Sex-specific proteomic analysis of epileptic brain tissues from Pten knockout mice and human refractory epilepsy

Abstract

Rationale: Epilepsy presents significant sex-based disparities in prevalence and manifestation. Epidemiological studies reveal that epilepsy is more prevalent in males, with lesional types being more common, whereas idiopathic generalized epilepsies are more frequently observed in females. These differences stress the importance of considering sex-specific factors in epilepsy diagnosis, treatment, and mechanistic research using preclinical models. To elucidate potential molecular differences that could explain these disparities and inform personalized treatment strategies, we conducted a proteomic analysis of epileptic brain tissues from both an experimental mouse model of genetic epilepsy and humans with drug-resistant epilepsy (DRE).

Methods: We employed mass spectrometry-based proteomic analysis on brain tissues from DRE patients and the Pten knockout (KO) mouse model of genetic epilepsy with focal cortical dysplasia. Mouse samples included hippocampi from adult wild-type (WT) and Pten KO mice (4-5 per group and sex). Human samples included the temporal cortex from 12 DRE adult patients (7 males, 5 females) and 5 non-epileptic (NE) controls (2 males, 3 females). Brain biopsies were collected with patients' informed consent under approved IRB protocols (Indiana University Health Biorepository). Proteomic profiles were analyzed using principal component analysis (PCA) along with volcano plots to identify significant changes in protein expression. The enrichment analysis of differentially expressed proteins was conducted by Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway.

Results: PCA revealed distinct clustering of brain proteomes between epilepsy and control cases in both human and mice, with 390 proteins showing significant differences in human and 437 proteins in mouse samples. These proteins are primarily associated with ion channels, synaptic processes, and neuronal energy regulation. In the mouse model, males have more pronounced proteomic changes than females, with enrichment in metabolic pathways and VEGF signaling pathway, indicating a more severe vascular permeability impairment in males. In human DRE cases, 118 proteins were significantly changed by comparing epileptic females to males. Pathway analysis revealed changes in metabolic pathways and the HIF-1 signaling pathway, indicating that altered neuronal activity and inflammation may lead to increased oxygen consumption.

Conclusion: These findings highlight differences between epilepsy and control brain samples in both humans and mice. Sex-specific analysis revealed distinct pathway enrichments between females and males, with males exhibiting a broader range of proteomic alterations. While these observations suggest potential sex-related differences in proteomic profiles, larger studies are needed to further validate these patterns. This exploratory work provides initial insights into possible underlying mechanisms of epilepsy and suggests that sex may be an important consideration in future epilepsy studies, though more comprehensive studies are required to establish therapeutic interventions.

Keywords: Cortex; Epilepsy; Proteomics; Pten; Sex; hippocampus.

Fig. 1.

Diagram of the experimental workflow. Mouse and human brain tissues were collected from control and epilepsy groups (Mouse: control, n = 9; epilepsy, n = 10; Human: control, n = 5; epilepsy, n = 12). Proteins were quantified using liquid chromatography-tandem mass spectrometry (LC-MS), followed by statistical analysis, functional enrichment, and pathway analysis to identify differentially expressed proteins and enriched pathways.

Fig. 2.

Proteomic differences in the hippocampus of Pten knockout (KO) and wildtype (WT) mice. (A) Principal component analysis (PCA) of Pten KO (red) and WT (blue) groups, showing distinct clustering based on differentially expressed proteins. Each data point represents an individual case. The PC2 bar plot indicates significant separation between groups. (B) Volcano plot displays protein expression differences for all identified proteins. Proteins highlighted in red showed significantly altered expression in Pten KO mice compared to WT mice. Significance was determined using Student’s t-test. P-values for significantly altered proteins are provided in the Supplementary Table 1. (C) Heatmap of scaled protein expression values (z-scores), color-coded according to the legend on the right, for significantly changed proteins used in clustering analysis. (D) Gene Ontology (GO) analysis of significantly altered proteins, including biological process (BP), cellular component (CC), and molecular function (MF). The top 10 terms are shown. (E) KEGG pathway analysis of significantly altered proteins. The top 20 pathways are shown. (F) Representative western blot showing GFAP protein levels, along with quantification for WT (n = 9; females: n = 5, males: n = 4) and Pten KO (n = 10; females: n = 5, males: n = 5) samples. Open circles represent female data points; closed circles represent male data points. *p < 0.05; **p < 0.01.

Fig. 3.

Sex-specific proteomic differences in Pten knockout (KO) and wildtype (WT) mice. (A–B) Volcano plots showing protein expression differences in all identified proteins. Proteins highlighted in red were significantly altered when comparing female KO (F-KO) with female WT (F-WT) (A), and male KO (M-KO) with male WT (M-WT) (B). Significance was determined using Student’s t-test. P-values for significantly altered proteins are provided in the Supplementary Table 1. (C–D) Heatmaps displaying scaled protein expression values (z-scores), color-coded according to the legend on the right, for significantly changed proteins in F-KO vs F-WT (C) and M-KO vs M-WT (D). (E–F) KEGG pathway analysis of significantly altered proteins in females (E) and males (F). (G) Fourteen upregulated proteins shared between females and males. (H) Twenty-one downregulated proteins shared between females and males. (I) Summary of overlapping proteins (14 upregulated (G) and 21 downregulated (H)).

Fig. 4.

Proteomic differences between human epilepsy and non-epileptic cases. (A) Principal component analysis (PCA) of epileptic (E, red) and non-epileptic (NE, blue) cases, showing distinct clustering based on protein expression profiles. Each point represents an individual case. The PC1 bar plot indicates significant separation between groups. (B) Volcano plot displaying protein expression differences for all identified proteins. Proteins highlighted in red showed significantly altered expression when comparing E and NE cases. Significance was determined using Student’s t-test. P-values for significantly altered proteins are provided in the Supplementary Table 1. (C) Heatmap showing scaled protein expression values (z-scores), color-coded according to the legend on the right, for significantly changed proteins used in clustering analysis. (D) Expression levels of selected proteins of interest with significant alterations in epilepsy. Each point represents an individual case. *p < 0.05; **p < 0.01. (E) Gene Ontology (GO) analysis of significantly altered proteins, including biological process (BP), cellular component (CC), and molecular function (MF). The top 10 terms are shown. (F) KEGG pathway analysis of significantly altered proteins. The top 10 pathways are shown.

Fig. 5.

Sex-specific proteomic differences in human epilepsy. (A) Principal component analysis (PCA) of the overall dataset. PCA of epileptic female (F-E, red) and epileptic male (M-E, blue) cases shows similar protein expression profiles. Each point represents an individual case. (B) PC1 and PC2 bar plots indicate no significant separation between F-E and M-E cases. (C) Volcano plot displays protein expression differences for all identified proteins. Proteins highlighted in red showed significantly altered expression when comparing F-E and M-E cases. Significance was determined using Student’s t-test. P-values for significantly altered proteins are provided in the Supplementary Table 1. (D) Heatmap of scaled protein expression values (z-scores), color-coded according to the legend on the right, for significantly changed proteins used in clustering analysis. (E) Expression levels of selected proteins of interest with significant sex-specific differences. Each point represents an individual case. *p < 0.05; **p < 0.01; ***p < 0.001. (F) KEGG pathway analysis of significantly altered proteins. The top 10 pathways are shown.

Fig. 6.

Overlapping significantly altered proteins in mouse and human epilepsy. (A) Twenty-five upregulated proteins were shared between human and mouse datasets. (B) Fourteen downregulated proteins are shared between human and mouse datasets. (C) Summary of overlapping proteins, including the 25 upregulated proteins shown in (A) and the 14 downregulated proteins shown in (B).