Peer review of: A single-cell atlas of the human substantia nigra reveals cell-specific pathways associated (2020)

REVIEWER COMMENTS 

Reviewer #1 (Remarks to the Author): 

This paper presents a single cell transcriptomic atlas for the human Substantia nigra. They 
sequenced thousands of cells in the cortex and nigra from 5 postmortem human brains and used 
LD score regression (LDSC) and Multi-marker Analysis of GenoMic Annotation (MAGMA) to analyze 
genetic risks for parkinson’s disease associated with expression patterns in specific cell types. They 
found association with dopamine neuron and oligodendrocyte-specific gene expression but no 
association with microglia and astrocytes, different from Alzheimer’s disease. In addition, they also 
report associations between nigra neuron expression pattern with several other psychiatric 
disorders. 

Given the paucity of data on human substantia nigra, the atlas from this study is highly valuable. 
In particular, they found a large difference in the relative proportions of neurons and glial cells 
between the cortex and nigra, indicating the importance of examining gene expression at the level 
of specific cell types rather than bulk brain tissues. However, I am not qualified to evaluate the 
analyses on disease association of the gene expression patterns to know whether the main 
conclusions are sufficiently supported. I will leave that to the other reviewers. 

Reviewer #3 (Remarks to the Author): 

In this work the Webber group generate a single cell atlas for Parkinson disease, by examining SN 
and cortical regions. The work is performed in a relatively modest number of samples. The results 
are mainly consistent with previous analysis of mouse single cell sequencing - suggesting that the 
majority of genetic risk loci have a functional context within DA neurons, interestingly the authors 
identified a distinct set within the oligodendrocyte population. That these are apparently distinct is 
one of the most interesting parts of this work. 

The manuscript is refreshingly simple, precise, and clear - and this leads me to have relatively 
little to suggest. 

Data availability - it would be wonderful to make these data available in a more useful way - GEO 
is a great step, but creating or leveraging an existing interactive portal would be useful for the PD 
community. I recognize this is not a small ask. 

Gene selection and models used. While MAGMA and LDSC are quite commonly used methods 
within this particular discipline, I believe that that work is of interest to the broader community - 
some simple discussion of how genes from risk loci are selected and what potential biases exist, 
would be useful. 

The authors mention that "We observed for the first time in human a significant association 
between PD genetic risk and genes with DaN-specific 
expression patterns" - this is true, but it may be worth contrasting this with the work previously 
done using available reference data (mouse) - the latest Nalls GWA and the work by Reynolds et 
al. 

Reviewer #4 (Remarks to the Author): 

In this work, Agarwal et. al present a single-cell transcriptomic atlas of the human substantia nigra 
(SN). The authors perform in-depth analyses of this atlas and identify cell type-specific 

associations between SN cell populations and risk genes for Parkinson’s disease (PD), as well as 
for a range of neuropsychiatric, neurologic, and other complex traits. The authors complement this 
atlas with a transcriptional characterization of matched cortical samples, similarly associating risk 
genes with specific cell types in this region. This work substantiates existing knowledge of PD 
etiology by demonstrating, for the first time in humans, an association between PD risk genes and 
transcriptomics of SN dopaminergic neurons. In addition, this work highlights novel associations 
between PD risk genes and oligodendrocyte-specific gene expression. These data allow for a 
critical evaluation of neuronal and glial mechanisms contributing to the progression of PD and 
other brain disorders. 

Overall, the key conclusions are well-supported by the data presented, which provide a valuable 
resource for the study of the SN in both homeostasis and disease. That said, additional 
commentary or revisions regarding comments below would strengthen the paper prior to 
publication. 

• On Line 84-86, it seems that the authors rely on GO terms to conclude that the astrocyte-1 sub-
cluster in SN samples is representative of A1 astrocytes, while the astrocyte-2 aligns with A2 
astrocytes (supplementary Table 2). However, A1 and A2 astrocytes are defined by very specific 
gene expression programs, and therefore overlapping genes should be shown or described. 
Alternatively, revising the text to omit this classification can be done. 

• According to Figure S1c, astrocyte-2 cells almost entirely come from the N2B patient sample. 
Similarly, microglia bias towards N3, N4B samples, and ODC-3 bias towards the N2B sample. Such 
big variations within the same region (sometimes from the sample patient as shown in Figure 
S12d) make it difficult to interpret the differences in proportions of cells between regions (line 
102). In addition to possible biological differences, technical variations or regional features 
reacting differently to experimental procedures may also contribute to the observed regional 
differences in neuronal and glial cell proportions. This caveat should be noted. 

• It is striking that the data show over 50% of post-mitotic cells are dividing (in G2M or S phases, 
Figure S1d), particularly in aged patient samples. This requires some explanation or validation. 

• Key example networks showing DaN (Figure S10) and ODC modules (e.g. M5 or M1) associated 
with PD should be displayed as a panel in Figure 4 rather than in a supplementary figure. 

Minor Comments: 

• Line 109-110: A more concise and clearer subheading is needed. 

• Line 97: Should refer to Figure 1d & e instead of Supplemental Figures 1d & e. 

• Figure S5 is missing an explanation of what the sizes of the circles mean in this graph. 

• The authors provide a clear description of their conditional analyses in the methods section, but 
a brief clarification in the main text would help readers more easily follow the conclusions drawn 
from these analyses (for ex: what does p < 0.05 mean in this context?). 

• The authors show that converging OPC/ODC genes are also associated with a range of 
psychiatric conditions. This should be mentioned in the abstract and/or introduction, as this is an 
important and novel result of this work.