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Droplet-based transcriptome profiling of individual synapses

Nature Biotechnology volume 41, pages 1332–1344 (2023)Cite this article

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Matters Arising to this article was published on 27 July 2023

Abstract

Synapses are crucial structures that mediate signal transmission between neurons in complex neural circuits and display considerable morphological and electrophysiological heterogeneity. So far we still lack a high-throughput method to profile the molecular heterogeneity among individual synapses. In the present study, we develop a droplet-based single-cell (sc) total-RNA-sequencing platform, called Multiple-Annealing-and-Tailing-based Quantitative scRNA-seq in Droplets, for transcriptome profiling of individual neurites, primarily composed of synaptosomes. In the synaptosome transcriptome, or ‘synaptome’, profiling of both mouse and human brain samples, we detect subclusters among synaptosomes that are associated with neuronal subtypes and characterize the landscape of transcript splicing that occurs within synapses. We extend synaptome profiling to synaptopathy in an Alzheimer’s disease (AD) mouse model and discover AD-associated synaptic gene expression changes that cannot be detected by single-nucleus transcriptome profiling. Overall, our results show that this platform provides a high-throughput, single-synaptosome transcriptome profiling tool that will facilitate future discoveries in neuroscience.

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Fig. 1: Overview of MATQ-Drop and the performance in species-mixing experiment.
Fig. 2: The mouse hippocampus cell atlas and synaptome atlas.
Fig. 3: The human hippocampus synaptome atlas.
Fig. 4: Nascent RNA-based cell atlas of the human hippocampus.
Fig. 5: AD-associated synaptopathy.
Fig. 6: The cell typing using only lncRNA species and the detection sensitivity comparison between 10x Chromium and MATQ-Drop.

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Data availability

The raw sequencing files are available in Gene Expression Omnibus (GEO) database under accession no. GSE199346.

The following public datasets were used in the present study for benchmark comparison: (1) DroNc-seq and Drop-seq on 3T3 cell line (https://singlecell.broadinstitute.org/single_cell/study/SCP128/dronc-seq-and-drop-seq-on-3t3-cell-line#study-download), (2) GEO accession no. GSE106678 and (3) 5k Adult Mouse Brain Nuclei Isolated with Chromium Nuclei Isolation Kit (10x Genomics, https://www.10xgenomics.com/resources/datasets/5k-adult-mouse-brain-nuclei-isolated-with-chromium-nuclei-isolation-kit-3-1-standard).

The mm10 genome can be accessed at https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20 and the Gencode gene annotation file at https://www.gencodegenes.org/mouse/release_M10.html. The hg19 genome can be accessed at https://www.ncbi.nlm.nih.gov/data-hub/genome/GCF_000001405.25 and the Gencode gene annotation file at https://www.gencodegenes.org/human/release_19.html.

Code availability

The analysis code customized for MATQ_Drop sequencing data is available at https://github.com/zonglab/MATQ_Drop.

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Acknowledgements

We thank the McNair family for their support. We also thank NIH NeuroBioBank for providing brain tissue samples. We thank H. Dierick and M. Xue for their helpful discussion. We thank S. Eshghjoo for providing the mouse samples. We thank other Zong lab members for their help in this project. FACS experiments were performed in the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the CPRIT Core Facility Support Award (no. CPRIT-RP180672), the NIH (grant nos. P30 CA125123, S10 RR024574 and S10 OD025251) and the assistance of J. M. Sederstrom. C.Z. is supported by a McNair Scholarship, NIH Director’s New Innovator Award (no. 1DP2EB020399) and the Behavioral Plasticity Research Institute (grant no. DBI-2021795). H.Z. is supported by the NIH (grant nos. RF1 AG020670, RF1 NS093652 and P01 AG066606).

Author information

Author notes

  1. Wenjian Cao & Qiangyuan Zhu

    Present address: Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou, China

  2. Yongcheng Wang

    Present address: Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou, China

Authors and Affiliations

  1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA

    Muchun Niu, Wenjian Cao, Qiangyuan Zhu, Jiayi Luo, Baiping Wang, Hui Zheng & Chenghang Zong

  2. Integrative Molecular and Biomedical Sciences Graduate Program, Baylor College of Medicine, Houston, TX, USA

    Muchun Niu

  3. Genetics and Genomics Graduate Program, Baylor College of Medicine, Houston, TX, USA

    Wenjian Cao

  4. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Yongcheng Wang

  5. Wyss Institute of Bioinspired Engineering, Harvard University, Cambridge, MA, USA

    Yongcheng Wang & David A. Weitz

  6. Cancer and Cell Biology Graduate Program, Baylor College of Medicine, Houston, TX, USA

    Jiayi Luo

  7. Huffington Center on Aging, Baylor College of Medicine, Houston, TX, USA

    Baiping Wang & Hui Zheng

  8. Department of Physics and School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA

    David A. Weitz

  9. Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, USA

    Chenghang Zong

  10. McNair Medical Institute, Baylor College of Medicine, Houston, TX, USA

    Chenghang Zong

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Contributions

C.Z. and D.A.W. designed the project. C.Z. and M.N. wrote the manuscript. M.N. and W.C. contributed to the MATQ-Drop chemistry development and hydrogel bead design and synthesis, and performed the major experiments. W.C., Y.W., Q.Z. and M.N. contributed to the microfluidics operation and microfluidics fabrication. J.L. performed the FACS and western blotting experiments. H.Z. and B.W. contributed mouse brain samples and AD mouse models. B.W. performed immunofluorescence staining experiments and related quantification. M.N. and C.Z. performed the bioinformatic analysis.

Corresponding authors

Correspondence to David A. Weitz or Chenghang Zong.

Ethics declarations

Competing interests

M.N., D.A.W. and C.Z. are cofounders and equity holders of Pioneer Genomics Inc. Baylor College of Medicine have submitted a patent application related to MATQ-Drop (US Provisional Patent Application serial no. 63/240339, 2 September 2021). The remaining authors declare no competing interests.

Peer review

Peer review information

Nature Biotechnology thanks Tibor Harkany and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–21, Texts 1 and 2, Content of Additional Supplementary Files and Source Data.

Reporting Summary

Supplementary Tables 1–7

Supplementary Table 1. Cluster annotation of mouse synaptosomes; Supplementary Table 2. Marker genes of each mouse synaptosome cluster; Supplementary Table 3. Cluster annotation of mouse nuclei; Supplementary Table 4. Marker genes of each mouse nucleus cluster; Supplementary Table 5. Differentially expressed genes between mouse synaptosomes and nuclei; Supplementary Table 6. Gene-based intron percentage in mouse synaptosomes and nuclei; Supplementary Table 7. List of genes whose transcripts are unspliced in mouse synapses.

Supplementary Tables 8–24

Supplementary Table 8. Information of frozen human brain tissues; Supplementary Table 9. Cluster annotation of human synaptosomes; Supplementary Table 10. Marker genes of each human synaptosome cluster; Supplementary Table 11. Differentially expressed genes between human HIsynapses and LO-synapses; Supplementary Table 12. GO-BP pathway enrichment of human HI-synapse enriched genes; Supplementary Table 13. GO-BP pathway enrichment of human LO-synapse enriched genes; Supplementary Table 14. Differentially expressed genes between human Nsynapses and other synapses; Supplementary Table 15. Differentially expressed genes between human LOsynapses and neuron-glia junctions; Supplementary Table 16. Nascent RNA-based cluster annotation of human brain nuclei; Supplementary Table 18. Annotation of human inhibitory neuron subtypes; Supplementary Table 19. Marker genes of each human inhibitory neuron subtypes; Supplementary Table 20. Differentially expressed genes between human HIsynapse subtypes and corresponding neuronal nucleus subtypes; Supplementary Table 21. GO-BP pathway enrichment of human synapse-enriched genes across four different neuronal subtypes; Supplementary Table 22. GO-BP pathway enrichment of human nucleus-enriched genes across four different neuronal subtypes; Supplementary Table 23. Gene-based intron percentage in human synaptosomes and nuclei; Supplementary Table 24. List of genes whose transcripts are unspliced in human synapses.

Supplementary Tables 25–27

Supplementary Table 25. Differentially expressed genes between 5xFAD and WT mouse nuclei, transcript-based; Supplementary Table 26. Differentially expressed genes between 5xFAD and WT mouse nuclei, exon-based; Supplementary Table 27. Differentially expressed genes between 5xFAD and WT mouse synaptosomes.

Supplementary Tables 28–31

Supplementary Table 28. LncRNA-based cluster annotation of human brain nuclei; Supplementary Table 29. Marker lncRNA genes of each lncRNA-based human nucleus cluster; Supplementary Table 30. LncRNA-based cluster annotation of mouse brain nuclei; Supplementary Table 31. Marker lncRNA genes of each lncRNA-based mouse nucleus cluster.

Supplementary Tables 32–34

Supplementary Table 32. Sequences of oligonucleotides used in barcoded hydrogel bead synthesis; Supplementary Table 33. Summary information of human sample library; Supplementary Table 34. Summary information of mouse sample library.

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Niu, M., Cao, W., Wang, Y. et al. Droplet-based transcriptome profiling of individual synapses. Nat Biotechnol 41, 1332–1344 (2023). https://doi.org/10.1038/s41587-022-01635-1

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