; methodology: L.W., K.P., L.Z., A.C.-S., S.G.-G., Jiani Li, P.L., Jingjing Li, J.M.G.-V.; resources: S.W., Y.P., E.J.H., E.A.W., M.F.P., R.K., N.S., A.A.P., A.R.K.; software: L.W., K.P.; supervision: X.P., J.M.G.-V., A.A.-B., Z.L., A.R.K.; visualization: L.W., K.P.; writing (original draft): L.W.; writing (review and editing): all authors.
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Extended data figures and tables
Extended Data Fig. 1 Isolation of PSDs from immature and mature human cortices.
a, Western blot analysis of different subcellular fractions of a GW23 sample demonstrating enrichment of PSD proteins and depletion of presynaptic SYP and cytoplasmic GAPDH in the PSD fraction. Enrichment of PSD proteins after fractionation was validated for all samples subjected to mass spectrometry analysis. b, Electron micrographs of the PSD fraction isolated from a GW23 sample (scale bar: 200 nm). Arrows denote structures resembling the PSD. The experiment was performed on one sample. c, Western blot analysis of purified PSDs from different age groups demonstrating changes in GRIN2B and DLG4 during development. The experiment was performed once. d, Correlation between PSD yield and synapse number of developing human prefrontal cortex. e, Venn diagram showing the overlap between Year18_22 samples in this study and the human PSD proteomes published in Roy et al., 2017 and Bayés et al., 2011. f, UpSet plot describing the number of identified proteins and their overlaps at each age group. g, PCA plots of the samples colored by various covariates. h, Variance explained by individual covariates (n = 1765 proteins). Boxplot center: median; hinges: the 25th and 75th percentiles; whiskers: 1.5 × inter-quartile range. i, Abundance patterns of GRIN2A, GRIN2B, DLG3, and DLG4. j, Colocalization of RPS6, CTNNB1, GDI1, or CFL1 with DLG4 in second-trimester human neocortex (n = 5, 5, 5 and 5 samples, scale bar: 10 μm or 5 μm as indicated in the figure). Data are presented as mean values ± s.e.m.
Extended Data Fig. 2 Examples of PSD protein abundance patterns in human cortical development.
a, Abundance patterns of representative PSD proteins. b, Examples of PSD protein paralogs that undergo reciprocal developmental changes. c, Abundance patterns of DBN1 and DBNL. d, Immunofluorescent intensity of DBN1 and DBNL at DLG4 loci in human neocortex (n = 3 and 3 samples, scale bar: 2 μm). Data are presented as mean values ± s.e.m. The P value was obtained from unpaired two-tailed t test; *P < 0.05. e, Original large field of view images for producing processed images in panel d (scale bar: 10 μm or 5 μm as indicated in the figure).
Extended Data Fig. 3 Protein modules of the developing human PSD.
a, Kernel density estimation of the null distributions of protein-protein interaction (PPI) numbers assuming no enrichment of PPI in individual modules; the vertical red lines indicate the observed PPI numbers in each module. The exact P value was derived from the one-tailed permutation test. b, SynGO biological pathway enrichment analysis of each module. Nominal P values from Fisher’s exact test were adjusted by the Benjamini and Hochberg method. c, PPI-co-abundance network of the four PSD modules. d, PPI-co-abundance network of each module highlighting proteins in enriched pathways. e, The normalized average shortest path lengths of pathways in individual modules. The asterisks denote that the average shortest path length is significantly shorter within pathway proteins than between pathway and non-pathway proteins. The P values were obtained from one-tailed Wilcoxon rank sum test; *P < 0.01.
Extended Data Fig. 4 Protein domains in PSD proteins.
a, Protein domains in individual PSD proteins. The rows are clustered based on the Jaccard distance. b, Abundance patterns of RhoGAPs and RhoGEFs not listed in Fig. 2e.
Extended Data Fig. 5 Changes in PSD composition during human V1 development.
a, Schematic illustrating the developmental stages of samples in the human V1 dataset. b, PCA plots of samples in the human V1 dataset colored by their age groups. c, Hierarchical clustering of the samples in the human V1 dataset based on proteins with differential abundance. d, Gene set enrichment analysis (GSEA) for individual age groups across species. NES, normalized enrichment score. Nominal P values were adjusted by the Benjamini and Hochberg method. e, Scaled abundance patterns (module eigengene values) of four protein modules in the human V1 dataset. f, Similarity matrices representing pairwise Pearson correlations between human PFC and human V1 samples.
Extended Data Fig. 6 Transcription of PSD proteins.
a, Preservation of human PSD modules in the bulk RNA-seq data. b, Uniform manifold approximation and projection (UMAP) plots showing the distribution of age groups and cell types in the single-nucleus RNA-seq data from developing human neocortex. c, UMAP plots showing the expression patterns of neuronal subtype-specific markers in the single-nucleus RNA-seq data from developing human neocortex. d, Standardized expression values of genes in the blue (n = 301 genes) and yellow (n = 218 genes) modules in individual neuronal subtypes of the adult human neocortex. Boxplot center: median; hinges: the 25th and 75th percentiles; whiskers: 1.5 × inter-quartile range. e, Standardized expression values of top TFs predicted to regulate the blue (95 genes) and yellow (97 genes) modules in individual neuronal subtypes of the adult human neocortex. Boxplot center: median; hinges: the 25th and 75th percentiles; whiskers: 1.5 × inter-quartile range. EN_IT, excitatory intratelencephalic neuron; EN_non-IT, excitatory non-intratelencephalic neuron; IN_MGE, inhibitory neuron derived from the medial ganglionic eminence; IN_CGE, inhibitory neuron derived from the caudal ganglionic eminence.
Extended Data Fig. 7 Changes in PSD composition during macaque and mouse neocortical development.
a, UpSet plot describing the number of identified proteins and their overlaps at each age group of macaque and mouse datasets. b, PCA plots of the macaque and mouse samples colored by their age groups. c, Hierarchical clustering of macaque and mouse samples based on proteins with differential abundance. d, Preservation of Human PSD modules in macaque and mouse PSD proteomic data. e, Gene set enrichment analysis (GSEA) for individual age groups across human V1, macaque, and mouse samples. NES, normalized enrichment score. Nominal P values were adjusted by the Benjamini and Hochberg method. f, Similarity matrices representing pairwise Pearson correlations between human V1, macaque, and mouse samples. g, Standardized abundance patterns of proteins in the four PSD modules (n = 127, 169, 159 and 178 proteins) across regions and species along the humanized age based on the human V1 dataset. Data are presented as mean values ± s.e.m. The macaque and mouse illustrations were created with BioRender.com.
Extended Data Fig. 8 RhoGEF levels and activities during synapse development.
a, Immunoblots and quantification of ARHGEF7 and PREX1 in the PSD and homogenate of developing human cortex (n = 4, 4 and 4 samples). Data are presented as mean values ± s.e.m. The P values were obtained from one-way ANOVA with Holm-Sidak’s multiple comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001. b, Immunoblots and quantification of ARHGEF7 and PREX1 in the PSD of developing mouse cortex (n = 2, 2, 2, 2, 2, 2 and 2 samples). Data are presented as mean values ± s.e.m. The P values were obtained from one-way ANOVA; ns, not significant. c, Original large field of view images for producing processed images in Fig. 4b (scale bar: 10 μm or 5 μm as indicated in the figure). d, Comparison of adult human PSD samples from postmortem brain tissues and neurosurgical biopsy tissues by western blot analysis (n = 4 and 3 samples). e, Immunoblots and quantification of ARHGEF7, PREX1, phospho-PAK, PAK, phospho-CFL1, and CFL1 in the synaptosomes of cultured primary human (n = 3 and 3 samples) and mouse cortical neurons (n = 3 and 3 samples) at indicated days in vitro (DIV). Data are presented as mean values ± s.e.m. The P values were obtained from one-way ANOVA with Holm-Sidak’s multiple comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant. The human and mouse illustrations were created with BioRender.com.
Extended Data Fig. 9 Manipulation of Rho GTPase regulators alters synapse development in human and mouse cortical neurons.
a, Original large field of view images for producing processed images in Fig. 4c (scale bar: 50 μm). b, Immunostaining of dendrites from primary mouse cortical neurons cultured 8 days in vitro. Neurons were transfected with mEGFP-C1 and vectors expressing mCherry, mCherry-ARHGEF7, or mCherry-RASGRF2 (n = 20, 20 and 20 neurons from 4 cultures, scale bar: 5 μm). Data are presented as mean values ± s.e.m. The P values were obtained from one-way ANOVA with Holm-Sidak’s multiple comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. c, Original large field of view images for producing processed images in panel b (scale bar: 50 μm). d, Quantification of mRNA levels of ARHGAP23 and SRGAP1 in HEK293T cells transfected with control shRNAs (shControl), two shRNAs targeting ARHGAP23 (shARGGAP23−1 and shARGGAP23−2), or two shRNAs targeting SRGAP1 (shSRGAP1−1 and shSRGAP1−2) (n = 3 cultures for all conditions). Data are presented as mean values ± s.e.m. The P values were obtained from one-way ANOVA with Holm-Sidak’s multiple comparisons test; ****P < 0.0001. e,f, Immunostaining of dendrites from primary human cortical neurons cultured six weeks in vitro. Neurons were transfected with mEGFP-C1 and vectors co-expressing turbo-RFP (tRGP) and shControl, shARGGAP23−1, shARGGAP23−2, shSRGAP1−1 or shSRGAP1−2 (n = 20, 22, 20, 20 and 20 neurons from 4 cultures, scale bar: 5 μm). Data are presented as mean values ± s.e.m. The P values were obtained from one-way ANOVA with Holm-Sidak’s multiple comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. g, Original large field of view images for producing processed images in panel e (scale bar: 50 μm). h, Original large field of view images for producing processed images in Fig. 4e (scale bar: 50 μm).
Extended Data Fig. 10 Association of PSD modules with cognitive functions and brain disorders.
a, PPI-co-abundance network of the turquoise module with activity-dependent proteins highlighted. b, Distribution of gnomAD synonymous Z-scores of genes in each category (n = 265, 313, 402, 224, 561, 1765 and 12892 genes). Boxplot center: median; hinges: the 25th and 75th percentiles; whiskers: 1.5 × inter-quartile range. The P values were obtained from Kruskal–Wallis test with Dunn’s multiple comparisons test; ns, not significant. c, Percentage of rare variants located at PSD module genes in subjects with or without neurodevelopmental disorders. d, PPI-co-abundance network of the turquoise module with genes carrying neurodevelopmental disorder-linked (at least 3) de novo missense variants or (at least 2) PTVs highlighted. e, PPI-co-abundance network of the brown module with genes carrying psychiatric disorder-linked common variants highlighted. f, PPI-co-abundance network of the yellow module with genes downregulated in psychiatric disorders highlighted. g, Volcano plots for misexpressed genes after the onset of psychiatric disorders in PSD modules. Red dots indicate differentially expressed genes with the Benjamini–Hochberg adjusted P values < 0.05.
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Wang, L., Pang, K., Zhou, L. et al. A cross-species proteomic map reveals neoteny of human synapse development. Nature 622, 112–119 (2023). https://doi.org/10.1038/s41586-023-06542-2
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DOI: https://doi.org/10.1038/s41586-023-06542-2
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Human neuronal maturation comes of age: cellular mechanisms and species differences
Nature Reviews Neuroscience (2023)
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