Single-cell RNA sequencing of CSF reveals neuroprotective RAC1+ NK cells in Parkinson's disease

Abstract

Brain infiltration of the natural killer (NK) cells has been observed in several neurodegenerative disorders, including Parkinson's disease (PD). In a mouse model of α-synucleinopathy, it has been shown that NK cells help in clearing α-synuclein (α-syn) aggregates. This study aimed to investigate the molecular mechanisms underlying the brain infiltration of NK cells in PD. Immunofluorescence assay was performed using the anti-NKp46 antibody to detect NK cells in the brain of PD model mice. Next, we analyzed the publicly available single-cell RNA sequencing (scRNA-seq) data (GSE141578) of the cerebrospinal fluid (CSF) from patients with PD to characterize the CSF immune landscape in PD. Results showed that NK cells infiltrate the substantia nigra (SN) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model mice and colocalize with dopaminergic neurons and α-syn. Moreover, the ratio of NK cells was found to be increased in the CSF of PD patients. Analysis of the scRNA-seq data revealed that Rac family small GTPase 1 (RAC1) was the most significantly upregulated gene in NK cells from PD patients. Furthermore, genes involved in regulating SN development were enriched in RAC1⁺ NK cells and these cells showed increased brain infiltration in MPTP-induced PD mice. In conclusion, NK cells actively home to the SN of PD model mice and RAC1 might be involved in regulating this process. Moreover, RAC1⁺ NK cells play a neuroprotective role in PD.

Keywords: NK cells; Parkinson’s disease; RAC1; brain infiltration; single-cell RNA sequencing.

Figure 1

NK cells colocalize with α-synuclein deposits in the SN of MPTP-induced PD model mice. (A) NK cells were stained using NKp46 antibody in green, and dopaminergic neurons were labelled by TH in red in the SN of MPTP-induced PD mice. (B) NK cells were stained using NKp46 antibody in green, and α-syn was labelled by TH in red in the SN of MPTP-induced PD mice. DNA was stained using DAPI. The results implicate that NK cells reside with α-syn deposits, and colocalized with dopaminergic neurons in the SN of MPTP-induced PD brain. (C) Protein level of NK marker was detected using NKp46 antibody via western blotting in the SN region of MPTP-induced PD and control mice. Increased NK cells were found as revealed by increased NKp46 level. **p<0.01 compared with control group.

Figure 2

scRNA-seq analysis of CSF cells in PD. (A) UMAP projection of 18,553 cells from all CSF cells. (B) UMAP projection of PD and HC groups, respectively. NK cells were labeled with ellipse tag, which were decreased in PD group. (C) Dot plot of cell type marker genes. CD4 T was marked using CD3 epsilon subunit of T-cell receptor complex (CD3E) and CD4 molecule (CD4); CD8 T was marked using CD3E, CD8a molecule (CD8A), and CD8b molecule (CD8B); naïve CD4 T was marked using CD3E, CD4, transcription factor 7 (TCF7), and C-C motif chemokine receptor 7 (CCR7); cycling T was marked CD3E, marker of proliferation Ki-67 (MKI67), and DNA topoisomerase II alpha (TOP2A); B cell was marked using CD79a molecule (CD79A), and membrane spanning 4-domains A1 (MS4A1); Macrophage (Mac) was marked using complement C1q A chain (C1QA), and complement C1q B chain (C1QB); Monocyte (Mono) was marked using S100 calcium binding protein A9 (S100A9), and S100 calcium binding protein A8 (S100A8); Natural killer (NK) was marked using natural cytotoxicity triggering receptor 1 (NCR1), and natural killer cell granule protein 7 (NKG7); Dendritic cell (DC) was marked using CD1e molecule (CD1E), and CD1c molecule (CD1C); Plasmacytoid dendritic cell (pDC) was marked using C-type lectin domain family 4 member C (CLEC4C), and interleukin 3 receptor subunit alpha (IL3RA). (D) Heatmap of top 3 DEGs in each cell type. These cell types could be well classified using these cell-type specific DEGs. (E) Cell type distribution in each group. (F) Cell type distribution in PD and HC group. The percent of Mac and NK was decreased in PD CSF.

Figure 3

The ratio of NK cells is increased in the CSF of PD patients. (A) Upset plot showed the intersection of DEGs among five main cell types. RAC1 was increased in PD group in all five cell types. (B) Violin plot depicts distributions of RAC1 in each cell type between PD and HC group. HC group was colored in orange and PD group was colored in green. RAC1 was increased in PD group across five main cell types. (C) TF activity was calculated using DoRothEA R package, and the top 10 most variable TFs were plotted. Cycling T cells showed the most active TF activity, while naïve T cells showed the least TF activity.

Figure 4

RAC1⁺ NK cells are associated with SN development. (A) Feature plot of RAC1 in NK cells. PD group showed increased RAC1 expression. (B) DEGs between PD and HC group in CSF NK cells. Upregulated DEGs were colored in orange, and downregulated DEGs were colored in green. RAC1, Rac family small GTPase 1; CD96, CD96 molecule; TMIGD2, transmembrane and immunoglobulin domain containing 2; RPS9, ribosomal protein S9; RPS18, ribosomal protein S18; RPS6, ribosomal protein S6; RPS26, ribosomal protein S26; RPL41, ribosomal protein S41; CD3E, CD3 epsilon subunit of T-cell receptor complex; RPS4Y1, ribosomal protein S4 Y-linked 1; KLF2, KLF transcription factor 2. (C) The NK cells were divided into RAC1_high and RAC1_low groups according to the median expression level of RAC1 calculated by AddModuleScore function in Seurat. 55% of NK cells from PD group were RAC1_high, while 42% of NK cells from HC were RAC1_high. (D) Upregulated DEGs in RAC1_high compared with RAC1_low group were enriched in SN development, neural nucleus development, and midbrain development biological process GO terms.

Figure 5

RAC1⁺ NK cells home to the SN of MPTP-induced PD model mice. (A) Immunofluorescence staining of NKp46 (green) and RAC1 (red) in the brain of control mice. Whole slice was shown. (B) Immunofluorescence staining of NKp46 (green) and RAC1 (red) in the brain of MPTP-induced PD mice. Whole slice was shown. (C) Immunofluorescence staining image of the SN region (labeled with ellipse tag) in control mice. (D) Enlarged image of the dashed rectangle in (C, E) Immunofluorescence staining image of the SN region (labeled with ellipse tag) in PD mice. (F) Enlarged image of the dashed rectangle in (E) Arrowhead showed the merged staining of NKp46 and RAC1. (G) The relative fluorescent intensity of NKp46 and RAC1/NKp46 colocalization were calculated. Increased NKp46 level and RAC1/NKp46 colocalization were observed in MPTP group. ***p<0.001 compared with control group.