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