Parkinson's disease (PD) and Lewy body dementia (LBD) are characterized by the degeneration of midbrain dopamine (DA) neurons in the substantia nigra pars compacta (SNpc). However, some SNpc neurons survive even in late stages, indicating differential vulnerability. Understanding the molecular characteristics of these vulnerable neurons is crucial for refining PD models and developing targeted therapies. Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool to study cell-type-specific changes in brain diseases. While previous studies have profiled the human SNpc at single-cell resolution, the number of DA neurons sampled has been limited, hindering the identification of molecularly defined subpopulations and robust cross-subject comparisons. This study aimed to overcome these limitations by developing a protocol for selective enrichment of DA neurons from postmortem human SNpc, enabling comprehensive transcriptional profiling and spatial localization to identify molecular features associated with vulnerability to PD.

Previous research has highlighted the selective vulnerability of DA neurons in PD, with some subpopulations being more susceptible than others. Studies using immunohistochemistry have identified variations in DA neuron subtypes based on the expression of markers like calbindin-D28k. These studies have suggested a ventral-to-dorsal gradient of vulnerability, with ventral tier neurons exhibiting greater susceptibility to degeneration. Recent advances in single-cell RNA sequencing technology and its application to post-mortem human brain tissue have provided valuable insights into cell-type specific changes in various neurological diseases. However, the relatively small number of DA neurons profiled in prior single-cell studies of the human SNpc has limited the depth of understanding of subtype-specific molecular characteristics and their relationship to disease vulnerability. This current study builds upon these previous efforts by employing advanced techniques to significantly increase the number of DA neurons profiled and employing spatial transcriptomics for precise localization of different subtypes.

The researchers developed a novel protocol based on fluorescence-activated nuclei sorting (FANS) to enrich DA neuron nuclei from postmortem human SNpc for snRNA-seq. They used *NR4A2* as a marker gene for DA neurons, identified through scRNA-seq of mouse midbrain. Nuclei were isolated from eight neurotypical donors and subjected to snRNA-seq, generating high-quality profiles. Clustering analysis revealed ten transcriptionally distinct DA neuron subpopulations. Slide-seq, a high-resolution spatial transcriptomics technology, was used to map the spatial distribution of these populations within the SNpc of a macaque brain. To investigate the selective vulnerability of DA neurons in PD, the researchers profiled additional nuclei from ten age- and postmortem-interval-matched individuals with PD or LBD. Integrative analysis of these datasets, along with neurotypical controls, identified proportionally altered subpopulations in PD/LBD. Single-molecule fluorescence in situ hybridization (smFISH) was employed to validate these findings and to determine the genetic loci influencing the selective vulnerability observed. Further analysis using MAGMA and stratified LD score regression examined the enrichment of familial and common variants associated with PD within specific DA neuron subpopulations. Finally, Gene Ontology (GO) enrichment analysis and gene set enrichment analysis (GSEA) were performed to identify molecular pathways and transcription factors involved in PD-associated degeneration.

The study identified ten transcriptionally distinct subpopulations of DA neurons in the human SNpc, characterized by expression of markers such as *SOX6* and *CALB1*. Spatial localization using Slide-seq revealed that these subtypes exhibit distinct dorsal-ventral distributions within the SNpc. A specific subtype, marked by *AGTR1* expression, showed a strong ventral localization and was highly susceptible to loss in PD. This vulnerable population displayed significant upregulation of TP53 and NR2F2 target genes, implicating these pathways in PD-associated neurodegeneration. Importantly, this *AGTR1*-expressing subtype showed the strongest enrichment for genes harboring both familial and common variants associated with PD, indicating a significant cell-intrinsic contribution to disease susceptibility. smFISH validation confirmed the selective depletion of the SOX6_AGTR1 subpopulation and enrichment of other subtypes in PD. GO analysis of genes differentially expressed in the vulnerable SOX6⁺ subtype identified significant enrichment for terms related to neuron death and Wnt signaling. GSEA analysis further identified 13 TFs with enriched targets in this subtype, including TP53 and NR2F2, which are known to modulate DA neuron loss in mouse models of PD. A primate-specific DA neuron population (CALB1_GEM) was identified, highlighting species-specific differences in DA neuron subtypes. Analysis of the dorsal striatum in neurotypical individuals showed enrichment of AD genetic risk in microglia, contrasting with the cell-intrinsic vulnerability observed in PD.

This study provides a comprehensive molecular taxonomy of human SNpc DA neurons, revealing distinct subtypes with varying vulnerabilities to PD. The strong enrichment of PD genetic risk within the most vulnerable subtype (SOX6_AGTR1) strongly supports the hypothesis that cell-intrinsic mechanisms play a crucial role in determining the selective vulnerability of DA neurons in PD. The identification of upregulated pathways (TP53 and NR2F2) provides potential therapeutic targets. The discovery of a primate-specific DA neuron population highlights species-specific differences that should be considered when developing and interpreting animal models of PD. The contrasting genetic risk enrichment in microglia for AD and in DA neurons for PD emphasizes the differences in disease mechanisms between these two neurodegenerative conditions. Future studies should investigate the functional role of the identified gene programs and TFs in mediating DA neuron death, potentially using gene editing or pharmacological manipulations to modify their expression or activity in cellular models.

This study provides a detailed molecular and spatial map of human SNpc DA neurons, identifying a specific subtype highly vulnerable to PD. The strong enrichment of PD risk genes in this vulnerable population underscores the importance of cell-intrinsic mechanisms in the pathogenesis of PD. The findings implicate specific molecular pathways and TFs, such as TP53 and NR2F2, as potential therapeutic targets. Future research should focus on validating these findings in larger cohorts and exploring the functional significance of the identified pathways in mediating DA neuron death.

The study relies on postmortem tissue, which might introduce biases related to the timing and circumstances of death. The spatial analysis was conducted on macaque tissue, which may not perfectly reflect the human SNpc. The identification of the AGTR1-expressing subtype as most vulnerable needs further validation in longitudinal studies tracking neuron loss in living subjects. The study focused on the SNpc and dorsal striatum, and further investigation in other brain regions is warranted to elucidate the broader pathological mechanisms of PD.