Oxidative stress and inflammation in a spectrum of epileptogenic cortical malformations: molecular insights into their interdependence

Oxidative stress (OS) occurs in brains of patients with epilepsy and coincides with brain inflammation, and both phenomena contribute to seizure generation in animal models. We investigated whether expression of OS and brain inflammation markers co-occurred also in resected brain tissue of patients with epileptogenic cortical malformations: hemimegalencephaly (HME), focal cortical dysplasia (FCD) and cortical tubers in tuberous sclerosis complex (TSC). Moreover, we studied molecular mechanisms linking OS and inflammation in an in vitro model of neuronal function. Untangling interdependency and underlying molecular mechanisms might pose new therapeutic strategies for treating patients with drug-resistant epilepsy of different etiologies. Immunohistochemistry was performed for specific OS markers xCT and iNOS and brain inflammation markers TLR4, COX-2 and NF-κB in cortical tissue derived from patients with HME, FCD IIa, IIb and TSC. Additionally, we studied gene expression of these markers using the human neuronal cell line SH-SY5Y in which OS was induced using H 2 O 2 . OS markers were higher in dysmorphic neurons and balloon/giant cells in cortex of patients with FCD IIb or TSC. Expression of OS markers was positively correlated to expression of brain inflammation markers. In vitro, 100 µM, but not 50 µM, of H 2 O 2 increased expression of TLR4, IL-1β and COX-2. We found that NF-κB signaling was activated only upon stimulation with 100 µM H 2 O 2 leading to upregulation of TLR4 signaling and IL-1β. The NF-κB inhibitor TPCA-1 completely reversed this effect. Our results show that OS positively correlates with neuroinflammation and is particularly evident in brain tissue of patients with FCD IIb and TSC. In vitro, NF-κB is involved in the switch to an inflammatory state after OS. We propose that the extent of OS can predict the neuroinflammatory state of the brain. Additionally, antioxidant treatments may prevent the switch to inflammation in neurons thus targeting multiple epileptogenic processes at once

Adverse prognosis of glioblastoma contacting the subventricular zone: Biological correlates

INTRODUCTION: The subventricular zone (SVZ) in the brain is associated with gliomagenesis and resistance to treatment in glioblastoma. In this study, we investigate the prognostic role and biological characteristics of subventricular zone (SVZ) involvement in glioblastoma. METHODS: We analyzed T1-weighted, gadolinium-enhanced MR images of a retrospective cohort of 647 primary glioblastoma patients diagnosed between 2005-2013, and performed a multivariable Cox regression analysis to adjust the prognostic effect of SVZ involvement for clinical patient- and tumor-related factors. Protein expression patterns of a.o. markers of neural stem cellness (CD133 and GFAP-δ) and (epithelial-) mesenchymal transition (NF-κB, C/EBP-β and STAT3) were determined with immunohistochemistry on tissue microarrays containing 220 of the tumors. Molecular classification and mRNA expression-based gene set enrichment analyses, miRNA expression and SNP copy number analyses were performed on fresh frozen tissue obtained from 76 tumors. Confirmatory analyses were performed on glioblastoma TCGA/TCIA data. RESULTS: Involvement of the SVZ was a significant adverse prognostic factor in glioblastoma, independent of age, KPS, surgery type and postoperative treatment. Tumor volume and postoperative complications did not explain this prognostic effect. SVZ contact was associated with increased nuclear expression of the (epithelial-) mesenchymal transition markers C/EBP-β and phospho-STAT3. SVZ contact was not associated with molecular subtype, distinct gene expression patterns, or markers of stem cellness. Our main findings were confirmed in a cohort of 229 TCGA/TCIA glioblastomas. CONCLUSION: In conclusion, involvement of the SVZ is an independent prognostic factor in glioblastoma, and associates with increased expression of key markers of (epithelial-) mesenchymal transformation, but does not correlate with stem cellness, molecular subtype, or specific (mi)RNA expression patterns

Distinct DNA Methylation Patterns of Subependymal Giant Cell Astrocytomas in Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is a monogenic disorder caused by mutations in either the TSC1 or TSC2 gene, two key regulators of the mechanistic target of the rapamycin complex pathway. Phenotypically, this leads to growth and formation of hamartomas in several organs, including the brain. Subependymal giant cell astrocytomas (SEGAs) are low-grade brain tumors commonly associated with TSC. Recently, gene expression studies provided evidence that the immune system, the MAPK pathway and extracellular matrix organization play an important role in SEGA development. However, the precise mechanisms behind the gene expression changes in SEGA are still largely unknown, providing a potential role for DNA methylation. We investigated the methylation profile of SEGAs using the Illumina Infinium HumanMethylation450 BeadChip (SEGAs n = 42, periventricular control n = 8). The SEGA methylation profile was enriched for the adaptive immune system, T cell activation, leukocyte mediated immunity, extracellular structure organization and the ERK1 & ERK2 cascade. More interestingly, we identified two subgroups in the SEGA methylation data and show that the differentially expressed genes between the two subgroups are related to the MAPK cascade and adaptive immune response. Overall, this study shows that the immune system, the MAPK pathway and extracellular matrix organization are also affected on DNA methylation level, suggesting that therapeutic intervention on DNA level could be useful for these specific pathways in SEGA. Moreover, we identified two subgroups in SEGA that seem to be driven by changes in the adaptive immune response and MAPK pathway and could potentially hold predictive information on target treatment response

Cellular localization of metabotropic glutamate receptors in cortical tubers and subependymal giant cell tumors of tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder associated with cortical malformations (cortical tubers) and the development of glial tumors (subependymal giant-cell tumors, SGCTs). Expression of metabotropic glutamate receptor (mGluR) subtypes is developmentally regulated and several studies suggest an involvement of mGluR-mediated glutamate signaling in the regulation of proliferation and survival of neural stem-progenitor cells, as well as in the control of tumor growth. In the present study, we have investigated the expression and cell-specific distribution of group I (mGluR1, mGluR5), group II (mGluR2/3) and group III (mGluR4 and mGluR8) mGluR subtypes in human TSC specimens of both cortical tubers and SGCTs, using immunocytochemistry. Strong group I mGluR immunoreactivity (IR) was observed in the large majority of TSC specimens in dysplastic neurons and in giant cells within cortical tubers, as well as in tumor cells within SGCTs. In particular mGluR5 appeared to be most frequently expressed, whereas mGluR1 alpha was detected in a subpopulation of neurons and giant cells. Cells expressing mGluR1 alpha and mGluR5, demonstrate IR for phospho-S6 ribosomal protein (PS6), which is a marker of the mammalian target of rapamycin (mTOR) pathway activation. Group II and particularly group III mGluR IR was less frequently observed than group I mGluRs in dysplastic neurons and giant cells of tubers and tumor cells of SGCTs. Reactive astrocytes were mainly stained with mGluR5 and mGluR2/3. These findings expand our knowledge concerning the cellular phenotype in cortical tubers and in SGCTs and highlight the role of group I mGluRs as important mediators of glutamate signaling in TSC brain lesions. Individual mGluR subtypes may represent potential pharmacological targets for the treatment of the neurological manifestations associated with TSC brain lesions. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserve

A neuropathological study of two autopsy cases of syndromic hemimegalencephaly

Hemimegalencephaly (HMEG) is a malformation of cortical development characterized by unilateral enlargement of the cerebral hemisphere, severe architectural and cellular abnormalities and association with intractable epilepsy. HMEG may represent an isolated lesion of the central nervous system, but may also be associated with several neurocutaneous syndromes. In the present study we discuss the neuropathological findings of two autopsy cases of HMEG associated with linear naevus sebaceous syndrome. Both cases showed the presence of linear naevus sebaceous on extensive areas of the face. The neurochemical profile of the glial and neuronal components in the affected hemisphere was determined using immunocytochemical markers and was compared with the unaffected contralateral hemisphere and normal control tissue. The observed cytomegalic neurones expressed receptors for distinct neurotransmitters, neuropeptides and growth factors. Analysis of components of the phosphoinositide 3-kinase pathway revealed expression of phospho-S6 ribosomal protein in cytomegalic neurones. Autopsy findings confirm the complexity of the histologic phenotypic manifestations in HMEG and proved useful in determining the spectrum of cytoarchitectural and neurochemical abnormalities, underlying the molecular pathogenesis and epileptogenesis of this brain malformatio

Calcinosis in juvenile dermatomyositis: a possible role for the vitamin K-dependent protein matrix Gla protein

Objectives. The aims of the present study were to investigate whether the calcification inhibitor matrix Gla protein (MGP) is expressed in muscle biopsies of patients with juvenile dermatomyositis (JDM), and whether different forms of MGP are differentially expressed in JDM patients with and without subcutaneous calcifications. Methods. Muscle tissue from six JDM patients (three without calcinosis, two with calcinosis and one recently diagnosed patient), four patients with muscular dystrophy, three patients with IBM and five normal histological control subjects was used for immunohistochemistry staining using novel antibodies to different conformations of MGP. Results. In the JDM patients, all forms of MGP [non-carboxylated MGP (ucMGP), carboxylated MGP (cMGP), non-phosphorylated MGP (serMGP) and phosphorylated MGP (pserMGP)] were more intensely stained in the perifascicular compared with the central muscle fibres. In addition, these MGP species were demonstrated in the pathological muscle fibres of IBM and dystrophy patients, but hardly in normal histological muscle tissue. In JDM patients with calcifications, only pserMGP was increased compared with those without calcifications. All forms of MGP were also found in various staining intensities in the microvasculature and macrophages of normal histological and disease biopsies. Conclusions. MGP was expressed at the site of muscle damage in JDM patients as well as in patients with muscular dystrophy and IBM. The difference in staining intensity of pserMGP appeared to distinguish between JDM patients with and without calcifications, whereas cMGP, the other functional form, was equally expressed