Role of Altered Expression, Activity and Sub-cellular Distribution of Various Histone Deacetylases (HDACs) in Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis.

Histone deacetylases (HDACs) have been described to have both neurotoxic and neuroprotective roles, and partly, depend on its sub-cellular distribution. HDAC inhibitors have a long history of use in the treatment of various neurological disorders including epilepsy. Key role of HDACs in GABAergic neurotransmission, synaptogenesis, synaptic plasticity and memory formation was demonstrated whereas very less is known about their role in drug-resistant epilepsy pathologies. The present study was aimed to investigate the changes in the expression of HDACs, activity and its sub-cellular distribution in mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) patients. For this study, surgically resected hippocampal tissue specimens of 28 MTLE-HS patients and 20 hippocampus from post-mortem cases were obtained. Real-time PCR was done to analyse the mRNA expression. HDAC activity and the protein levels of HDACs in cytoplasm as well as nucleus were measured spectrophotometrically. Further, sub-cellular localization of HDACs was characterized by immunofluorescence. Significant upregulation of HDAC1, HDAC2, HDAC4, HDAC5, HDAC6, HDAC10 and HDAC11 mRNA were observed in MTLE-HS. Alterations in the mRNA expression of glutamate and gamma-aminobutyric acid (GABA) receptor subunits have been also demonstrated. We observed significant increase of HDAC activity and nuclear level of HDAC1, HDAC2, HDAC5 and HDAC11 in the hippocampal samples obtained from patients with MTLE-HS. Moreover, we found altered cytoplasmic level of HDAC4, HDAC6 and HDAC10 in the hippocampal sample obtained from patients with MTLE-HS. Alterations in the level of HDACs could potentially be part of a dynamic transcription regulation associated with MTLE-HS. Changes in cytoplasmic level of HDAC4, 6 and 10 suggest that cytoplasmic substrates may play a crucial role in the pathophysiology of MTLE-HS. Knowledge regarding expression pattern and sub-cellular distribution of HDACs may help to devise specific HDACi therapy for epilepsy.

Altered expression of activating transcription factor 3 in the hippocampus of patients with mesial temporal lobe epilepsy-hippocampal sclerosis (MTLE-HS).

Aim of the study: Activating Transforming factor 3 (ATF3) is a stress induced gene and closely associated with neuro-inflammation while Transforming growth Factor Beta (TGFß) signalling is also reported to be involved in neuro-inflammation and hyper-excitability associated with drug resistant epilepsy. Animal model studies indicate the involvement of ATF3 and TGFß receptors to promote epileptogenesis. Human studies also show that TGFß signalling is activated in MTLE-HS. However, lack of studies on ATF3 and TGFßRI expression in MTLE-HS patients exists. We hypothesize that ATF3 and TGFßRI might be expressed in hippocampi of patients with MTLE-HS and playing role in epileptogenesis.Materials & methods: Protein expression of ATF3 and TGFßRI was performed by western blotting. Localisation of ATF3 was performed by immunohistochemistry and immunoflorescence.Results: Protein expression of ATF3 and TGFßRI was significantly up-regulated in hippocampi of patients as compared to controls. Also ATF3 IR was significantly expressed in hippocampi of patients and ATF3 was expressed predominantly in cytoplasm as compared to nucleus. No correlation was found between ATF3 expression and epilepsy duration and seizure frequency.Conclusions: ATF3 and TGFßRI are both important players in neuro-inflammation and might potentiate epileptogenesis in these patients.

α7 nicotinic receptors contributes to glutamatergic activity in the hippocampus of patients with mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS).

Hyperglutamatergic activity in the hippocampus is a major feature of patients with mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS). Here we investigated whether tonic α7 nicotinic receptor (nAChR) activity could contribute to enhanced glutamatergic activity in the hippocampus of patients with MTLE-HS. Results showed that frequency and amplitude of glutamatergic events recorded from pyramidal neurons in the hippocampal samples obtained from patients with MTLE-HS were altered by α7 nAChR antagonist, methyllycaconitine, suggesting α7 nAChRs may influence hyperexcitability in MTLE-HS.

Synaptic roles of cyclin-dependent kinase 5 & its implications in epilepsy.

There is an urgent need to understand the molecular mechanisms underlying epilepsy to find novel prognostic/diagnostic biomarkers to prevent epilepsy patients at risk. Cyclin-dependent kinase 5 (CDK5) is involved in multiple neuronal functions and plays a crucial role in maintaining homeostatic synaptic plasticity by regulating intracellular signalling cascades at synapses. CDK5 deregulation is shown to be associated with various neurodegenerative diseases such as Alzheimer's disease. The association between chronic loss of CDK5 and seizures has been reported in animal models of epilepsy. Genetic expression of CDK5 at transcriptome level has been shown to be abnormal in intractable epilepsy. In this review various possible mechanisms by which deregulated CDK5 may alter synaptic transmission and possibly lead to epileptogenesis have been discussed. Further, CDK5 has been proposed as a potential biomarker as well as a pharmacological target for developing treatments for epilepsy.

Viral nanoparticle-encapsidated enzyme and restructured DNA for cell delivery and gene expression.

Packaging specific exogenous active proteins and DNAs together within a single viral-nanocontainer is challenging. The bacteriophage T4 capsid (100 × 70 nm) is well suited for this purpose, because it can hold a single long DNA or multiple short pieces of DNA up to 170 kb packed together with more than 1,000 protein molecules. Any linear DNA can be packaged in vitro into purified procapsids. The capsid-targeting sequence (CTS) directs virtually any protein into the procapsid. Procapsids are assembled with specific CTS-directed exogenous proteins that are encapsidated before the DNA. The capsid also can display on its surface high-affinity eukaryotic cell-binding peptides or proteins that are in fusion with small outer capsid and head outer capsid surface-decoration proteins that can be added in vivo or in vitro. In this study, we demonstrate that the site-specific recombinase cyclic recombination (Cre) targeted into the procapsid is enzymatically active within the procapsid and recircularizes linear plasmid DNA containing two terminal loxP recognition sites when packaged in vitro. mCherry expression driven by a cytomegalovirus promoter in the capsid containing Cre-circularized DNA is enhanced over linear DNA, as shown in recipient eukaryotic cells. The efficient and specific packaging into capsids and the unpackaging of both DNA and protein with release of the enzymatically altered protein-DNA complexes from the nanoparticles into cells have potential in numerous downstream drug and gene therapeutic applications.

RNA-seq analysis of hippocampal tissues reveals novel candidate genes for drug refractory epilepsy in patients with MTLE-HS.

Array-based profiling studies have shown implication of aberrant gene expression patterns in epileptogenesis. We have performed transcriptome analysis of hippocampal tissues resected from patients with MTLE-HS using RNAseq approach. Healthy tissues from tumour margins obtained during tumour surgeries were used as non-epileptic controls. RNA sequencing was performed using standard protocols on Illumina HiSeq 2500 platform. Differential gene expression analysis of the RNAseq data revealed 56 significantly regulated genes in MTLE patients. Gene cluster analysis identified 3 important hubs of genes mostly linked to, neuroinflammation and innate immunity, synaptic transmission and neuronal network modulation which are supportive of intrinsic severity hypothesis of pharmacoresistance. This study identified various genes like FN1 which is central in our analysis, NEUROD6, RELN, TGFßR2, NLRP1, SCRT1, CSNK2B, SCN1B, CABP1, KIF5A and antisense RNAs like AQP4-AS1 and KIRREL3-AS2 providing important insight into the understanding of the pathophysiology or genomic basis of drug refractory epilepsy due to MTS.

Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation.

Viral genome packaging into capsids is powered by high-force-generating motor proteins. In the presence of all packaging components, ATP-powered translocation in vitro expels all detectable tightly bound YOYO-1 dye from packaged short dsDNA substrates and removes all aminoacridine dye from packaged genomic DNA in vivo. In contrast, in the absence of packaging, the purified T4 packaging ATPase alone can only remove up to ∼1/3 of DNA-bound intercalating YOYO-1 dye molecules in the presence of ATP or ATP-γ-S. In sufficient concentration, intercalating dyes arrest packaging, but rare terminase mutations confer resistance. These distant mutations are highly interdependent in acquiring function and resistance and likely mark motor contact points with the translocating DNA. In stalled Y-DNAs, FRET has shown a decrease in distance from the phage T4 terminase C terminus to portal consistent with a linear motor, and in the Y-stem DNA compression between closely positioned dye pairs. Taken together with prior FRET studies of conformational changes in stalled Y-DNAs, removal of intercalating compounds by the packaging motor demonstrates conformational change in DNA during normal translocation at low packaging resistance and supports a proposed linear "DNA crunching" or torsional compression motor mechanism involving a transient grip-and-release structural change in B form DNA.

Presurgical epileptogenic network analysis: A way to enhance epilepsy surgery outcome.

Accurate localization of the "epileptogenic zone (EZ)" is an important issue in epilepsy surgery. The EZ is not discrete and focal; in fact, the epileptogenic networks can spread ictal activity to different regions of the brain. Changes in network characteristics and functional connectivity are shown to be associated with epilepsy. Seizures are thought to represent a hyper-synchronous state and presumable changes in synchronization between different brain regions underlie the mechanisms of seizure spread. Although presurgical evaluation of the epileptogenic network analysis can be carried out using existing investigative techniques like electroencephalogram (EEG), video-EEG, magnetic resonance imaging, single-photon emission computed tomography, and magnetoencephalography, advanced imaging techniques such as optical intrinsic spectroscopy, auto-fluorescence imaging, voltage sensitive dye imaging, and calcium imaging have the advantage of better spatiotemporal resolution over a large area of cortex. Understanding the wide-scale dynamic networks by analyzing the changes in the synchronization patterns using advanced imaging techniques will be instrumental in the presurgical analysis of the epileptogenic network and better localization of the EZs in the future.

Quantification of Neuronal Dendritic Spine Density and Lengths of Apical and Basal Dendrites in Temporal Lobe Structures Using Golgi-Cox Staining.

The objective of this chapter is to provide an overview of the methods used to investigate the connectivity and structure of the nervous system. These methods allow neuronal cells to be categorized according to their location, shape, and connections to other cells. The Golgi-Cox staining gives a thorough picture of all significant neuronal structures found in the brain that may be distinguished from one another. The most significant characteristic is its three-dimensional integrity since all neuronal structures may be followed continuously from one part to the next. Successions of sections of the brain's neurons are seen with the Golgi stain. The Golgi method is used to serially segment chosen brain parts, and the resulting neurons are produced from those sections.

Quantification of Neuroinflammatory Markers in Blood, Cerebrospinal Fluid, and Resected Brain Samples Obtained from Patients.

Cytokines have the potential to be the ideal biomarkers to track the onset and progression of immune-mediated diseases, study the development of novel therapeutic strategies, and they can serve as outcome parameters due to their crucial role in the regulation of immune and inflammatory responses. It is vital to keep track of the entire cytokine spectrum due to the complex interactions, pleiotropic effects, and redundancy in the cytokine network. The multiplex immunoassay (MIA) is, therefore, the best method for achieving that goal. This chapter addresses the key methodological processes of this technique, such as sample preparation, antibody coupling to beads, and assay procedure.

Role of non-receptor tyrosine kinases in epilepsy: significance and potential as therapeutic targets.

INTRODUCTION: Epilepsy is a chronic neurological condition characterized by a persistent propensity for seizure generation. About one-third of patients do not achieve seizure control with the first-line treatment options, which include >20 antiseizure medications. It is therefore imperative that new medications with novel targets and mechanisms of action are developed. AREAS COVERED: Clinical studies and preclinical research increasingly implicate Non-receptor tyrosine kinases (nRTKs) in the pathogenesis of epilepsy. To date, several nRTK members have been linked to processes relevant to the development of epilepsy. Therefore, in this review, we provide insight into the molecular mechanisms by which the various nRTK subfamilies can contribute to the pathogenesis of epilepsy. We further highlight the prospective use of specific nRTK inhibitors in the treatment of epilepsy deriving evidence from existing literature providing a rationale for their use as therapeutic targets. EXPERT OPINION: Specific small-molecule inhibitors of NRTKs can be employed for the targeted therapy as already seen in other diseases by examining the precise molecular pathways regulated by them contributing to the development of epilepsy. However, the evidence supporting NRTKs as therapeutic targets are limiting in nature thus, necessitating more research to fully comprehend their function in the development and propagation of seizures.

The multifaceted role of Wnt canonical signalling in neurogenesis, neuroinflammation, and hyperexcitability in mesial temporal lobe epilepsy.

Epilepsy is a neurological disorder characterised by unprovoked, repetitive seizures caused by abnormal neuronal firing. The Wnt/ß-Catenin signalling pathway is involved in seizure-induced neurogenesis, aberrant neurogenesis, neuroinflammation, and hyperexcitability associated with epileptic disorder. Wnt/ß-Catenin signalling is crucial for early brain development processes including neuronal patterning, synapse formation, and N-methyl-d-aspartate receptor (NMDAR) regulation. Disruption of molecular networks such as Wnt/ß-catenin signalling in epilepsy could offer encouraging anti-epileptogenic targets. So, with a better understanding of the canonical Wnt/-Catenin pathway, we highlight in this review the important elements of Wnt/-Catenin signalling specifically in Mesial Temporal Lobe Epilepsy (MTLE) for potential therapeutic targets.

Integrated Proteomics and Protein Co-expression Network Analysis Identifies Novel Epileptogenic Mechanism in Mesial Temporal Lobe Epilepsy.

Over 50 million people worldwide are affected by epilepsy, a common neurological disorder that has a high rate of drug resistance and diverse comorbidities such as progressive cognitive and behavioural disorders, and increased mortality from direct or indirect effects of seizures and therapies. Despite extensive research with animal models and human studies, limited insights have been gained into the mechanisms underlying seizures and epileptogenesis, which has not translated into significant reductions in drug resistance, morbidities, or mortality. To better understand the molecular signaling networks associated with seizures in MTLE patients, we analyzed the proteome of brain samples from MTLE and control cases using an integrated approach that combines mass spectrometry-based quantitative proteomics, differential expression analysis, and co-expression network analysis. Our analyses of 20 human brain tissues from MTLE patients and 20 controls showed the organization of the brain proteome into a network of 9 biologically meaningful modules of co-expressed proteins. Of these, 6 modules are positively or negatively correlated to MTLE phenotypes with hub proteins that are altered in MTLE patients. Our study is the first to employ an integrated approach of proteomics and protein co-expression network analysis to study patients with MTLE. Our findings reveal a molecular blueprint of altered protein networks in MTLE brain and highlight dysregulated pathways and processes including altered cargo transport, neurotransmitter release from synaptic vesicles, synaptic plasticity, proteostasis, RNA homeostasis, ion transport and transmembrane transport, cytoskeleton disorganization, metabolic and mitochondrial dysfunction, blood micro-particle function, extracellular matrix organization, immune response, neuroinflammation, and cell signaling. These insights into MTLE pathogenesis suggest potential new candidates for future diagnostic and therapeutic development.

Cold atmospheric plasma (CAP) treatment increased reactive oxygen and nitrogen species (RONS) levels in tumor samples obtained from patients with low-grade glioma.

Low-grade gliomas (LGGs) are a heterogeneous group of tumors with an average 10-year survival rate of 40%-55%. Current treatment options include chemotherapy, radiotherapy, and gross total resection (GTR) of the tumor. The extent of resection (EOR) plays an important role in improving surgical outcomes. However, the major obstacle in treating low-grade gliomas is their diffused nature and the presence of residual cancer cells at the tumor margins post resection. Cold Atmospheric Plasma (CAP) has shown to be effective in targeted killing of tumor cells in various glioma cell lines without affecting non-tumor cells through Reactive Oxygen and Nitrogen Species (RONS). However, no study on the effectiveness of CAP has been carried out in LGG tissues till date. In this study, we applied helium-based CAP on tumor tissues resected from LGG patients. Our results show that CAP is effective in promoting RONS accumulation in LGG tissues when CAP jet parameters are set at 4 kV voltage, 5 min treatment time and 3 lpm gas flow rate. We also observed that CAP jet is more effective in thinner slice preparations of tumor as compared to thick tumor samples. Our results indicate that CAP could prove to be an effective adjunct therapy in glioma surgery to target residual cancer cells to improve surgical outcome of patients with low-grade glioma.

Epigenetics of neurological diseases.

Higher-order DNA structure and gene expression are governed by epigenetic processes like DNA methylation and histone modifications. Abnormal epigenetic mechanisms are known to contribute to the emergence of numerous diseases, including cancer. Historically, the chromatin abnormalities were only considered to be limited to discrete DNA sequences and were thought to be associated with rare genetic syndrome however, recent discoveries have pointed to genome-wide level changes in the epigenetic machinery which has contributed to a better knowledge of the mechanisms underlying developmental and degenerative neuronal problems associated with diseases such as Parkinson's disease, Huntington's disease, Epilepsy, Multiple sclerosis, etc. In the given chapter we describe the epigenetic alterations seen in various neurological disorders and further discuss the influence of these epigenetic changes on developing novel therapies.

Epigenetics in epilepsy.

Epilepsy affects over 50 million individuals globally, making it the most prevalent chronic and serious neurological condition. A precise therapeutic strategy is complicated by poor understanding of the pathological changes in epilepsy thus, 30% of TLE patients are resistant to drug therapy. In the brain, epigenetic processes translate information from transient cellular impulses and adjustments in neuronal activity into long-lasting impacts on gene expression. Research suggests that epigenetic processes can be manipulated in the future to treat or prevent epilepsy as epigenetics has been shown to have a profound influence on how genes are expressed in epilepsy. As well as being potential biomarkers for epilepsy diagnosis, epigenetic changes can also be used as prognostic indicators of treatment response. In this chapter, we review the most recent findings in several molecular pathways linked with the pathogenesis of TLE that are controlled by epigenetic mechanisms highlighting their potential utility as biomarkers for upcoming treatment strategies.

RNA Sequencing of Intraoperative Peritumoral Tissues Reveals Potential Pathways Involved in Glioma-Related Seizures.

Tumor-induced changes in the peritumoral neocortex play a crucial role in generation of seizures. This study aimed to investigate the molecular mechanisms potentially involved in peritumoral epilepsy in low-grade gliomas (LGGs). Intraoperative peritumoral brain tissues resected from LGG patients with seizures (pGRS) or without seizures (pGNS) were used for RNA sequencing (RNA-seq). Comparative transcriptomics was performed to identify differentially expressed genes (DEGs) in pGRS compared to pGNS using deseq2 and edgeR packages (R). Gene set enrichment analysis (GSEA) using Gene Ontology terms and Kyoto Encyclopedia of Genes & Genomes (KEGG) pathways was performed using the clusterProfiler package (R). The expression of key genes was validated at the transcript and protein levels in the peritumoral region using real-time PCR and immunohistochemistry, respectively. A total of 1073 DEGs were identified in pGRS compared to pGNS, of which 559 genes were upregulated and 514 genes were downregulated (log2 fold-change ≥ 2, padj < 0.001). The DEGs in pGRS were highly enriched in the "Glutamatergic Synapse" and "Spliceosome" pathways, with increased expression of GRIN2A (NR2A), GRIN2B (NR2B), GRIA1 (GLUR1), GRIA3 (GLUR3), GRM5, CACNA1C, CACNA1A, and ITPR2. Moreover, increased immunoreactivity was observed for NR2A, NR2B, and GLUR1 proteins in the peritumoral tissues of GRS. These findings suggest that altered glutamatergic signaling and perturbed Ca2+ homeostasis may be potential causes of peritumoral epilepsy in gliomas. This explorative study identifies important genes/pathways that merit further characterization for their potential involvement in glioma-related seizures.

Dendritic reorganization in the hippocampus, anterior temporal lobe, and frontal neocortex of lithium-pilocarpine induced Status Epilepticus (SE).

Status Epilepticus (SE) is a distributed network disorder, which involves the hippocampus and extra-hippocampal structures. Epileptogenesis in SE is tightly associated with neurogenesis, plastic changes and neural network reorganization facilitating hyper-excitability. On the other hand, dendritic spines are known to be the excitatory synapse in the brain. Therefore, dendritic spine dynamics could play an intricate role in these network alterations. However, the exact reason behind these structural changes in SE are elusive. In the present study, we have investigated the aforementioned hypothesis in the lithium-pilocarpine treated rat model of SE. We have examined cytoarchitectural and morphological changes using hematoxylin-eosin and Golgi-Cox staining in three different brain regions viz. CA1 pyramidal layer of the dorsal hippocampus, layer V pyramidal neurons of anterior temporal lobe (ATL), and frontal neocortex of the same animals. We observed macrostructural and layer-wise alteration of the pyramidal layer mainly in the hippocampus and ATL of SE rats, which is associated with sclerosis in the hippocampus. Sholl analysis exhibited partial dendritic plasticity in apical and basal dendrites of pyramidal cells as compared to the saline-treated weight-/age-matched control group. These findings indicate that region-specific alterations in dendritogenesis may contribute to the development of independent epileptogenic networks in the hippocampus, ATL, and frontal neocortex of SE rats.

Differential Levels of Tryptophan-Kynurenine Pathway Metabolites in the Hippocampus, Anterior Temporal Lobe, and Neocortex in an Animal Model of Temporal Lobe Epilepsy.

Glutamate-receptor-mediated hyperexcitability contributes to seizure generation in temporal lobe epilepsy (TLE). Tryptophan-kynurenine pathway (TKP) metabolites regulate glutamate receptor activity under physiological conditions. This study was designed to investigate alterations in the levels of TKP metabolites and the differential regulation of glutamatergic activity by TKP metabolites in the hippocampus, anterior temporal lobe (ATL), and neocortex samples of a lithium-pilocarpine rat model of TLE. We observed that levels of tryptophan were reduced in the hippocampus and ATL samples but unaltered in the neocortex samples. The levels of kynurenic acid were reduced in the hippocampus samples and unaltered in the ATL and neocortex samples of the TLE rats. The levels of kynurenine were unaltered in all three regions of the TLE rats. The magnitude of reduction in these metabolites in all regions was unaltered in the TLE rats. The frequency and amplitude of spontaneous excitatory postsynaptic currents were enhanced in hippocampus ATL samples but not in the neocortex samples of the TLE rats. The exogenous application of kynurenic acid inhibited glutamatergic activity in the slice preparations of all these regions in both the control and the TLE rats. However, the magnitude of reduction in the frequency of kynurenic acid was higher in the hippocampus (18.44 ± 2.6% in control vs. 30.02 ± 1.5 in TLE rats) and ATL (16.31 ± 0.91% in control vs. 29.82 ± 3.08% in TLE rats) samples of the TLE rats. These findings suggest the differential regulation of glutamatergic activity by TKP metabolites in the hippocampus, ATL, and neocortex of TLE rats.

Differential glutamate receptor expression and function in the hippocampus, anterior temporal lobe and neocortex in a pilocarpine model of temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most common form of intractable epilepsy where hyperactive glutamate receptors may contribute to the complex epileptogenic network hubs distributed among different regions. This study was designed to investigate the region-specific molecular alterations of the glutamate receptors and associated excitatory synaptic transmission in pilocarpine rat model of TLE. We recorded spontaneous excitatory postsynaptic currents (EPSCs) from pyramidal neurons in resected rat brain slices of the hippocampus, anterior temporal lobe (ATL) and neocortex. We also performed mRNA and protein expression of the glutamate receptor subunits (NR1, NR2A, NR2B, and GLUR1-4) by qPCR and immunohistochemistry. We observed significant increase in the frequency and amplitude of spontaneous EPSCs in the hippocampal and ATL samples of TLE rats than in control rats. Additionally, the magnitude of the frequency and amplitude was increased in ATL samples compared to that of the hippocampal samples of TLE rats. The mRNA level of NR1 was upregulated in both the hippocampal as well as ATL samples and that of NR2A, NR2B were upregulated only in the hippocampal samples of TLE rats than in control rats. The mRNA level of GLUR4 was upregulated in both the hippocampal as well as ATL samples of TLE rats than in control rats. Immunohistochemical analysis demonstrated that the number of NR1, NR2A, NR2B, and GLUR4 immuno-positive cells were significantly higher in the hippocampal samples whereas number of NR1 and GLUR4 immuno-positive cells were significantly higher in the ATL samples of the TLE rats than in control rats. This study demonstrated the region-specific alterations of glutamate receptor subunits in pilocarpine model of TLE, suggesting possible cellular mechanisms contributing to generation of independent epileptogenic networks in different temporal lobe structures.