Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disease characterized by recurrent seizures. The pathogenic mechanisms underlying mTLE involve defects in post-transcriptional regulation of gene expression. So far, transcriptome profiles from epileptic tissue have been generated using whole cells, thereby lacking information on RNA localization and function at a subcellular level. In line with this, we have previously observed by in situ hybridization that a few microRNAs (miRNAs) display subcellular mis-localization with aberrant enrichment in the nucleus in human hippocampal mTLE tissue samples (Kan et al., 2012). To further investigate the possible mechanisms leading to the mis-localization of miRNAs, we set out to understand the compartment-specific total RNA (coding and non-coding) profile of human mTLE tissue samples. For this, we have successfully established a protocol to isolate cytoplasmic and nuclear compartments from human hippocampal tissue. After confirming the purity of the isolated cell compartments, we performed total RNA-sequencing (RNA-seq) on five resected hippocampal (HC) mTLE (no hippocampal sclerosis (non-HS)) samples and five HC postmortem control samples. Similarly, six neo-cortical (Cx) tissue samples from mTLE non-HS and HS International League Against Epilepsy (ILAE) Type 1, or mTLE+HS, samples were compared with six Cx postmortem controls. Our dataset provides a comprehensive overview of compartment-specific transcriptomic profiles of pharmacoresistant mTLE patient HC and Cx tissue, which in further studies can be used to investigate disease mechanisms.

Aims Oxidative stress is evident in resected epileptogenic brain tissue of patients with developmental brain malformations related to mammalian target of rapamycin activation: tuberous sclerosis complex (TSC) and focal cortical dysplasia type IIb (FCD IIb). Whether chronic activation of anti‐oxidant pathways is beneficial or contributes to pathology is not clear. Methods We investigated oxidative stress markers, including haem oxygenase 1, ferritin and the inflammation associated microRNA‐155 in surgically resected epileptogenic brain tissue of TSC ( n = 10) and FCD IIb ( n = 8) patients and in a TSC model ( Tsc1 GFAP−/− mice) using immunohistochemistry, in situ hybridization, real‐time quantitative PCR and immunoblotting. Using human foetal astrocytes we performed an in vitro characterization of the anti‐oxidant response to acute and chronic oxidative stress and evaluated overexpression of the disease‐relevant pro‐inflammatory microRNA‐155. Results Resected TSC or FCD IIb tissue displayed higher expression of oxidative stress markers and microRNA‐155. Tsc1 GFAP−/− mice expressed more microRNA‐155 and haem oxygenase 1 in the brain compared to wild‐type, preceding the typical development of spontaneous seizures in these animals. In vitro , chronic microRNA‐155 overexpression induced haem oxygenase 1, iron regulatory elements and increased susceptibility to oxidative stress. Overexpression of iron regulatory genes was also detected in patients with TSC, FCD IIb and Tsc1 GFAP−/− mice. Conclusion Our results demonstrate that early and sustained activation of anti‐oxidant signalling and dysregulation of iron metabolism are a pathological hallmark of FCD IIb and TSC. Our findings suggest novel therapeutic strategies aimed at controlling the pathological link between both processes.

AIM:Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) control proteolysis within the extracellular matrix (ECM) of the brain. Dysfunction of this enzymatic system due to brain inflammation can disrupt the blood-brain barrier (BBB) and has been implicated in the pathogenesis of epilepsy. However, this has not been extensively studied in the epileptogenic human brain. METHODS:We investigated the expression and cellular localization of major MMPs (MMP2, MMP3, MMP9 and MMP14) and TIMPs (TIMP1, TIMP2, TIMP3 and TIMP4) using quantitative real-time polymerase chain reaction (RT-PCR) and immunohistochemistry in resected epileptogenic brain tissue from patients with tuberous sclerosis complex (TSC), a severe neurodevelopmental disorder characterized by intractable epilepsy and prominent neuroinflammation. Furthermore, we determined whether anti-inflammatory microRNAs, miR146a and miR147b, which can regulate gene expression at the transcriptional level, could attenuate dysregulated MMP and TIMP expression in TSC tuber-derived astroglial cultures. RESULTS:We demonstrated higher mRNA and protein expression of MMPs and TIMPs in TSC tubers compared to control and perituberal brain tissue, particularly in dysmorphic neurons and giant cells, as well as in reactive astrocytes, which was associated with BBB dysfunction. More importantly, IL-1β-induced dysregulation of MMP3, TIMP2, TIMP3 and TIMP4 could be rescued by miR146a and miR147b in tuber-derived TSC cultures. CONCLUSIONS:This study provides evidence of dysregulation of the MMP/TIMP proteolytic system in TSC, which is associated with BBB dysfunction. As dysregulated MMP and TIMP expression can be ameliorated in vitro by miR146a and miR147b, these miRNAs deserve further investigation as a novel therapeutic approach.

BACKGROUND:The proteasome is a multisubunit enzyme complex involved in protein degradation, which is essential for many cellular processes. During inflammation, the constitutive subunits are replaced by their inducible counterparts, resulting in the formation of the immunoproteasome.METHODS:We investigated the expression pattern of constitutive (β1, β5) and immunoproteasome (β1i, β5i) subunits using immunohistochemistry in malformations of cortical development (MCD; focal cortical dysplasia (FCD) IIa and b, cortical tubers from patients with tuberous sclerosis complex (TSC), and mild MCD (mMCD)). Glial cells in culture were used to elucidate the mechanisms regulating immunoproteasome subunit expression.RESULTS:Increased expression was observed in both FCD II and TSC; β1, β1i, β5, and β5i were detected (within cytosol and nucleus) in dysmorphic neurons, balloon/giant cells, and reactive astrocytes. Glial and neuronal nuclear expression positively correlated with seizure frequency. Positive correlation was also observed between the glial expression of constitutive and immunoproteasome subunits and IL-1β. Accordingly, the proteasome subunit expression was modulated by IL-1β in human astrocytes in vitro. Expression of both constitutive and immunoproteasome subunits in FCD II-derived astroglial cultures was negatively regulated by treatment with the immunomodulatory drug rapamycin (inhibitor of the mammalian target of rapamycin (mTOR) pathway, which is activated in both TSC and FCD II).CONCLUSIONS:These observations support the dysregulation of the proteasome system in both FCD and TSC and provide new insights on the mechanism of regulation the (immuno)proteasome in astrocytes and the molecular links between inflammation, mTOR activation, and epilepsy.

Recent evidence supports the activation of mechanisms underlying cellular ageing and neurodegeneration in developmental lesions associated with epilepsy. The present study examined the ongoing cell injury and vulnerability to neuronal degeneration in glioneuronal tumours (GNT).

AIM:Gangliogliomas (GGs) and dysembryoplastic neuroepithelial tumours (DNTs) represent the most common histological entities within the spectrum of glioneuronal tumours (GNTs). The wide variability of morphological features complicates histological classification, including discrimination from prognostically distinct diffuse low-grade astrocytomas (AIIs). This study was performed to increase our understanding of these tumours. METHODS:We studied chromosomal copy number aberrations (CNAs) by genome-wide sequencing in a large cohort of GNTs and linked these to comprehensive histological analysis and clinical characteristics. One hundred fourteen GNTs were studied: 50 GGs and 64 DNTs. Also, a data set of CNAs from 38 diffuse AIIs was included. RESULTS:The most frequent CNAs in both GGs and DNTs were gains at chromosomes 5 and 7, often concurrent, and gain at chromosome 6. None of the CNAs was linked to histological subtype, immunohistochemical features or to clinical characteristics. Comparison of AIIs and diffuse GNTs revealed that gain at whole chromosome 5 is only observed in GNTs. CNA patterns indicative of chromothripsis were detected in three GNTs. CONCLUSION:We conclude that GNTs with diverse morphologies share molecular features, and our findings support the need to improve classification and differential diagnosis of tumour entities within the spectrum of GNTs, as well as their distinction from other gliomas.

Purpose: miR21, miR146, and miR155 represent a trio of microRNAs which has been shown to play a key role in the regulation of immune and inflammatory responses. In the present study, we investigated the differential expression and clinical significance of these three miRNAs in glioneuronal tumors (gangliogliomas, GGs) which are characterized by prominent activation of the innate immune response.Methods: The expression levels of miR21, miR146, and miR155 were evaluated using Taqman PCR in 34 GGs, including 15 cases with sufficient amount of perilesional cortex. Their expression was correlated with the tumor features and the clinical history of epilepsy. In addition, in situ hybridization was used to evaluate their cellular distribution in both tumor and peritumoral cortex.Results: Increased expression of miR146a was observed in both tumor and peritumoral cortex compared to control samples. miR146a was detected in both neuronal and astroglial cells. Tumor and peritumoral miR146a expression was negatively correlated with frequency of seizures and the density of activated microglial cells. Neuronal and astroglial expression was observed for both miR21 and miR155 with increased expression of miR21 within the tumor and miR155 in the peritumoral region. Negative correlations were observed between the miRNA levels and the expression of putative targets within the astroglial component of the tumor.Conclusion: We report a differential regulation of three miRNAs, known to be related to inflammation, in both tumor and peritumoral cortex of patients with GG. Moreover, our findings suggest a functional relationship between miR146a expression and epilepsy, either directly in epileptogenesis or as modulation of seizure activity.

Objective: Removal of brain tissue showing high frequency oscillations (HFOs; ripples: 80-250 Hz and fast ripples: 250-500 Hz) in preresection electrocorticography (preECoG) in epilepsy patients seems a predictor of good surgical outcome. We analyzed occurrence and localization of HFOs in intra-operative preECoG and postresection electrocorticography (postECoG).Methods: HFOs were automatically detected in one-minute epochs of intra-operative ECoG sampled at 2048 Hz of fourteen patients. Ripple, fast ripple, spike, ripples on a spike (RoS) and not on a spike (RnoS) rates were analyzed in pre- and postECoG for resected and nonresected electrodes.Results: Ripple, spike and fast ripple rates decreased after resection. RnoS decreased less than RoS (74% vs. 83%; p = 0.01). Most fast ripples in preECoG were located in resected tissue. PostECoG fast ripples occurred in one patient with poor outcome. Patients with good outcome had relatively high postECoG RnoS rates, specifically in the sensorimotor cortex.Conclusions: Our observations show that fast ripples in intra-operative ECoG, compared to ripples, may be a better biomarker for epileptogenicity. Further studies have to determine the relation between resection of epileptogenic tissue and physiological ripples generated by the sensorimotor cortex.Significance: Fast ripples in intra-operative ECoG can help identify the epileptogenic zone, while ripples might also be physiological. (C) 2014 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights.

BRAF V600E mutations have been recently reported in glioneuronal tumors (GNTs). To evaluate the expression of the BRAF V600E mutated protein and its association with activation of the mammalian target of rapamycin (mTOR) pathway, immunophenotype and clinical characteristics in GNTs, we investigated a cohort of 174 GNTs. The presence of BRAF V600E mutations was detected by direct DNA sequencing and BRAF V600E immunohistochemical detection. Expression of BRAF-mutated protein was detected in 38/93 (40.8%) gangliogliomas (GGs), 2/4 (50%) desmoplastic infantile gangliogliomas (DIGs) and 23/77 (29.8%) dysembryoplastic neuroepithelial tumors (DNTs) by immunohistochemistry. In both GGs and DNTs, the presence of BRAF V600E mutation was significantly associated with the expression of CD34, phosphorylated ribosomal S6 protein (pS6; marker of mTOR pathway activation) in dysplastic neurons and synaptophysin (P<0.05). In GGs, the presence of lymphocytic cuffs was more frequent in BRAF-mutated cases (31 vs. 15.8%; P=0.001). The expression of both BRAF V600E and pS6 was associated with a worse postoperative seizure outcome in GNT (P<0.001). Immunohistochemical detection of BRAF V600E-mutated protein may be valuable in the diagnostic evaluation of these glioneuronal lesions and the observed association with mTOR activation may aid in the development of targeted treatment involving specific pathogenic pathways.

Recent data support the involvement of the endocannabinoid signaling in early brain development, as well as a key role of cannabinoid receptors (CBR) in pathological conditions associated with unbalanced neuronal excitability and inflammation. Using immunocytochemistry, we explored the expression and cellular pattern of CBR 1 and 2 (CB1 and CB2) during prenatal human cortical development, as well as in focal malformations of cortical development associated with intractable epilepsy (focal cortical dysplasia; cortical tubers in patients with the tuberous sclerosis complex and glioneuronal tumors). Strong CB1 immunoreactivity was detected in the cortical plate in developing human brain from the earliest stages tested (gestational week 9) and it persisted throughout prenatal development. Both cannabinoid receptors were not detected in neural progenitor cells located in the ventricular zone. Only CB1 was expressed in the subventricular zone and in Cajal–Retzius cells in the molecular zone of the developing neocortex. CB2 was detected in cells of the microglia/ macrophage lineage during development. In malformations of cortical development, prominent CB1 expression was demonstrated in dysplastic neurons. Both CBR were detected in balloon/giant cells, but CB2 appeared to be more frequently expressed than CB1 in these cell types. Reactive astrocytes were mainly stained with CB1, whereas cells of the microglia/ macrophage lineage were stained with CB2. These findings confirm the early expression pattern of cannabinoid receptors in the developing human brain, suggesting a function for CB1 in the early stages of corticogenesis. The expression patterns in malformations of cortical PS Emanuele 10-12.indd 218 19-12-12 13:26 219 development highlight the role of cannabinoid receptors as mediators of the endocannabinoid signaling and as potential pharmacological targets to modulate neuronal and glial cell function in epileptogenic developmental pathologies.Introduction The endogenous cannabinoid signaling system, including the endogenous ligands (endocannabinoids, eCB) and their receptors has been suggested to play a critical role during brain development (for review see [Fernandez-Ruiz et al., 2000] and [Fernandez-Ruiz et al., 2004]). Increasing evidence indicates that developmental exposure to cannabinoids may induce subtle and long-lasting neurofunctional alterations (for review see; Trezza et al., 2008). Moreover, several studies show expression of cannabinoid receptor 1 (CB1) and their endogenous ligands early during brain development in rodents ( [Berrendero et al., 1998], [Buckley et al., 1998], [Fernandez-Ruiz et al., 1999] and [Vitalis et al., 2008]). In particular, high levels of CB1 mRNA expression were observed in the cerebral cortex and in the subventricular zone (SVZ), ( [Berrendero et al., 1998], [Fernandez-Ruiz et al., 2000], [Mulder et al., 2008] and [Vitalis et al., 2008]). Abundant levels of CB1 mRNA and CB1 binding have been also detected in early prenatal stages in human brain ( [Glass et al., 1997] and [Mato et al., 2003]). Experimental evidence, in vivo and in vitro, further supports the role of this system in the process of neural development, regulating neural progenitor proliferation and migration, axonal elongation, synaptogenesis and myelinogenesis ( [Fernandez-Ruiz et al., 1999], [Fernandez-Ruiz et al., 2000], [Fernandez-Ruiz et al., 2004], [Fride, 2004], [Gomez et al., 2008a], [Mulder et al., 2008] and [Vitalis et al., 2008]). The ubiquitous abundance of eCB and their receptors in the CNS, together with the complexity of the eCB signal transduction pathways, may also suggest a critical role for the cannabinoid receptors (CBR) in various physiological and pathological conditions in the postnatal and adult brain. Thus, depending on the cellular localization and the signal transduction pathways, CBR have been shown to exert neuroprotective actions and regulate both glutamatergic and GABAergic synaptic transmission (for reviews see [Pacher et al., 2006] and [Onaivi, 2009]). In addition, evidence exists to support the possible involvement of the cannabinoid system in a number of neurological conditions, including epilepsy ( [Lutz, 2004] and [Armstrong et al., 2009]). Dysregulation of the eCB system, with alterations in the expression of CB1, has been reported in both human and experimental temporal lobe epilepsy (TLE; [Falenski et al., 2007], [Falenski et al., 2009] and [Ludanyi et al., 2008]). Recent studies also demonstrate the existence of eCB-mediated neuron-astrocyte communication, supporting the potential role of cannabinoid receptors expressed by glial cells in both physiological and pathological processes (Navarrete and Araque, 2008). Moreover the cannabinoid system exerts immunomodulatory effects and has been suggested as potential pharmacological target in pathological conditions associated with brain inflammation ( [Sheng et PS Emanuele 10-12.indd 219 19-12-12 13:26 220 al., 2005] and [Marchalant et al., 2007]). The expression pattern and cellular localization of CBR (CB1 and CB2) during corticogenesis in human brain remains uncharacterized. Furthermore, there is no information about CB1 and cannabinoid receptor 2 (CB2) distribution in human developmental pathologies associated with epilepsy. To gain further insight into the role of CBR in both normal and abnormal corticogenesis, we studied the expression of CB1 and CB2 in the developing human cerebral cortex and in focal malformations of cortical development (MCD) associated with intractable epilepsy. Materials and methods Human material The subjects included in this study were obtained from the databases of the Department of Neuropathology of the Academic Medical Center (University of Amsterdam; UvA) in Amsterdam, the Netherlands, the Service Histologie-Embryologie-Cytogénétique Hôpital NeckerEnfants malades, Paris, France (Dr. F. Encha-Razavi and Dr. M. Sinico) and the University Medical Center in Utrecht (UMCU). Informed consent was obtained for the use of brain tissue and for access to medical records for research purposes. Tissue was obtained and used in a manner compliant with the Declaration of Helsinki. The developmental expression of CB1 and CB2 was evaluated at the following ages: 9, 10, 13, 16, 17, 20, 22, 23, 25, 29, 31, 36 and 40 gestational weeks (GW) obtained from spontaneous or medically induced abortions with appropriate maternal written consent for brain autopsy. Normal-appearing control cortex/white matter and hippocampus was obtained at autopsy from pediatric patients (3 weeks, 7 months, 8 years) and from 6 young adult patients (male/female: 3/3; mean age 31; range 14-35), without a history of seizures or other neurological diseases. For comparison with the autopsy specimens, we also included 3 surgical cases of glioneuronal tumors (gangliogliomas; GG) that contained sufficient amount of perilesional tissue (mean age 35). Three autopsy specimens from patients with multiple sclerosis (MS) were also included in the study. All autopsies were performed within 12 hours after death. Expression of CB1 and CB2 in focal epileptogenic lesions was examined in 28 surgical specimens (6 focal cortical dysplasia, FCD; 6 cortical tubers; 6 gaglioglioma, GG; 6 dysembryoplastic neuroepithelial tumor, DNT; and 4 subependymal giant-cell astrocytomas, SEGA; Table1). All FCD cases included in this study fulfilled the histopathological criteria for FCD type IIB, containing dysmorphic neurons (DNs) and balloon cells (BCs) (Palmini et al., 2004). The histopathological features of the cortical tuber specimens included abnormal cortical laminar architecture, DNs, giant cells (GCs) and astrogliosis (Mizuguchi and Takashima, 2001, DiMaPS Emanuele 10-12.indd 220 19-12-12 13:26 221 rio, 2004). For the GG and DNT and SEGA we used the revised WHO classification of tumors of the central nervous system (Louis et al., 2007). The clinical characteristics derived from the patient’s medical records are summarized in Table 1. Tissue preparation Tissue was fixed in 10% buffered formalin and embedded in paraffin. Paraffin-embedded tissue was sectioned at 6 μm, mounted on pre-coated glass slides (StarFrost, Waldemar Knittel Glasbearbeitungs GmbH, Braunschweig, Germany) and used for immunocytochemical staining as described below. Antibody characterization Antibodies specific for glial fibrillary acidic protein (GFAP; polyclonal rabbit, DAKO, Glostrup, Denmark; 1:4000), vimentin (mouse clone V9; DAKO; 1:1000), neuronal nuclear protein (NeuN; mouse clone MAB377; Chemicon, Temecula, CA, USA; 1:2000), neurofilament (NF, SMI311; Sternberger Monoclonals, Lutherville, MD; 1:1000), human leukocyte antigen (HLA)-DP, DQ, DR (mouse clone CR3/43; DAKO, Glostrup, Denmark; 1:400) and microtubuleassociated protein 2 (MAP2; mouse clone HM2; Sigma, St Louis, MO; 1:100) were used in the routine immunocytochemical analysis. For the detection of CB1, we used the anti-human CB1 polyclonal rabbit antibody from Affinity BioReagents (Rockford, IL, USA; PA1-743; raised against a fusion protein containing the first 99 amino acid residues from human CB1; 1:200) and the anti-human CB1 polyclonal rabbit from Abcam (Cambridge, MA, USA) raised against a synthetic peptide, corresponding to C terminal amino acids 461-472 of Human Table 1. Summary of clinical details of epilepsy cases studied according to pathology Pathology type (PM or S) Number of cases Mean age at surgery (years/range) Localization Mean duration of epilepsy (years/range) FCD IIB 6 25.8 (14-43) Temporal(5) Frontal (1) 17.3 (5-22) Cortical Tubers (TSC) 6 17.8 (5 – 35) Frontal (3) Temporal (2) Parietal (1) 13.5 (2.8 – 34) GG 6 32 (16-49) Temporal 16.1 (12-26) DNT 6 31 (18-38) Temporal 15.6 (2-22) SEGA 4 15.1 (8-23) LV 7.3 (2-20) HS: Hippocampal Sclerosis; FCD: Focal Cortical Dysplasia; TSC:Tuberous Sclerosis; GG: ganglioglioma; DNT: dysembryoplastic neuroepithelial tumor; LV: lateral ventricle. SEGA: subependymal giant-cell ast

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