Molecular profiling of human substantia nigra identifies diverse neuron types associated with vulnerability in Parkinson's disease

Abstract

Parkinson's disease (PD) is characterized pathologically by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Whether cell types beyond DA neurons in the SN show vulnerability in PD remains unclear. Through transcriptomic profiling of 315,867 high-quality single nuclei in the SN from individuals with and without PD, we identified cell clusters representing various neuron types, glia, endothelial cells, pericytes, fibroblasts, and T cells and investigated cell type-dependent alterations in gene expression in PD. Notably, a unique neuron cluster marked by the expression of RIT2, a PD risk gene, also displayed vulnerability in PD. We validated RIT2-enriched neurons in midbrain organoids and the mouse SN. Our results demonstrated distinct transcriptomic signatures of the RIT2-enriched neurons in the human SN and implicated reduced RIT2 expression in the pathogenesis of PD. Our study sheds light on the diversity of cell types, including DA neurons, in the SN and the complexity of molecular and cellular changes associated with PD pathogenesis.

Fig. 1.. Cellular diversity in the SN from patients with PD and control samples.

(A) Flow chart of the experimental procedure and data processing. Barcoded single-nucleus suspension was prepared using frozen SN samples from PD and control subjects followed by RNA sequencing. Sequencing data were quality controlled and classified into cell clusters, which were annotated with known cell-type markers. Downstream analyses include cell composition changes, immunofluorescence (IF) staining, DEG identification, and cell communication alterations. (B) UMAP plot showing cell clusters. Ast, astrocytes; Neu, neurons; Mic, microglia; Oli, oligodendrocytes; OPC, oligodendrocyte progenitor cells; End, endothelial cells; Fib, fibroblast-like cells; Per, pericytes; T, T cell. (C) Expression pattern of known brain cell-type marker genes in the control cells. (D) Pie-chart for the fractions of major cell types in the human SN. (E) Sequenced cell distribution represented by disease status in each cluster.

Fig. 2.. Evidence of RIT2⁺ neuronal populations in health and PD brain.

(A) Fraction of c9 neurons in one cohort containing PD and the controls. (B) Distribution of the fraction of c9 neurons in PD and the controls shown in (A). P value was computed by one-tailed Wilcoxon rank sum test. (C) IHC staining of the postmortem tissue of the human SN with anti-TH (blue, left) and -RIT2 (purple, right) antibodies. Enclosed areas are SN pars compacta (SNpc). Scale bars, 4 mm and 100 μm in magnified images. (D) IF staining of the postmortem tissue of the human SN with anti-RIT2 (red) and anti-TH (green) antibodies. Yellow arrows, NM⁺RIT2⁺TH⁺neurons; yellow arrowheads, NM⁺RIT2⁺TH⁻neurons); blue arrowheads, NM⁻RIT2⁺TH⁻neurons. DA neurons contain neuromelanin (bright-field images). Scale bar, 100 μm. (E) Quantification of the fractions of RIT2⁺ among NM⁻ neurons and RIT2⁺TH⁺ and RIT2⁺TH⁻ among NM⁺ neurons from five unaffected controls. (F) Quantification of the number of RIT2⁺ among NM⁻ neurons and RIT2⁺TH⁺ and RIT2⁺TH⁻ among NM⁺ neurons in the SNpc of the control (n = 5) and PD (n = 5). P values were calculated by unpaired two-tailed Student’s t test. (G) RNAscope in situ hybridization assay in mouse brain. Rit2⁺Th⁺ and Rit2⁺Th⁻ cells were mapped on the schematic images of mouse brain. Scale bars, 1 mm.

Fig. 3.. scRNA-seq and immunostaining analysis of human midbrain organoids.

(A) UMAP visualization of the single-cell clustering from the hiPSC-derived midbrain organoid (day 40). (B and C) UMAP visualization of the TH and RIT2 gene expression. (D) Comparison between the cluster markers between the human organoids and the SN of the control samples. The minimum adjusted P value was set at 1E-20 for visualization purpose. (E and F) IF staining of midbrain organoid with anti-RIT2 and anti-TH antibodies. White arrows indicate a RIT2⁺TH⁺ cell population, and white arrowheads indicate a RIT2⁺TH⁻ cell population. Scale bars, 100 μm (E) and 10 μm (F).

Fig. 4.. Subclustering analysis of clusters c6 and c7 and identification of subtypes of DA neurons.

(A to F) Subclustering analysis of clusters c6 [(A) to (C)] and c7 [(D) to (F)]. [(A) and (D)] UMAP plot for subclusters. [(B) and (E)] Expression pattern of known brain cell–type marker genes. [(C) and (F)] Cell fraction distribution represented by disease status in each subcluster. (G) UMAP plots for the expression of selected marker genes in the neuron clusters c6, c7, and c9. (H) Comparison of cluster markers between the organoids and DA neuron subclusters of human SN (control). The minimum adjusted P value was set at 1 × 10⁻²⁰ for visualization purpose.

Fig. 5.. Cell type–specific DEGs between PD and the control.

(A) Number count of up- (UP) and down-regulated (DN) DEGs in each cluster. (B) Heatmap of top canonical pathways enriched for up- and down-regulated genes in each cluster. (C) Top DEGs involved in the indicated pathways in each cluster. (D) Comparison of DEGs identified in bulk tissue–based meta-analysis and snRNA-seq. FC, fold change.

Fig. 6.. Cell type–specific expression and DEG of PD-associated genes and risk loci.

(A) Heatmap for cell type–specific expression (left) and DEG (right) of PARK family genes in PD. (B) Number of GWAS loci related genes enriched (top) and differentially expressed (bottom) in each cell type. Dark gray indicated genes unique to each cell type, and light gray indicated genes shared among cell clusters. (C) Expression patterns of selected PD GWAS loci-related genes in different cell clusters. (D) Heatmap for the log2 fold change of differential expression of selected PD GWAS loci genes in each cluster.

Fig. 7.. Altered cell communication networks in PD.

(A) Differential LR interactions between PD and the controls. Blue lines and red lines indicated decreased and increased interactions, respectively. The line width was proportional to the difference. (B) Distribution of cell cluster based on their relative changes in the incoming and outgoing signaling strengths between PD and control. (C) Information flow changes of major signaling pathways between PD and control in each cell cluster. Dashed boxes highlighted gain (red) or loss (blue) of signaling in specific cell clusters. (D) Chord diagram of EPHA and CDH signaling in control and PD.