Abstract
Major depressive disorder represents a serious public health challenge worldwide; however, the underlying cellular and molecular mechanisms are mostly unknown. Here, we profile the dorsolateral prefrontal cortex of female cynomolgus macaques with social stress-associated depressive-like behaviors using single-nucleus RNA-sequencing and spatial transcriptomics. We find gene expression changes associated with depressive-like behaviors mostly in microglia, and we report a pro-inflammatory microglia subpopulation enriched in the depressive-like condition. Single-nucleus RNA-sequencing data result in the identification of six enriched gene modules associated with depressive-like behaviors, and these modules are further resolved by spatial transcriptomics. Gene modules associated with huddle and sit alone behaviors are expressed in neurons and oligodendrocytes of the superficial cortical layer, while gene modules associated with locomotion and amicable behaviors are enriched in microglia and astrocytes in mid-to-deep cortical layers. The depressive-like behavior associated microglia subpopulation is enriched in deep cortical layers. In summary, our findings show cell-type and cortical layer-specific gene expression changes and identify one microglia subpopulation associated with depressive-like behaviors in female non-human primates.
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Data availability
All sequencing data generated by this study are available in the Gene Expression Omnibus as a SuperSeries (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE201687).
Code availability
The code for analysis and generating figures can be found at https://github.com/JWu-brainstudy/Macaca-sn-ana2022 and at https://doi.org/10.5281/zenodo.8015827 (ref. 100).
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Acknowledgements
This work was supported by the National Key R&D Program of China (2017YFA0505700 to P.X.), Projects of International Cooperation and Exchanges NSFC (81820108015 to P.X.), Nonprofit Central Research Institute Fund of Chinese Academy of Medical Sciences (2019PT320002 to P.X.), the Natural Science Foundation Project of China (81971296 and 82171523 to P.Z., 82101596 to J.P., 82201688 to J.W., 82201683 to L.L.), Chongqing Science and Technology Commission (cstc2019 jcyjjqX0009 to P.Z., cstc2021 jscx-msxm0026 to J.W.), Program for Youth Innovation in Future Medicine, Chongqing Medical University to P.Z., China Postdoctoral Science Foundation (2020TQ0393 to L.L., 2021TQ0396 and 2021MD703928 to H.Z., 2021MD693926 to H.W., 2022MD713717 to J.W.), Chongqing Talents Plan for Young Talents (CQYC202105017 to P.Z.) and institutional funds from the State University of New York (SUNY) Upstate Medical University. This paper is subject to the SUNY Open Access Policy. We thank J. Hu (ShanghaiTech University), T. F. Yuan (Shanghai Jiao Tong University), J. Yang and Y. He (Beijing Anding Hospital, Capital Medical University) for technical advice and helpful discussions.
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P.X. and P.Z. designed the experiments. J.W., Y.H., H.Z., K.C. and H.W. collected the dIPFC of macaques. J.W., Y.L., Y.H. and J.P. performed the sc- and snRNA-seq analysis. J.W., Y.L., Y.H. and L.L. performed the ST analysis. J.W., Y.L., Y.H., X.T., Y.L. and Q.W. observed animal behaviors. J.W. and P.Z. drafted the manuscript. P.X., P.Z., J.W., S.W.P., M.-L.W., J.L., C.N. and G.T. revised the manuscript for intellectual content.
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Extended data
Extended Data Fig. 1 Schematic of macaque behavioral observation.
a-b, conflicts were recorded with two webcams on either side of the enclosure. The top three populations with the highest confliction behaviors were screened for further social rank evaluation; detailed depressive-like behaviors were recorded with three webcams to eliminate the coverage holes. The depressive-like behaviors of individuals in the top 3 and bottom 4 were analyzed. A trough was located in the middle of each enclosure. Each enclosure measures 8.0 m × 3.0 m × 3.0 m (L×W×H). c, the social rank was stable with or without male presence calculated by David’s score. Two-sided Pearson correlation test, error bands represent the 95% confidence interval of linear model.
Extended Data Fig. 2 Nuclei clustering, cell type annotation, and cell-specific genes.
a, QC analysis of snRNA-seq; 8,750 doublets and 136,231 single nuclei were identified. b, heatmap indicated the enrichment of expressional signatures between macaque and human single-nuclei clusters in dlPFC. All the nuclei clusters were identified using graph-based clustering method through Seurat. The annotation of macaque cell types was displayed at the top, and the annotation of human cell types was displayed at the right of the heatmap. The cell type annotation of human dlPFC clusters was independently performed and previously published34. c, dissected UMAP plot showed a coincident distribution of nuclei clustering of in the three groups. d, Venn diagram showed only 5 overlapped DEGs between neuron and glial cells. e, Venn diagram showed 12 overlapped DEGs in 6 cell types and 207 unique DEGs. f, KEGG enrichment analysis showed differential involved pathways of cell-specific DEGs.
Extended Data Fig. 3 DEGs and associated signaling pathways in each cell type.
a, volcano plot of DEGs. DEGs were identified with the Wilcox rank-sum test (two-sided) in R package Seurat, and the significance threshold was logFC > 0.25, FDR < 0.05, and min.pct > 0.1. b, KEGG enrichment analysis in each cell type. Blue and red bars indicated the proportion of up and down-regulated DEGs in involved pathways (Hyper geometric test). c, The number of specific activated receptor–ligand pairs in dlPFC of macaques. The activated pairs in DLs were significantly higher (~4 fold) than in controls. Microglia involved the primary (54%, 56/104) DLs-specific communications. The background communication pairs were identified shared pairs between DLs and controls.
Extended Data Fig. 4 Depressive pattern and rank pattern DEGs involved in different processes.
a, DEG with D-pattern (depressive pattern, left) showed differential expression in the DLs and non-DLs groups (RES and controls), which was specifically associated with depressive-like behaviors. DEG with R-pattern (rank pattern, right) showed differential expression between high rank (controls) and low rank (DL and RES), which was specifically associated with social stress. b-c, enrichment analysis showed different involved pathways between D-pattern and R-pattern. D-pattern DEGs were mainly contributed by Mic and involved the pro-inflammatory pathways. R-pattern DEGs were mainly contributed by Ast and involved the synthesis of neurotransmitter.
Extended Data Fig. 5 Microglial subpopulations were not significantly activated by dissociation enzyme and considered as ‘artifactual’.
a, the number (left) or proportion (right) of microglia were not significantly different between these three groups. b, boxplot showed a significantly increased PIMID proportion in DLs (Controls, n = 7; DLs, n = 7; RES, n = 5. box=25-75th percentiles, whiskers=Tukey, horizontal line in box=median). c, activation score based on the exAM signature gene modules and plotted on UMAP coordinates. d, expression of exAM gene markers in microglia subpopulations. 10 genes (SOCS3, CCL3, CCL4, KLF2, HIST2H2AA1, HIST1H4I, HSPA1A, HSPA1B, HIST1H2BC, HIST1H1C) were not expressed in any microglia. e, scoring results of microglia subpopulations based on the activation score in each subpopulation.
Extended Data Fig. 6 PIMID expression signatures.
a, normalized expression of 4 marker genes of PIMID. b, overview of dlPFC slices. Each slice contained clear cortical structures, including edges, grey matter and white matter. n = 5 per group. c, Immunofluorescence staining and quantification of IQGAP2+ microglia in dlPFC. IQGAP2+IBA1+ colocalization cells were identified as Mic03 cells. IQGAP2 was identified as a gene marker of Mic03 (FDR = 1.095 e-212) in sn-RNA data. IBA1 was a widely used microglial marker. The IQGAP2+ microglia were identified following a criterion of IQGAP2+IBA1+ cells with clear microglial features including long processes and a central soma. Two representative cells were photographed at 40X magnification, IQGAP2+IBA1+(left, yellow arrow) and IBA1+ microglia (right, white arrow).
Extended Data Fig. 7 Functional profiling of PIMID.
a, activated and inactivated gene sets in PIMID, identified by QUSAGE. b-c, KEGG (b) and GO (c) enrichment of PIMID marker genes indicated pro-inflammatory processes (Hyper geometric test). GO terms, BP, biological process; MF, molecular function; CC, cell component.
Extended Data Fig. 8 Digestion-obtained microglia exhibited expression signatures and differences between groups similar to microglial nuclei.
a-b, UMAP visualization showing clustering of 12,374 digested living cells, colored by unsupervised cluster (a) and annotated cell types (b). c, gene markers for annotating major cell types. d, digested microglial cluster8 (Sc08) and microglial nuclei cluster3 (Mic03, also called PIMID) were highly aligned in coordinate space (highlighted by red circles), also see Fig. 4j. e, mapping relationship between the subpopulations of microglial cells and nuclei. The color depth indicates the odds ratio (OR). Sc08 and Sn03 were highly identical (FDR = 1.21×10−134, OR = 18.19, Fisher exact test, two-sided). f, QUSAGE analysis shows the pro-inflammatory profiles both in subcluster 8 using enzymic digestion. g-i, a higher amount and proportion of Sc08 were detected in DLs in digested microglial cells (n = 1 per group).
Extended Data Fig. 9 Unsupervised clustering of dlPFC ST hybridization spots showed layer distribution.
a, HE staining of dlPFC ST slices (Controls, n = 3; DLs, n = 2; RESs, n = 3). The boxes indicated the hybridized spot area (6.5 mm×6.5 mm). b-i, spot clusters showed layer distribution. Left, the merged plots of HE-stained image and in situ clusters. Right, the UMAP plots of each cluster.
Extended Data Fig. 10 Depressive-like behavioral and spatial location of cell-type-specific WGCNA modules.
a-f, the top heatmap indicates the behavioral association of Ast(a), Mic(b), Oli(c), Exn(d), Int(e) and OPC(f). The color (blue to red) indicates the coefficient between the expression of gene modules and depressive-like behaviors (*p < 0.05, #p < 0.01, Pearson correlation, two-sided); the bottom heatmap indicates the enrichment of gene modules in the dlPFC ST regions, color depth indicates the Fisher exact test odds ratio. g, a table summarizing depressive-like behaviors and associated gene modules. Check marks indicate whether a gene module identified in cell types significantly correlated with depressive-like behaviors. Red check marks indicate the depressive-like behavior-associated gene modules were also spatially specific in some ST regions.
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Wu, J., Li, Y., Huang, Y. et al. Integrating spatial and single-nucleus transcriptomic data elucidates microglial-specific responses in female cynomolgus macaques with depressive-like behaviors. Nat Neurosci 26, 1352–1364 (2023). https://doi.org/10.1038/s41593-023-01379-4
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DOI: https://doi.org/10.1038/s41593-023-01379-4
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