Transcriptomic atlas of midbrain dopamine neurons uncovers differential vulnerability in a Parkinsonism lesion model

Reviewer #1 (Public Review):

In this study by Yaghmaeian Salmani et al., the authors performed single-nuclei RNA sequencing of a large number of cells (>70,000) in the ventral midbrain. The authors focused on cells in the ventral tegmental area (VTA) and substantia nigra (SN), which contain heterogeneous cell populations comprising dopaminergic, GABAergic, and glutamatergic neurons. Dopamine neurons are known to consist of heterogeneous subtypes, and these cells have been implicated in various neuropsychiatric diseases. Thus, identifying specific marker genes across different dopamine subpopulations may allow researchers in future studies to develop dopamine subtype-specific targeting strategies that could have substantial translational implications for developing more specific therapies for neuropsychiatric diseases.

A strength of the authors' approach compared to previous work is that a large number of cells were sequenced, which was achieved using snRNA-seq, which the authors found to be superior compared to scRNA-seq for reducing sampling bias. A weakness of the study is that relatively little new information is provided as the results are largely consistent with previous studies (e.g., Poulin et al., 2014). Nevertheless, it should be noted that the authors found some more nuanced subdivisions in several genetically identified DA subtypes.

Lastly, the authors performed molecular analysis of ventral midbrain cells in response to 6-OHDA exposure, which leads to the degeneration of SN dopamine neurons, whereas VTA dopamine neurons are largely unaffected. Based on this analysis, the authors identified several candidate genes that may be linked to neuronal vulnerability or resilience.

Overall, the authors present a comprehensive mouse brain atlas detailing gene expression profiles of ventral midbrain cell populations, which will be important to guide future studies that focus on understanding dopamine heterogeneity in health and disease.

Reviewer #2 (Public Review):

In the manuscript by Salmani et al., the authors explore the transcriptomic characterization of dopamine neurons in order to explore which neurons are particularly vulnerable to 6-OHDA-induced toxicity. To do this they perform single nucleus RNA sequencing of a large number of cells in the mouse midbrain in control animals and those exposed to 6-OHDA. This manuscript provides a detailed atlas of the transcriptome of various types of ventral midbrain cells - though the focus here is on dopaminergic cells, the data can be mined by other groups interested in other cell types as well. The results in terms of cell type classification are largely consistent with previous studies, though a more nuanced picture of cellular subtypes is portrayed here, a unique advantage of the large dataset obtained. The major advance here is exploring the transcriptional profile in the ventral midbrain of animals treated with 6-OHDA, highlighting potential candidate genes that may influence vulnerability. This approach could be generalizable to investigate how various experiences and insults alter unique cell subtypes in the midbrain, providing valuable information about how these stimuli impact DA cell biology and which cells may be the most strongly affected.

Overall, the manuscript is relatively heavy on characterization and comparatively light on functional interpretation of findings. This limits the impact of the proposed work. It also isn't clear what the vulnerability factors may be in the neurons that die. Beyond the characterization of which neurons die - what is the reason that these neurons are susceptible to lesion? Also, the interpretation of these findings is going to be limited by the fact that 6-OHDA is an injectable, and the effects depend on the accuracy of injection targeting and the equal access of the toxin to access all cell populations. Though the site of injection (MFB) should hit most/all of the forebrain-projecting DA cells, the injection sites for each animal were not characterized (and since the cells from animals were pooled, the effects of injection targeting on the group data would be hard to determine in any case).

I am also not clear why the authors don't explore more about what the genes/pathways are that differentiate these conditions and why some cells are particularly vulnerable or resilient. For example, one could run GO analyses, weighted gene co-expression network analysis, or any one of a number of analysis packages to highlight which genes/pathways may give rise to vulnerability or resilience. Since the manuscript is focused on identifying cells and gene expression profiles that define vulnerability and resilience, there is much more that could have been done with this based on the data that the authors collected.

Another limitation of this study as presented is the missed opportunity to integrate it with the rich literature on midbrain dopamine (and non-dopamine) neuron subtypes. Many subtypes have been explored, with divergent functions, and can usually be distinguished by either their projection site, neurotransmitter identity, or both. Unfortunately, the projection site does not seem to track particularly well with transcriptomic identities, aside from a few genes such as DAT or the DRD2 receptor. However, this could have been more thoroughly explored in this manuscript, either by introducing AAVretro barcodes through injection into downstream brain sites, or through existing evidence within their sequencing dataset. There are likely clear interpretations from some of that literature, some of which may be more exciting than others. For example, the authors note that vGluT2-expressing cells were part of the resilient territory. This might be because this is expressed in medially-located DA cells and not laterally-located ones, which tends to track which cells die and which don't.

It is not immediately clear why the authors used a relaxed gate for mCherry fluorescence in Figure 1. This makes it difficult to definitively isolate dopaminergic neurons - or at least, neurons with a DAT-Cre expression history. While the expression of TH/DAT should be able to give a fairly reliable identification of these cells, the reason for this decision is not made clear in the text.