Chronic activation of anti-oxidant pathways and iron accumulation in epileptogenic malformations.

J C Baayen,W Hecke,J J Anink,J D Mills,E A Vliet,F E Jansen,E Aronica,T S Zimmer,N R Rensing,P C Rijen,M Wong,A Korotkov,S Idema,W G Spliet,A Arena,G Ciriminna

doi:10.1111/nan.12596

Abstract

AimsOxidative 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.MethodsWe 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.ResultsResected 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.ConclusionOur 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.

Highlights

A hallmark seen in resected brain tissue of patients suffering from acquired forms of epilepsy, such as temporal lobe epilepsy (TLE), is the excessive generation of reactive oxygen species (ROS) that leads to oxidative stress (OS) [1,2,3]
As our in vitro data suggested that prolonged activation of anti-oxidant pathways and haem oxygenase 1 (HO-1) activity induced ferritin, likely due to iron release, we investigated ferritin and other markers of iron metabolism in resected epileptogenic brain tissue of patients with focal cortical dysplasia (FCD) IIb, tuberous sclerosis complex (TSC) as well as in Tsc1GFAPÀ/À mice
We report evidence that TSC and FCD IIb are characterized by OS, chronic Nrf-2 activation and provide indications that redox state and iron metabolism are altered in both these developmental malformations related to mammalian target of rapamycin (mTOR) dysregulation

Summary

Introduction

A hallmark seen in resected brain tissue of patients suffering from acquired forms of epilepsy, such as temporal lobe epilepsy (TLE), is the excessive generation of reactive oxygen species (ROS) that leads to oxidative stress (OS) [1,2,3]. We showed for the first time strong expression of OS markers, in particular the light chain and catalytic cysteine/glutamate antiporter of the xc system (SLC7A11 referred to as xCT), in resected brain tissue of patients suffering from epileptogenic developmental malformations, namely focal cortical dysplasia (FCD) type IIb and tuberous sclerosis complex (TSC) [4] Histopathological hallmarks of both pathologies include cortical dyslamination, presence of dysmorphic neurones and large, improperly developed immature cells called balloon cells in FCD IIb or giant cells in TSC [5,6]. We were interested in changes in iron metabolism due to its potentially pathogenic interaction with ROS and because mTOR is a master regulator of a variety of metabolic processes To this extent, we used surgically resected brain tissue from patients with drug-resistant epilepsy due to FCD IIb or TSC and a mouse epilepsy model based on conditional Tsc deletion in glial fibrillary acidic protein (GFAP) expressing cells (Tsc1GFAPÀ/À mice) to investigate these interactions. We overexpressed miR155 in vitro in human foetal astrocytes to investigate the mechanistic link between the factors involved

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Ethical approval