Elsevier

Neurobiology of Disease

Volume 77, May 2015, Pages 127-140
Neurobiology of Disease

SOX11 identified by target gene evaluation of miRNAs differentially expressed in focal and non-focal brain tissue of therapy-resistant epilepsy patients

https://doi.org/10.1016/j.nbd.2015.02.025Get rights and content

Highlights

  • A miRNA screen in focal and non-focal brain tissue of MTLE patients was conducted.

  • Hsa-miR-34c-5p and hsa-miR-212-3p are strongly differentially regulated.

  • Hsa-miR-34c-5p interferes with 3′-UTR sequences of GABRA3, GRM7 and GABBR2.

  • Hsa-miR-212-3p interferes with 3′-UTR sequences of SOX11, MECP2, ADCY1 and ABCG2.

  • miR-212-3p/132-3p controls Sox11 expression during neuronal maturation.

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally control the expression of their target genes via RNA interference. There is increasing evidence that expression of miRNAs is dysregulated in neuronal disorders, including epilepsy, a chronic neurological disorder characterized by spontaneous recurrent seizures. Mesial temporal lobe epilepsy (MTLE) is a common type of focal epilepsy in which disease-induced abnormalities of hippocampal neurogenesis in the subgranular zone as well as gliosis and neuronal cell loss in the cornu ammonis area are reported. We hypothesized that in MTLE altered miRNA-mediated regulation of target genes could be involved in hippocampal cell remodeling. A miRNA screen was performed in hippocampal focal and non-focal brain tissue samples obtained from the temporal neocortex (both n = 8) of MTLE patients. Out of 215 detected miRNAs, two were differentially expressed (hsa-miR-34c-5p: mean increase of 5.7 fold (p = 0.014), hsa-miR-212-3p: mean decrease of 76.9% (p = 0.0014)). After in-silico target gene analysis and filtering, reporter gene assays confirmed RNA interference for hsa-miR-34c-5p with 3′-UTR sequences of GABRA3, GRM7 and GABBR2 and for hsa-miR-212-3p with 3′-UTR sequences of SOX11, MECP2, ADCY1 and ABCG2. Reporter gene assays with mutated 3′-UTR sequences of the transcription factor SOX11 identified two different binding sites for hsa-miR-212-3p and its primary transcript partner hsa-miR-132-3p. Additionally, there was an inverse time-dependent expression of Sox11 and miR-212-3p as well as miR-132-3p in rat neonatal cortical neurons. Transfection of neurons with anti-miRs for miR-212-3p and miR-132-3p suggest that both miRNAs work synergistically to control Sox11 expression. Taken together, these results suggest that differential miRNA expression in neurons could contribute to an altered function of the transcription factor SOX11 and other genes in the setting of epilepsy, resulting not only in impaired neural differentiation, but also in imbalanced neuronal excitability and accelerated drug export.

Introduction

Epilepsy is a common chronic neurological disorder affecting more than 50 million people of all ages worldwide (Sisodiya, 2007). The neuronal hyperexcitabilty associated with seizures may be explained by an imbalance in excitatory and inhibitory signal transmission. However, the cellular and molecular mechanisms underlying increased susceptibility to recurrent seizures are not well understood. Mesial temporal lobe epilepsy (MTLE) is a common type of focal epilepsy often associated with pharmacoresistance and a histopathological finding of mesial temporal sclerosis (MTS) (Blumcke et al., 2007, Engel, 2001). Histopathological findings in a rat MTLE model and in human hippocampal specimens have demonstrated an increase in neurogenesis of dentate granule cells accompanied by a dispersed dentate granule cell layer and the ectopic location of granule cells in the hilus after acute seizures (Blumcke et al., 2001, Parent et al., 1997, Parent et al., 2006, Thom, 2004) which may contribute to excitatory circuitry in the hilus and CA3 region. In contrast, in the chronic phase of epilepsy with spontaneous recurrent seizures and signs of gliosis and neuronal cell loss in the cornu ammonis area, the number of neuronal stem cells (NSCs) seems not to change dramatically. However, the correct differentiation of NSC progenies into mature neurons appears to be impaired (Hattiangady and Shetty, 2008, Kuruba et al., 2009), potentially leading to areas of increased excitability that contribute to the development, maintenance and progression of epilepsy.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally fine-tune the expression of their target genes by interfering with the mRNA 3′-UTR region, resulting in mRNA degradation or inhibition of protein translation. miRNAs are involved in many important cellular processes including proliferation, differentiation, and maintenance of tissue specificity. The process of adult neurogenesis is associated with a miRNA-controlled sequence of altered expression of specific transcription factors and other proteins (Faigle and Song, 2013, Liu et al., 2010, Szulwach et al., 2010). Moreover, there is increasing evidence that expression of miRNAs is dysregulated in neuronal disorders, including Alzheimer's disease, depression, schizophrenia and bipolar disorder (Jin et al., 2013). Recent reports from screening studies in animal epilepsy models and from human specimens of MTLE patients show that miRNA expression is also altered in epilepsy (Dogini et al., 2013, Jimenez-Mateos and Henshall, 2013). Furthermore, epilepsy-dependent alterations in miRNA expression change between the acute post-seizure phase and the chronic, often intractable phase of spontaneous recurrent seizures (Bot et al., 2013, Song et al., 2011).

The current study is based on the hypothesis that hippocampal neuronal reorganization in the chronic phase of MTLE is associated with altered miRNA-mediated regulation of target genes predisposing the brain to recurrent epileptic seizures. After screening genome-wide miRNA expression in hippocampal focal and non-focal brain tissue from the temporal neocortex of MTLE patients who underwent temporal lobectomy, a reporter-gene assay was used to functionally investigate the interaction between dysregulated miRNAs and predicted target genes potentially involved in hippocampal cellular remodeling during epileptogenesis. Bioinformatic filtering for potential target genes included a hypothesis-free as well as a phenotype-guided approach. Candidate miRNAs and their functionally confirmed target genes identified in this study imply that differential hippocampal miRNA expression could contribute to altered function of several genes in the chronic stage of MTLE, resulting not only in impaired neural differentiation, but also in imbalanced neuronal excitability and accelerated drug export. Moreover, the results suggest that certain miRNAs act synergistically to control the expression of their target gene, emphasizing the complexity of miRNA networks in epilepsy.

Section snippets

Human tissue material

Eight hippocampal focal and eight non-focal brain tissue samples obtained from the temporal neocortex of 10 MTLE patients (six males and four females, mean age: 37.3 +/− 11.5 years), who underwent temporal lobectomy were screened for miRNA expression. The study protocol was approved by Committee on Human Research (CHR) at University of California, San Francisco, and informed consent was obtained from all participants. Demographic data, diagnosis, histopathological findings and history of

miRNA screen in human brain tissue

Out of 754 miRNAs on a TaqMan® low density array, 215 miRNAs were detected in all 16 (8 hippocampal focal, 8 temporal neocortical non-focal) human brain tissue samples. miRNAs with the highest median overall expression levels (top 3% of all detected miRNAs) were hsa-miR-125b-5p, hsa-miR-218-5p, hsa-miR-150-5p, hsa-miR-30c-5p, hsa-miR-24-3p, and hsa-miR-99b-5p. These miRNAs were not differentially expressed between focal and non-focal tissue. PCA of the expression of the 215 miRNAs revealed

Discussion

Neurogenesis persists in the subventricular zone (SVZ) of the temporal ventricles and the subgranular zone (SGZ) of the hippocampus throughout adulthood. In the SGZ, learning and physical exercise can stimulate neurogenesis, whereas neurotransmitters, growth factors, cytokines, and hormones stage-specifically influence proliferation of neural stem cell derived progenitor cells and their differentiation into mature neurons (Masiulis et al., 2011, Ming and Song, 2011). Moreover, in certain

Acknowledgements

This work was supported by a fellowship from DFG (Ha 6112/1-1) and NIH grant GM61390.

We thank Prof. Edmund Maser and Michael Kisiella, Institute of Toxicology and Pharmacology of Natural Scientists, University of Kiel, for providing access to Transfac® software tool. We also thank Huizi Wu as well as Britta Schwarten and Micheline Neubert for technical assistance. We thank the Institute of Clinical Molecular Biology in Kiel for providing Sanger sequencing as supported in part by the DFG Cluster

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