Studies of biomarkers in temporal lobe epilepsy.

Refractory temporal lobe epilepsy associated with hippocampal sclerosis (TLE-HS) affects about 30% of TLE patients, where antiepileptic drugs are not effective in controlling seizures. These patients become candidates for surgical treatment which is effective in only 60-70% of cases. In addition, surgical treatment causes memory and cognitive impairments as well as psychopathological disturbance. Therefore, the aim of this study is to investigate potential biomarkers in surgically resected sclerotic TLE-HS (n = 49) and non-spiking superior temporal gyrus samples (TLE-STG; n = 25) from TLE patients and post-mortem hippocampi (PMC; n = 10), in order to increase our understanding of refractory TLE pathophysiology and help in identifying new potential drug targets for treatment of TLE patients. GABA b receptor subunits were investigated in TLE-HS, TLE-STG and PMC tissue by quantitative real time polymerase chain reaction (qRT-PCR) and quantitative western blot (WB) techniques. Alterations in expressions of SGK1, SCN4B, IP3R1 and SYNPR were investigated in TLE-HS, TLE-STG and PMC specimens by qRT-PCR and WB. The transcriptome profiling of TLE-HS, TLE-STG and PMC samples was done by microarray analysis (MA). MA was followed by functional annotation clustering analysis (FAC) of the MA differentially expressed genes (DEGs). Genes from FAC analysis were further investigated by qRT-PCR. MA Aquaporin (AQP1, 3, 4, 5, 8, 9, 11) expressions were further validated by qRT-PCR. Expression of the inhibitory GABA b2 receptor subunit was significantly up regulated in TLE-HS compared to PMC but its expression was reduced in TLE-HS compared to TLE-STG.The expression of SCN4B, IP3R1 and SYNPR, which are involved in regulating neuronal excitability, were significantly reduced in TLE-HS compared to TLE-STG and were significantly increased compared to PMC. The expression of SGK1 mRNA was significantly increased in TLE-HS compared to both TLE-STG and PMC. MA analysis revealed 1821 genes were significantly up regulated and 1511 genes were significantly down regulated in TLE-HS compared to TLE-STG and PMC. The first cluster from FAC analysis of DEGs showed that the up regulated inflammatory genes such as cytokines had the highest enrichment score. The qRT-PCR data showed that expression of IL-1B, IL-18, Fas, ICAM-1, CCL2, CCL4, CXCL1, CXCL2, CXCL12, CXCR4 and CX3CR1 were significantly higher in TLE-HS compared to TLE-STG and PMC therefore validating MA data. AQP1 and -4, which are involved in water homeostasis, were significantly up regulated in TLE-HS compared to TLE-STG. AQP11 expression was significantly reduced in TLE-HS while AQP3, -5, -8 and -9 were not significantly altered in TLE-HS compared to TLE-STG and PMC. The significant dysregulation of biomarker expression investigated in this study indicate that different biological processes such as neuronal excitability, neuronal and astrocytic energy metabolism, neurogenesis, apoptosis, neuroinflammation, intracellular calcium and water homeostasis are affected in the epileptogenic TLE-HS tissue. These biomarkers seem to be associated with TLE-HS pathophysiology. Furthermore, they highlight the role of neuronal, astrocytic, microglia and endothelial cell dysfunction in TLE-HS pathology. In conclusion, the biomarkers investigated increased our understanding of biological processes affected in TLE-HS pathophysiology and they represent potential drug targets for refractory TLE-HS. However, further research is still needed to understand the temporal and spatial changes of those genes and their proteins during TLE

The Sodium Channel B4-Subunits are Dysregulated in Temporal Lobe Epilepsy Drug-Resistant Patients

Temporal lobe epilepsy (TLE) is the most common type of partial epilepsy referred for surgery due to antiepileptic drug (AED) resistance. A common molecular target for many of these drugs is the voltage-gated sodium channel (VGSC). The VGSC consists of four domains of pore-forming α-subunits and two auxiliary β-subunits, several of which have been well studied in epileptic conditions. However, despite the β4-subunits’ role having been reported in some neurological conditions, there is little research investigating its potential significance in epilepsy. Therefore, the purpose of this work was to assess the role of SCN4β in epilepsy by using a combination of molecular and bioinformatics approaches. We first demonstrated that there was a reduction in the relative expression of SCN4B in the drug-resistant TLE patients compared to non-epileptic control specimens, both at the mRNA and protein levels. By analyzing a co-expression network in the neighborhood of SCN4B we then discovered a linkage between the expression of this gene and K+ channels activated by Ca2+, or K+ two-pore domain channels. Our approach also inferred several potential effector functions linked to variation in the expression of SCN4B. These observations support the hypothesis that SCN4B is a key factor in AED-resistant TLE, which could help direct both the drug selection of TLE treatments and the development of future AED

Transcriptome analysis suggests a role for the differential expression of cerebral aquaporins and the MAPK signalling pathway in human temporal lobe epilepsy

Epilepsies are common disorders of the central nervous system (CNS), affecting up to 2% of the global population. Pharmaco-resistance is a major clinical challenge affecting about 30% of temporal lobe epilepsy (TLE) patients. Water homeostasis has been shown crucial for regulation of neuronal excitability. The control of water movement is achieved through a family of small integral membrane channel proteins called aquaporins (AQPs). Despite the fact that changes in water homeostasis occur in sclerotic hippocampi of people with TLE, the expression of AQPs in the epileptic brain is not fully characterised. This study uses microarray and ELISA methods to analyse the mRNA and protein expression of the human cerebral AQPs in sclerotic hippocampi (TLE-HS) and adjacent neocortex tissue (TLE-NC) of TLE patients. The expression of AQP1 and AQP4 transcripts was significantly increased, while that of the AQP9 transcript was significantly reduced in TLE-HS compared to TLE-NC. AQP4 protein expression was also increased while expression of AQP1 protein remained unchanged, and AQP9 was undetected. Microarray data analysis identified 3,333 differentially regulated genes and suggested the involvement of the MAPK signalling pathway in TLE pathogenesis. Proteome array data validated the translational profile for 26 genes and within the MAPK pathway (e.g. p38, JNK) that were identified as differentially expressed from microarray analysis. ELISA data showed that p38 and JNK inhibitors decrease AQP4 protein levels in cultured human primary cortical astrocytes. Elucidating the mechanism of selective regulation of different AQPs and associated regulatory proteins may provide a new therapeutic approach to epilepsy treatment. This article is protected by copyright. All rights reserved

Quantitative expression and localization of GABAB receptor protein subunits in hippocampi from patients with refractory temporal lobe epilepsy

This study investigates GABAB protein expression and mRNA levels in three types of specimens. Two types of specimens from patients with temporal lobe epilepsy (TLE), secondary to hippocampal sclerosis, sclerotic hippocampal samples (TLE-HS), and tissue from the structurally preserved non-spiking ipsilateral superior temporal gyrus (TLE-STG) removed from the same patient during epilepsy surgery; and third specimen is hippocampal tissue from individuals with no history of epilepsy (post-mortem controls, PMC). mRNA expression of GABAB subunits was quantified in TLE-HS, TLE-STG and PMC specimens by qRT-PCR. Qualitative and quantitative Western blot (WB) and immunohistochemistry techniques were employed to quantify and localize GABAB proteins subunits. qRT-PCR data demonstrated an overall decrease of both GABAB1 isoforms in TLE-HS compared to TLE-STG. These results were mirrored by the WB findings. GABAB2 mRNA and protein were significantly reduced in TLE-HS samples compared to TLE-STG; however they appeared to be upregulated in TLE-HS compared to the PMC samples. Immunohistochemistry (IHC) showed that GABAB proteins were widely distributed in PMC and TLE-HS hippocampal sections with regional differences in the intensity of the signal. The higher expression of mature GABAB protein in TLE-HS than PMC is in agreement with previous studies. However, these findings could be due to post-mortem changes in PMC specimens. The TLE-STG samples examined here represent a better 'control' tissue compared to TLE-HS samples characterized by lower than expected GABAB expression. This interpretation provides a better explanation for previous functional studies suggesting reduced inhibition in TLE-HS tissue due to attenuated GABAB currents. [Abstract copyright: Copyright © 2017. Published by Elsevier Ltd.

The Sodium Channel B4-Subunits are Dysregulated in Temporal Lobe Epilepsy Drug-Resistant Patients

Temporal lobe epilepsy (TLE) is the most common type of partial epilepsy referred for surgery due to antiepileptic drug (AED) resistance. A common molecular target for many of these drugs is the voltage-gated sodium channel (VGSC). The VGSC consists of four domains of pore-forming α-subunits and two auxiliary β-subunits, several of which have been well studied in epileptic conditions. However, despite the β4-subunits’ role having been reported in some neurological conditions, there is little research investigating its potential significance in epilepsy. Therefore, the purpose of this work was to assess the role of SCN4β in epilepsy by using a combination of molecular and bioinformatics approaches. We first demonstrated that there was a reduction in the relative expression of SCN4B in the drug-resistant TLE patients compared to non-epileptic control specimens, both at the mRNA and protein levels. By analyzing a co-expression network in the neighborhood of SCN4B we then discovered a linkage between the expression of this gene and K+ channels activated by Ca2+, or K+ two-pore domain channels. Our approach also inferred several potential effector functions linked to variation in the expression of SCN4B. These observations support the hypothesis that SCN4B is a key factor in AED-resistant TLE, which could help direct both the drug selection of TLE treatments and the development of future AEDs