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Item Proteomic analysis of human epileptic neocortex predicts vascular and glial changes in epileptic regions(2018-04) Keren-Aviram, Gal; Dachet, Fabian; Bagla, Shruti; Balan, Karina; Loeb, Jeffrey A.Epilepsy is a common neurological disorder, which is not well understood at the molecular level. Exactly why some brain regions produce epileptic discharges and others do not is not known. Patients who fail to respond to antiseizure medication (refractory epilepsy) can benefit from surgical removal of brain regions to reduce seizure frequency. The tissue removed in these surgeries offers an invaluable resource to uncover the molecular and cellular basis of human epilepsy. Here, we report a proteomic study to determine whether there are common proteomic patterns in human brain regions that produce epileptic discharges. We analyzed human brain samples, as part of the Systems Biology of Epilepsy Project (SBEP). These brain pieces are in vivo electrophysiologically characterized human brain samples withdrawn from the neocortex of six patients with refractory epilepsy. This study is unique in that for each of these six patients the comparison of protein expression was made within the same patient: a more epileptic region was compared to a less epileptic brain region. The amount of epileptic activity was defined for each patient as the frequency of their interictal spikes (electric activity between seizures that is a parameter strongly linked to epilepsy). Proteins were resolved from three subcellular fractions, using a 2D differential gel electrophoresis (2D-DIGE), revealing 31 identified protein spots that changed significantly. Interestingly, glial fibrillary acidic protein (GFAP) was found to be consistently down regulated in high spiking brain tissue and showed a strong negative correlation with spike frequency. We also developed a two-step analysis method to select for protein species that changed frequently among the patients and identified these proteins. A total of 397 protein spots of interest (SOI) were clustered by protein expression patterns across all samples. These clusters were used as markers and this analysis predicted proteomic changes due to both histological differences and molecular pathways, revealed by examination of gene ontology clusters. Our experimental design and proteomic data analysis predicts novel glial changes, increased angiogenesis, and changes in cytoskeleton and neuronal projections between high and low interictal spiking regions. Quantitative histological staining of these same tissues for both the vascular and glial changes confirmed these findings, which provide new insights into the structural and functional basis of neocortical epilepsy.Item Toward resolving the human neocortex epileptic proteome(Montana State University - Bozeman, College of Letters & Science, 2013) Keren-Aviram, Gal; Chairperson, Graduate Committee: Edward DratzEpilepsy is a common and often devastating neurological disorder, which is not well understood at the molecular level. Exactly why some brain regions produce epileptic discharges and others do not is not known. Patients who fail to respond to antiseizure medication can benefit from surgical removal of brain regions that produce epileptic activities. The tissue removed in these surgeries offers an invaluable resource to uncover the molecular and cellular basis of human epilepsy. Here, we report a proteomic study, as part of a Systems Biology of Epilepsy Project, which utilizes in vivo electrophysiologically-characterized human brain samples from the neocortex of 6 patients with refractory epilepsy, to determine whether there are common proteomic patterns in human brain regions that produce epileptic discharges. This study is unique in that comparison of protein expression was made within same patient, between nearby epileptic and non-epileptic (or less epileptic) brain regions, as defined by their interictal (between seizure) spike frequencies. Protein spots were resolved from three subcellular fractions, using two-dimensional differential-in-gel-electrophoresis, revealing 31 spots that changed significantly and were identified by liquid-chromatography tandem mass-spectrometry. Interestingly, glial fibrillary acidic protein was found to be consistently down regulated in high spiking brain tissue and glial fibrillary acidic protein levels showed strong negative correlation with spiking frequency. We next developed a two-step analysis method to select for frequently changing spots among the patients and identified 397 of those proteins. Spots of interest were clustered by protein expression patterns across all samples. This analysis predicted proteomic changes due to both histological differences and molecular pathways by examination of gene ontology clusters. Our experimental design and proteomic data analysis predicts novel glial and vascular changes and changes in cytoskeleton and neuronal projections that provide new insights into the structural and functional basis of neocortical epilepsy.Item Human Brain Proteomics in the Systems Biology of Epilepsy Project (SBEP)(2013-03) Keren-Aviram, Gal; Dratz, EdwardEpilepsy is a neurological disorder that manifests as recurrent seizures. Many cases are resistant to antiepileptic drugs and may benefit from surgical procedures to identify and remove the epileptic foci. The project takes advantage of unique human surgical specimens, removed from electrophysiologically mapped brains to compare electrically active brain to adjacent quieter normal regions of the same individuals. We present a pilot proteomic study and initial integration with the project’s database of clinical, histological, genomic and metabolomic information. We used Differential in Gel Electrophoresis (DiGE) to compare protein abundances in three fractionated cellular compartments of six patients. About 4400 protein isoform spots were resolved for each patient and the identities of a subset of 400 significantly changing spots was determined by LC-MS/MS. Hierarchical clustering of the spot expression patterns was used to group changing proteins, followed by gene ontology enrichment analysis. Combination of the two analysis tools allowed for enhanced interpretation of the changes in cellular processes taking place in the tissues that trigger seizures. Changes in cell populations and increased vascularity, predicted from the proteome, were validated by histology. Integrating the findings to develop human epilepsy models seeks to deepen understanding of the disorder and suggest new drug targets.