Single-nucleus transcriptomic analysis of human dorsal root ganglion neurons

  1. Minh Q Nguyen
  2. Lars J von Buchholtz
  3. Ashlie N Reker
  4. Nicholas JP Ryba
  5. Steve Davidson

  • Curated by eLife

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    Evaluation Summary:

    In this work, Minh Nguyen and colleagues performed single nuclear RNAseq of human dorsal root ganglion (DRG) neurons and classified them into 15 clusters. A bioinformatic comparison to mouse lumbar DRG single nucleus sequencing results is also described. The importance of reporting the single nucleus or single cell molecular profiles of human DRG cannot be overstated. Proper molecular targeting of therapeutics requires knowing this information. Given that the field is just starting to understand the human specific molecular signature of primary somatosensory neurons using single cell/nuclear RNAseq, this study is important and timely, providing one of the first gene expression databases of individual human DRG neurons.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors)

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Abstract

Somatosensory neurons with cell bodies in the dorsal root ganglia (DRG) project to the skin, muscles, bones, and viscera to detect touch and temperature as well as to mediate proprioception and many types of interoception. In addition, the somatosensory system conveys the clinically relevant noxious sensations of pain and itch. Here, we used single nuclear transcriptomics to characterize transcriptomic classes of human DRG neurons that detect these diverse types of stimuli. Notably, multiple types of human DRG neurons have transcriptomic features that resemble their mouse counterparts although expression of genes considered important for sensory function often differed between species. More unexpectedly, we identified several transcriptomic classes with no clear equivalent in the other species. This dataset should serve as a valuable resource for the community, for example as means of focusing translational efforts on molecules with conserved expression across species.

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  1. Version published to 10.7554/elife.71752 on eLife
  2. eLife

    Evaluation Summary:

    In this work, Minh Nguyen and colleagues performed single nuclear RNAseq of human dorsal root ganglion (DRG) neurons and classified them into 15 clusters. A bioinformatic comparison to mouse lumbar DRG single nucleus sequencing results is also described. The importance of reporting the single nucleus or single cell molecular profiles of human DRG cannot be overstated. Proper molecular targeting of therapeutics requires knowing this information. Given that the field is just starting to understand the human specific molecular signature of primary somatosensory neurons using single cell/nuclear RNAseq, this study is important and timely, providing one of the first gene expression databases of individual human DRG neurons.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors)

  3. eLife

    Reviewer #1 (Public Review):

    In this work, Minh Nguyen and colleagues performed single nuclear RNAseq of human dorsal root ganglion neurons and classified human DRG neurons into 15 clusters. By comparing the transcriptomic features between human and mouse DRG neurons, they found that there are similar as well as human specific DRG neuron cell-types. Based on this analysis, they assigned the human DRG neuron clusters into different functional groups. In addition, the authors provided multiplexed ISH data to support their sequencing and classification results. Overall, this study is straightforward and the quantify of data is good. Given that the field is just starting to understand the human specific molecular signature of primary somatosensory neurons using single cell/nuclear RNAseq, this study is valuable and provides one of the first gene expression databases of individual human DRG neurons. Below are our critiques and suggestions for this manuscript.

    Comments:

    1. The authors enriched neuronal nuclei by NeuN antibody selection. However, no direct evidence was provided to show this method can unbiasedly select all types of DRG neurons. If it is not the case, this NeuN selection/enrichment might introduce the bias to some types of neurons and lose some other types.

    2. The authors separated the non-neuronal from the neuronal nuclei in analysis, based on marker genes PRP1, MBP and APOE. From figure supplement 1, however, we still see the expression of APOE in quite a lot of selected neuronal nuclei. One possibility is that these transcriptomes are from more than one nucleus as the authors hinted in the figure legend. It would be the best if the authors could give an estimation about the percentage of multiple nuclei from their sequencing result. Since this is one of the first set of papers performing human DRG neuron single RNAseq, adding discussion about the strength and potential technical caveats of this method would be beneficial for the field.

    3. The authors should provide a figure or figure panels to show the sequencing quality and depth. For example, the reads number, mapped rates, detected genes numbers, etc. Again, this would facilitate evaluation/comparison of different human DRG neuron sequencing methods and data.

    4. The sequencing results show that Nppb is expressed in H10 and H11. Please add fluorescent in situ to validate the point.

    5. The authors compared the human and mouse transcriptome data. For a fair comparison and for the readers' information, the author may need to provide some basic information about the two sequencing results, such as the cell number, sequence depth, sex, etc. A table like this would be nice.

    6. In Fig. 4, they authors proposed that "transcriptomically related classes of human DRG neurons are spatially clustered in the ganglion" by showing staining of two sections. They provided some quantification in the supplementary figure. Overall, the data in supporting this claim is not strong, and it is not clear whether this is a human specific phenomenon or generally true in other species. Similar trends were noticed for the mouse small diameter vs large-diameter neurons as well, but no careful quantification and careful comparison were made. Depending on how strongly the authors want to claim the "spatial clustering" model, the authors may need additional quantification/modeling and perform similar analysis with mouse DRG neurons.

  4. eLife

    Reviewer #2 (Public Review):

    In this study, the authors attempted to generate the classification of the human DRG neurons, and compare it to the organization of the mouse DRGs in an effort to better understand the species differences, and to help advance the translational aspect of pain research.

    This is an interesting and timely study. It is also perform by some of the labs that either pioneered the use of human tissue for translational pain research (Davidson), or the identification of markers of sensory neurons (Ryba).

    Overall, the authors achieved their goal and present a detailed description of the clusters of human sensory neurons. The results support their conclusions.
    The database produced by this work will be of great help to the community of sensory physiologists.

    One caution that readers must bear in mind is that access to human neurons is difficult, and the sample sizes remain unfortunately low. For instance, one mouse DRH snRNAseq study referred to in this work is based on 141,000 nuclei. The human data, comes from around 1,800 nuclei. Therefore, the work provides a great clue to the clusters of sensory neurons in human DRGs, but this clustering may be refined down the road when more data becomes available.

  5. eLife

    Reviewer #3 (Public Review):

    The manuscript by Nguyen et al describes the assignment of neuronal cell types to human L4 or L5 DRG based on single nucleus sequencing and bioinformatic analyses of freshly isolated samples (1837 neuronal nuclei and an average of 2839 genes per nucleus). A bioinformatic comparison to mouse lumbar DRG single nucleus sequencing results by Renthal et al is also described. Additionally, the manuscript describes results of Hi-Plex RNAscope performed on L4 or L5 DRG from the same donors using 9 different probes. Lastly, the authors make an observation for a potential organization of NEFH and SCN10A expressing neurons in the DRG in situ that has not been reported in mice. The study uses DRG from one male and five female donors age 34-55. The importance of obtaining single nucleus or single cell molecular profiles from humans cannot be overstated. Properly directed therapeutic translation requires knowing this information. The authors have immediate access to freshly removed human tissue which is critical for obtaining high quality samples. Preceding this submission is the submission of a human single DRG gene expression study performed in situ in BioRxiv and the publication of a single nucleus study of macaque DRG. This study was only cursorily compared to those studies. A bioinformatic comparison to the macaque study would have a lot of value and a more in depth comparison to the other human study would also add value. The authors have created a searchable database free and easily accessible to researchers.

  6. eLife

    Reviewer #4 (Public Review):

    This research fills a valuable gap in our understanding of neural cell populations. There is immense complexity in the neuron subtype landscape of the dorsal root gangion (DRG). Profiling had been previously conducted in mouse, but not within human. Providing the data and analysis of the human DRG is a valuable resource because substantial differences in cell populations and expression programs can exist between mouse and human. Any research that is focussed on the translational potential of a gene or pathway should verity its conservation across species.

    However, additional evidence is required to support a major claim of the manuscript: that there are mouse-specific and human-specific neuron subtypes. This claim is based on two major pieces of evidence. First, cluster comparison and co-clustering identify some cell populations that are species specific. Although this approach is suggestive, it is not definitive. Clustering separates populations of cells based major axes of variability, but those axes may not perfectly align across conditions or species. For example, excitatory cortical neurons may vary based upon cortical layer or whether they originate from the primary or secondary visual cortex. It is possible that one source of variation is stronger in one species and another source of variation is stronger in another species, leading to differences in clustering. The co-clustering may overcome of those limitations, but if the differences across species and experimental parameters are large enough, then even cells that come from the same population may not align. The in situ hybridization experiments also provide some support for species specificity, but it the overlap of specific markers could be confounded when the expression of individual marker genes evolve while the overall cell population remains consistent.

  7. Version published to 10.1101/2021.07.02.450845v1 on bioRxiv