Integrating Proteomics and Transcriptomics Reveals the Potential Pathways of Hippocampal Neuron Apoptosis in Dravet Syndrome Model Mice

Abstract

An important component contributing to the onset of epilepsy is the death of hippocampal neurons. Several studies have shown that Dravet syndrome model mice: Scn1a KO mice have a high number of apoptotic neurons following seizures, but the precise mechanism underlying this remains unclear. The aim of this research was to elucidate the potential molecular mechanism of neuronal apoptosis in Scn1a KO mice by integrating proteomics and transcriptomics, with the ultimate goal of offering better neuroprotection. We found that apoptotic processes were enriched in both proteomic and transcriptomic GO analyses, and KEGG results also indicated that differential proteins and genes play a role in neurotransmission, the cell cycle, apoptosis, and neuroinflammation. Then, we examined the upstream and downstream KGML interactions of the pathways to determine the relationship between the two omics, and we found that the HIF-1 signaling pathway plays a significant role in the onset and apoptosis of epilepsy. Meanwhile, the expression of the apoptosis-related protein VHL decreased in this pathway, and the expression of p21 was upregulated. Therefore, this study suggests that VHL/HIF-1α/p21 might be involved in the apoptosis of hippocampal neurons in Scn1a KO mice.

Keywords: Dravet syndrome; HIF-1α; Scn1a mice; VHL; neuron apoptosis; p21; proteomics; transcriptomics.

Figure 1

Scn1a KO (heterozygous) mice and WT mice were identified using PCR.

Figure 2

A representative EEG recording of seizure activity in the KO and WT mice.

Figure 3

Compared with WT mice, KO mice exhibited a loss of neurons in the hippocampus. (a) Representative Nissl staining images of CA3 and CA1 in mouse hippocampal regions. (b) Statistical plot of the number of Nissl staining-positive cells in the CA3 region. (c) Statistical plot of the number of Nissl staining-positive cells in the CA1 region. (d) Representative TUNEL staining images of CA3 and CA1 in the hippocampi of the mice. (e) Statistical plot of the number of TUNEL staining-positive cells in the CA3 region. (f) Statistical plot of the number of TUNEL staining-positive cells in the CA1 region. All data are expressed as the mean ± SEM (n = 3). * p < 0.05 and ** p < 0.01. Scale bar = 100 μm.

Figure 3

Compared with WT mice, KO mice exhibited a loss of neurons in the hippocampus. (a) Representative Nissl staining images of CA3 and CA1 in mouse hippocampal regions. (b) Statistical plot of the number of Nissl staining-positive cells in the CA3 region. (c) Statistical plot of the number of Nissl staining-positive cells in the CA1 region. (d) Representative TUNEL staining images of CA3 and CA1 in the hippocampi of the mice. (e) Statistical plot of the number of TUNEL staining-positive cells in the CA3 region. (f) Statistical plot of the number of TUNEL staining-positive cells in the CA1 region. All data are expressed as the mean ± SEM (n = 3). * p < 0.05 and ** p < 0.01. Scale bar = 100 μm.

Figure 4

Details of the protein identification and multivariate statistical analyses grounded in proteomic research. (a) Principal component analysis (PCA). (b) Volcano map of differentially expressed proteins. (c) Clustering heatmap of differentially expressed proteins. (d) Gene Ontology (GO) annotation classification statistics chart of the top 30 downregulated differential proteins. (e) Gene Ontology (GO) annotation classification statistics chart of the top 30 upregulated differential proteins. (f) KEGG enrichment analysis bubble map of the top 20 downregulated differential proteins. (g) KEGG enrichment analysis bubble map of the top 20 upregulated differential proteins.

Figure 5

Validation of DEPs in proteomic data through WB experiments. (a–d) Representative Western blot images and statistical plots for Ace, Aqp1, Vhl, and Folr1. All data are expressed as the mean ± SEM (n = 3). * p < 0.05 and ** p < 0.01.

Figure 6

Details of the protein identification and multivariate statistical analyses grounded in transcriptome research. (a) PCA score plots of hippocampal tissue samples between KO and WT mice. (b) Volcano map of differentially expressed genes. (c) Cluster heatmap of differentially expressed genes. (d) GO enrichment map of downregulated differential genes. (e) GO enrichment map of upregulated differential genes. (f) KEGG enrichment map of downregulated differential genes. (g) KEGG enrichment map of upregulated differential genes.

Figure 7

Validation of transcriptomic data through real-time quantitative polymerase chain reaction experiments. (a–e) Representative RT-PCR statistical plot for Cartpt, Sprrla, Tgm1, Trh, and Etv3l. All data are expressed as the mean ± SEM. ** p < 0.01 and *** p < 0.001.

Figure 8

Integrated information from proteomic and transcriptomics analyses. (a) Venn diagram of proteomic and transcriptomic association analysis. (b) Distribution map of the top 30 associated elements. (c) KGML interaction network diagram: level 1. (d) KGML interaction network diagram: level 2.

Figure 9

KEGG map (mmu04066). The boxes represent genes/proteins (genes on the left, and proteins on the right), and the circles represent metabolites. Color gradients are used to show specific expressions.

Figure 10

Expression of DGPs and DGEs in the HIF-1 signaling pathway. (a–c) Representative Western blot images and statistical plots of P21 and HIF-1α. (d) Representative RT-qPCR statistical plot of P21. All data are expressed as the mean ± SEM. * p < 0.05 and ** p < 0.01. **** p < 0.0001.