Generation of a snRNAseq dataset from the mouse midbrain

A) Schematic design of the study, unilateral injection of low doses of 6-OHDA (0.7, 1.5 µg/µl) in the medial forebrain bundle (MFB), followed by dissection, nuclei isolation and enrichment via FANS, before library preparation, sequencing, and data analysis. B) Transgenic, Cre-dependent mCherryTRAP mouse model. DatCre and TrapCfllines were intercrossed to generate DatCre/+; TrapCfl/flmice. C) Immunohistochemical staining of the mouse ventral midbrain coronal section, showing the overlap of TH and mCherry, scalebar 100 μm. D) UMAP projection of the all-nuclei dataset, with cluster color-coding. E) Hierarchical dendrogram of the identified clusters and their descriptions with the same color-coding.

Midbrain DA dataset, cell types and composition

A) UMAP projection of the dopaminergic dataset, with numerical labels of the 71 clusters and color-coded for the major cell types. B) Typical dopaminergic markers plotted in UMAP separately, with the top 1 percentile of the normalized gene expression range clipped for better visualization. C) Hierarchical dendrogram of clusters from the dopaminergic nuclei dataset, with the cell-type enriched markers as a dotplot and cell-type color-coding. mODC = mature oligodendrocyte, HyDA = hypothalamic dopaminergic.

Dopaminergic territories and neighborhoods

A) UMAP projection of the dopaminergic dataset with territory color-coding and size. B) Co-expression of Sox6 and Calb1 plotted in UMAP, roughly delineating SNc and VTA respectively. C) Hierarchical dendrograms with labels and color-coded territories and their respective neighborhoods. Dotplot showing territory (TER) and neighborhood (NH) specific markers.

Mapping of mDA territories in the adult mouse midbrain

A) A color-coded UMAP projection of the seven mDA territories and of the mostly-lesion (ML) clusters, with individual enriched genes plotted on smaller UMAPs. B-D) Immunohistochemical staining with antibodies indicated in the ventral midbrain. E) Fluorescent RNA in situ hybridization with Tacr3 probe combined with immunohistochemistry. Insets are shown as higher magnification on the right. F) Schematic presentation of the localization of the seven mDA territories across the midbrain, with the most anterior section uppermost. SNc, Substantia Nigra pars compacta, SNcL, Substantia Nigra lateral, SNcV, Substantia Nigra ventral, SNcM, Substantia Nigra medial, VTAR, Ventral Tegmental Area rostral part, PBP, parabrachial pigmented nucleus, PN, paranigral nucleus, PIF, parainterfascicular nucleus, PAG, periaqueductal grey, RRF, retrorubral field, RLi, rostral linear nucleus, CLi, caudal linear nucleus of the raphe, IF, interfascicular nucleus, RN, red nucleus, IPN, interpeduncular nucleus, PN, pontine nucleus, fr, fasciculus retroflexus, ml, medial lemniscus. Scalebars are 200 μm.

Normalized cell loss across territories and neighborhoods in mDA dataset

A, B) UMAP projection of the dopaminergic dataset nuclei from the intact (un-lesioned) and the lesioned hemisphere, respectively. C) Calculated normalized cell loss for territories, visualized as percentages. D) Pairwise comparison of normalized cell loss across territories. E) Calculated normalized cell loss for neighborhoods, visualized as percentages. Comparisons were made between neighborhoods within each territory. See methods for the statistical tests used. F) Percentages of nuclei in each major cell type from either intact or lesioned samples. ns = not significant, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. For the Conover-Iman test; p-value = P(T ≥ |t|), and null hypothesis (H0) was rejected at p ≤ α/2, which is 0.025. Error bars show standard deviation (SD).

Vulnerability and resilience modules in territories, neighborhoods, and the ML clusters

A-B) Violin plots of the vulnerability module across territories and neighborhoods, with percentage of normalized cell loss shown at the bottom per territory and neighborhood, respectively. C-D) Violin plots of the resilience module across territories and neighborhoods, with percentage of normalized cell loss shown at the bottom per territory and neighborhood, respectively. Pairwise comparisons of vulnerability module scores across territories and neighborhoods, respectively. E) Pairwise comparisons of resilience module scores across neighborhoods. F) Vulnerability module gene list, ranked by average Log2FC. G) Resilience module gene list, ranked by average Log2FC. Summary statistics center lines are mean values. na = not applicable. ns = not significant. See Methods for the statistical tests used.

Transcriptional kinship and enriched markers of ML clusters

A) UMAP projection of the dopaminergic dataset nuclei from the intact (non-lesioned) hemisphere. B) UMAP projection of the dopaminergic dataset nuclei from the lesioned hemisphere. ML clusters are marked with dashed circles in A and B. C) UMAP projection of the integrated dopaminergic nuclei dataset, color-coded for the territory IDs of the merged dataset. D, E) Sox6 and Calb1 expression plotted in intact and lesioned nuclei respectively. F) Same UMAP projection as in C, with ML clusters nuclei highlighted. G-H) Bar graphs showing the numbers and percentages of ML clusters nuclei per territory of the integrated dataset, respectively. More than 80% of ML nuclei cluster in Sox6 and Pcsk6 territories of the integrated data. I) Mostly lesioned (ML) clusters enriched markers plotted in UMAP. J-M) ML-enriched markers in I, detected by fluorescent RNA in situ hybridization, combined with TH and mCherry immunohistochemistry in lesioned versus untreated tissue. Scalebar = 50 µm.

Integration of human and mouse datasets

A, B) UMAP projections of the integrated dopaminergic human (control + diseased) and mouse (intact + lesioned) nuclei, split per condition. This integrated dataset includes 25,003 human nuclei and 33,052 mouse nuclei. C) UMAP projections of the human and mouse nuclei grouped by condition. Dataset was generated using the Seurat canonical correlation analysis (CCA). Ctrl = control neurotypical, PD = Parkinson’s disease, LBD = Lewy body dementia, intact = non-lesioned.