Ferroptosis-related factors in the substantia nigra are associated with Parkinson's disease

Abstract

Ferroptosis is an iron-dependent, lipid peroxidation-driven cell death pathway, while Parkinson's disease (PD) patients exhibit iron deposition and lipid peroxidation in the brain. Thus, the features of ferroptosis highly overlap with the pathophysiological features of PD. Despite this superficial connection, the possible role(s) of ferroptosis-related (Fr) proteins in dopaminergic neurons and/or glial cells in the substantia nigra (SN) in PD have not been examined in depth. To explore the correlations between the different SN cell types and ferroptosis at the single-cell level in PD patients, and to explore genes that may affect the sensitivity of dopaminergic neurons to ferroptosis, we performed in silico analysis of a single cell RNA sequence (RNA-seq) set (GSE178265) from the Gene Expression Omnibus (GEO) database. We identified differentially expressed genes (DEGs) in the different cell types in the human SN, and proceeded to perform enrichment analysis, constructing a protein-protein interaction network from the DEGs of dopaminergic neurons with the Metascape database. We examined the intersection of Fr genes present in the FerrDb database with DEGs from the GSE178265 set to identify Fr-DEGs in the different brain cells. Further, we identified Fr-DEGs encoding secreted proteins to implicate cell-cell interactions in the potential stimulation of ferroptosis in PD. The Fr-DEGs we identified were verified using the bulk RNA-seq sets (GSE49036 and GSE20164). The number of dopaminergic neurons decreased in the SN of PD patients. Interestingly, non-dopaminergic neurons possessed the fewest DEGs. Enrichment analysis of dopaminergic neurons' DEGs revealed changes in transmission across chemical synapses and ATP metabolic process in PD. The secreted Fr-DEGs identified were ceruloplasmin (CP), high mobility group box 1 (HMGB1) and transferrin (TF). The bulk RNA-seq set from the GEO database demonstrates that CP expression is increased in the PD brain. In conclusion, our results identify CP as a potential therapeutic target to protect dopaminergic neurons by reducing neurons' sensitivity to ferroptosis.

Figure 1

Cell types and proportions in the SN of PD patients and the control group (CN). A shows the combining multiple samples together for statistical analysis. A total of 184,673 cells from 45 CN people were screened in the control group, and the whole number of each cell types was calculated to the total cell population (184,673 cells). While 135,344 cells from 34 PD patients were screened in the PD group, in which the proportion of each cell types was calculated to the whole population (135,344 cells). B is the statistical analysis of the proportion of cells in each individual sample with T-TEST, there are 45 CN samples and 34 PD patients used for statistical analysis, but the samples with data of 0% were excluded in charts. **p < 0.01.

Figure 2

Results of DEG analysis of each cell type in PD. Red dots represent up-regulated genes; black dots represent down-regulated genes. Average log2FC indicates average log2-fold change, and Thr_signi represents the threshold value and significance. The five most up-regulated genes and the five most down-regulated genes in each cell type are provided.

Figure 3

Fr-DEGs in various cell types.

Figure 4

Hierarchical clustering results and clustering trees for functional enrichment of dopaminergic neuron DEGs. Only the p-value information is shown in the histogram, which can be viewed against the dendrogram.

Figure 5

PPI network and MCODE component analysis results. Genes that do not interact with other genes in the list of DEGS are not provided. The PPI network shows genes that have cooperative relationships with other genes, and also possess MCODE components. MCODE reveals closely connected molecular complex regions in the PPI network.