Proteomic Differences in the Hippocampus and Cortex of Epilepsy Brain Tissue

Abstract

Epilepsy is a common neurological disorder affecting over 70 million people worldwide, with a high rate of pharmaco-resistance, diverse comorbidities including progressive cognitive and behavioral disorders, and increased mortality from direct (e.g., Sudden Unexpected Death in Epilepsy [SUDEP], accidents, drowning) or indirect effects of seizures and therapies. Extensive research with animal models and human studies provides limited insights into the mechanisms underlying seizures and epileptogenesis, and these have not translated into significant reductions in pharmaco-resistance, morbidities or mortality. To help define changes in molecular signaling networks associated with epilepsy, we examined the proteome of brain samples from epilepsy and control cases. Label-free quantitative mass spectrometry (MS) was performed on the hippocampal CA1-3 region, frontal cortex, and dentate gyrus microdissected from epilepsy and control cases (n=14/group). Epilepsy cases had significant differences in the expression of 777 proteins in the hippocampal CA1-3 region, 296 proteins in the frontal cortex, and 49 proteins in the dentate gyrus in comparison to control cases. Network analysis showed that proteins involved in protein synthesis, mitochondrial function, G-protein signaling, and synaptic plasticity were particularly altered in epilepsy. While protein differences were most pronounced in the hippocampus, similar changes were observed in other brain regions indicating broad proteomic abnormalities in epilepsy. Among the most significantly altered proteins, G-protein Subunit Beta 1 (GNB1) was one of the most significantly decreased proteins in epilepsy in all regions studied, highlighting the importance of G-protein subunit signaling and G-protein–coupled receptors (GPCRs) in epilepsy. Our results provide insights into the molecular mechanisms underlying epilepsy, which may allow for novel targeted therapeutic strategies.

Proteomics and Transcriptomics of the Hippocampus and Cortex in SUDEP and High-Risk SUDEP Patients

“…30 Myelin damage may occur after prolonged seizures and its loss may promote further seizure activity. 31 We found that the mature oligodendrocyte marker myelin basic protein (MBP) is decreased in epilepsy patients compared to non-epilepsy control patients, 18 and it is decreased in the hippocampus of an animal model of epilepsy. 32 However, we found no further decrease in MBP expression in SUDEP or high-risk SUDEP patients when compared to controls in this study, nor was MBP different in our recent RNAseq analysis between MTLE and non-epilepsy controls.…”

“…Group sizes were determined based on the number of patients with significant findings as previously reported, [14][15][16] including our earlier studies in epilepsy patients with similar methods. 17,18 Laser Capture Microdissection for Proteomics: FFPE brain tissue blocks containing either hippocampus (lateral geniculate nucleus level) 19 or superior frontal gyrus were sectioned at 8 µm and collected onto laser capture microdissection (LCM) compatible PET slides (Leica). Sections were stained with cresyl violet to localize regions of interest for LCM 20 and air dried overnight in a loosely closed container.…”

“…Label-free quantitative MS Proteomics: Label-free quantitative MS assessed differential protein expression, as described previously. 18,2122 FFPE cuts were incubate in 50 mM ammonium bicarbonate (ABC) solution containing 20% (v/v) acetonitrile (ACN) for 1h at 95 o C followed by 2h at 65 o C. Disulfide bonds were reduced with 10 mM DTT (1h at 57 o C) and alkylated with 30 mM IAA (45 min at RT in the dark). Proteins were enzymatically digested into peptides with 300 ng of trypsin (sequencing grade, Promega) overnight at RT.…”

“…Immunohistochemistry: Immunohistochemistry was performed to validate the identified protein of interest, ermin (ERMN) as previously described. 18,27 Briefly, FFPE sections (8 µm) were deparaffinized and rehydrated through a series of xylenes and ethanol dilutions. Heat-induced antigen retrieval was performed with 10 mM sodium citrate, 0.05% triton-x 100; pH6.…”

iTRAQ-Based Proteomic Analysis of Dentate Gyrus in Temporal Lobe Epilepsy With Hippocampal Sclerosis

“…Two previous studies have performed proteomics on the DG region of epilepsy patients: Liu et al ( 47 ) compared the proteomes of basal and dispersed granule cells in the hippocampus of MTLE patients with GCD (not including surgical or post-mortem healthy controls), and found that upregulated proteins in dispersed samples were involved in developmental cellular migratory processes. Pire et al ( 48 ) performed proteomic analysis of the hippocampal CA1-3 region, frontal cortex, and DG samples from epilepsy (without specific classification) and control cases, the pathway analysis of altered proteins in the DG involved in mitochondrial dysfunction showed the most enrichment. Interestingly, by analyzing the gene expression profiles of dentate granule cells from surgically resected hippocampal specimens from patients with MTLE with and without HS, Griffin et al ( 19 ) demonstrated that differentially expressed genes were associated with the oxidative phosphorylation pathway, which is generally consistent with our findings.…”