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 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 significant 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 alterations, suggesting more extensive proteomic alterations. This study offers valuable insights into potential underlying mechanisms of epilepsy and underscores the importance of considering sex as a key factor in epilepsy research and therapeutic development.