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The heterogeneity in various aspects of Parkinson's disease (PD) is increasingly recognized as an important aspect in understanding the condition, with ethnicity and race being one of the major causes of heterogeneity that affects the risk and symptoms of PD onset. While there have been numerous reports related to PD in East Asia, there has been a lack of contribution from single-cell (or nucleus) transcriptome studies, which have been making significant contributions to understanding PD. In this study, we profiled nuclei extracted from the substantia nigra (SN) of confirmed pathological PD and control patients in South Korea, revealing 8 different cell types through cluster analysis. Monocle-based pseudotime analysis identified two disease-associated trajectories for each astrocyte and microglia and identified genes that differentiate them. Interestingly, we uncovered the inflammatory intervention in the early PD-associated transition in microglia and identified the molecular features of this intermediate state of microglia. In addition, gene regulatory networks (GRNs) based on TENET analysis revealed the detrimental effect of an HSPA5-led module in microglia and MSRB3- and HDAC8- led modules specifying the two different astrocyte trajectories. In SN neurons, we observed high heterogeneity and population changes, a decrease in dopaminergic and glutamatergic neurons and a proportional increase in GABAergic neurons. By deconvolution in spatial transcriptome obtained the PD sample, we confirmed spatiotemporal heterogeneity of neuronal subpopulations and PD-associated progressive gliosis specific to dopaminergic nuclei, SN and ventral tegmental areas (VTAs). In conclusion, our approach has enabled us to identify the genetic and spatial characterization of neurons and to demonstrate different glial fates in PD. These findings advance our molecular understanding of cell type-specific changes in Korean PD progression, providing an important foundation for predicting and validating interventions or drug effects for future treatments.

case control design

single nucleus RNA sequencing

The substantia nigra (SN) was macro-dissected from the frozen midbrain slice using a carving knife by an experienced neuropathologist. Dissection was performed with particular care to avoid inclusion of adjacent structures, including the red nucleus and surrounding white matter. The dissection strategy aimed to encompass the full medial-lateral extent of the SN to maximize representation of nigral cellular diversity.

Nuclei were isolated using a modified 10x Genomics protocol (CG000124), stained with 7-AAD, and sorted by FACS. cDNA was generated, amplified using Chromium Next GEM Single Cell 3' Kits v3.1 (Dual Index).

Size selection was used to optimize cDNA amplicon size. Libraries were constructed with Chromium Next GEM Single Cell 3' Kits v3.1 (Dual Index) protocol.

Prepared libraries were sequenced on Illumina HiSeq X.

Raw single-nucleus RNA-seq count data were processed using the Seurat R package. Low-quality nuclei were removed based on gene expression and mitochondrial transcript content, and doublets were identified and excluded using DoubletFinder. Gene expression data were normalized, highly variable genes were identified, and samples were integrated using an anchor-based method to correct for batch effects. The integrated data were scaled with regression of technical covariates, followed by dimensionality reduction, clustering, and marker-based cell-type annotation to generate an analysis-ready dataset. Detailed analysis parameters and code are described in the associated publication and supplementary materials.