Transcriptome and proteome profiling of neural stem cells from the human subventricular zone in Parkinson's disease

Abstract

It is currently accepted that the human brain has a limited neurogenic capacity and an impaired regenerative potential. We have previously shown the existence of CD271-expressing neural stem cells (NSCs) in the subventricular zone (SVZ) of Parkinson's disease (PD) patients, which proliferate and differentiate towards neurons and glial cells in vitro. To study the molecular profile of these NSCs in detail, we performed RNA sequencing and mass spectrometry on CD271⁺ NSCs isolated from human post-mortem SVZ and on homogenates of the SVZ. CD271⁺ cells were isolated through magnetic cell separation (MACS). We first compared the molecular profile of CD271⁺ NSCs to the SVZ homogenate from control donors and then compared CD271⁺ cells to CD11b⁺ microglia. These results confirmed their neural stem cell identity. Finally we compared controls and PD patients to establish a specific molecular profile of NSCs and the SVZ in PD. While our transcriptome analysis did not identify any differentially expressed genes in the SVZ between control and PD patients, our proteome analysis revealed several proteins that were differentially expressed in PD. Some of these proteins are involved in cytoskeletal organization and mitochondrial function. Transcriptome and proteome analyses of NSCs from PD revealed changes in the expression of genes and proteins involved in metabolism, transcriptional activity and cytoskeletal organization. Our data suggest that NSCs may transit into a primed-quiescent state, that is in an "alert" non-proliferative phase in PD. Our results not only confirm pathological hallmarks of PD (e.g. impaired mitochondrial function), but also show that the NSCs from SVZ undergo significant changes at both transcriptome and proteome level following PD.

Keywords: Human SVZ; Neural stem cells; Parkinson’s disease; Proteomics; Quiescence; RNA sequencing; Transcriptomics.

Fig. 1

Transcriptome profiling of neural stem cells of the human SVZ. a Scheme of experimental set-up. b PCA plot showing distribution of SVZ and CD271⁺ NSCs from both control and PD donors. c Differentially expressed genes (in red) visualized in an MA plot for CD271⁺ cells vs SVZ cells from control donors (p-value < 0.01, adj p-value < 0.01). d Panther pathway analysis of differentially expressed genes between CD271⁺ cells and SVZ cells (p-value < 0.05). e Panther pathway analysis of genes upregulated in CD271⁺ cells when compared to SVZ homogenate (p-value < 0.05). f PCA plot showing the distribution of CD271⁺ cells and CD11b⁺ cells. g MA plot showing differentially expressed genes (in red) between CD271⁺ cells and CD11b⁺ cells. CTRL = control; PD = Parkinson’s disease

Fig. 2

Differential gene expression in CD271⁺ cells following PD. a-b MA plots showing no differentially expressed transcripts between SVZ from controls and PD patients (a) and CD271⁺ NSCs from controls and PD patients (b) (p-value < 0.01; adj p-value < 0.05). c Panther pathway analysis highlights changes in serotonin (i.e. 5-hydroxytryptamine) degradation and dopamine signaling (p-value < 0.05). d GO analysis of upregulated genes in CD271⁺ NSCs showing changes in oxidoreductase activity and transcriptional activity. e GO biological process analysis of downregulated genes highlights cilium movement and assembly. f GO molecular function analysis of downregulated transcripts showing dysregulation of channel function, TGF-β binding and cytoskeletal organization. Cntr = control; PD = Parkinson’s disease

Fig. 3

Expression of genes involved in NSC activation state remains unaltered after PD. a-c Relative expression of markers for qNSCs and aNSCs (a), lineage progression (b) and proliferation (c). Data shown are TPM-normalized read counts. d Quantification of the number of S100β⁺ and FGFR3⁺ cells in the human SVZ (control and PD). e Representative images of FGFR3⁺ cells in the human SVZ. Cntr = control; PD = Parkinson’s disease. Data are presented as mean ± SEM. Scale bar = 10 μm

Fig. 4

Proteomics analysis reveals differential protein expression in CD271⁺ NSCs of PD patients. a Schematic of experimental set-up. b Vulcano plot showing differentially expressed proteins in CD271⁺ NSCs of PD patients (red = downregulated; green = upregulated) (p-value < 0.05; logFC > 0.1). c Panther pathway analysis showing changes in signaling pathways involved in metabolism and PD (p-value < 0.05). d GO analysis (biological process) further highlights dysregulation of metabolic processes. e Protein-protein interaction (PPI) analysis showing the top 10 proteins to interact with downregulated proteins

Fig. 5

Changes in protein expression in the SVZ of PD patients. a Vulcano plot showing differentially expressed proteins in the SVZ of PD patients (p < 0.05; logFC > 0.1). b Panther pathway analysis showing changes in signaling pathways involved in “Parkinson’s disease”, “glycolysis” and “ATP synthesis” (p-value < 0.5). c GO analysis (biological process) of downregulated proteins suggests dysregulation in “protein translocation machinery”. d Protein-protein interaction (PPI) analysis showing the top 10 proteins to interact with downregulated proteins. e GO analysis (biological process) of upregulated proteins shows an increase in proteins involved in “mitochondria function”