Proteomic analysis of human epileptic neocortex predicts vascular and glial changes in epileptic regions.

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 These brain pieces are in vivo electrophysiologically characterized human brain samples withdrawn from the neocortex of six patients with refractory epilepsy.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.

Predicting novel histopathological microlesions in human epileptic brain through transcriptional clustering.

Although epilepsy is associated with a variety of abnormalities, exactly why some brain regions produce seizures and others do not is not known. We developed a method to identify cellular changes in human epileptic neocortex using transcriptional clustering. A paired analysis of high and low spiking tissues recorded in vivo from 15 patients predicted 11 cell-specific changes together with their 'cellular interactome'. These predictions were validated histologically revealing millimetre-sized 'microlesions' together with a global increase in vascularity and microglia. Microlesions were easily identified in deeper cortical layers using the neuronal marker NeuN, showed a marked reduction in neuronal processes, and were associated with nearby activation of MAPK/CREB signalling, a marker of epileptic activity, in superficial layers. Microlesions constitute a common, undiscovered layer-specific abnormality of neuronal connectivity in human neocortex that may be responsible for many 'non-lesional' forms of epilepsy. The transcriptional clustering approach used here could be applied more broadly to predict cellular differences in other brain and complex tissue disorders.

Identification of C-terminal phosphorylation sites of N-formyl peptide receptor-1 (FPR1) in human blood neutrophils.

Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 Mr) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu(312)-Arg(322) and Arg(323)-Lys(350)) and extracellular FPR1 peptide (Ile(191)-Arg(201)) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala(323)-Lys(350) only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr(325), Ser(328), Thr(329), Thr(331), Ser(332), Thr(334), and Thr(339). No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC50 = 33 ± 8 to 74 ± 21 nM. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of FPR1 phosphorylation can be monitored with C-terminal tail FPR1-phosphospecific antibodies.