Promoter modification and gene silencing of neuropeptide genes in animal models and in human epilepsy

About 1% of the population has epilepsy, and approx. 30% of the patients lack response to currently available antiepileptic drug treatment. Onset and progression of spontaneous drug-resistant seizure activity remains, however, difficult to predict and determine in affected patients, irrespective of their epileptogenic condition, i.e., traumatic brain injury, temporal lobe (hippocampal) sclerosis or genetic inheritance. The objective of this CRP is to characterize common epigenetic pathomechanisms of epileptogenesis and, thereby, to identify novel targets for pharmacotherapy. Animal models and investigations of human specimens have shown alterations in the expression patterns of a variety of genes, including neuropeptides. This altered expression pattern of specific neuropeptides is characterized by rapid onset and can be long lasting. For some of these changes an endogenous anticonvulsive role has been suggested. The underlying mechanisms regulating altered expression of peptides in epilepsy are yet not understood. Recent evidence has identified chromatin remodeling at gene promoter regions as a key control mechanism of gene expression and may, therefore, partly mediate acute and chronic effects of epileptic seizures on gene activity. In particular methylation of DNA and modifications of histones, including acetylation, methylation and phosphorylation are identified modifications resulting in altered gene transcription. The project part will use an animal model of temporal lobe epilepsy and tissue from temporal lobe epilepsy patients to investigate such epigenetic mechanisms involved in the expression of the neuropeptides dynorphin, neuropeptide Y and neurokinin B. We will investigate the expression patterns of epigenetically acting key enzymes, including histone deacetylases and DNA methyl-transferases in animal models of epileptic seizures compared to specimens from TLE patients. In particular altered DNA methylation and histone acetylation of promoter regions of neuropeptides (neuropeptide Y, dynorphin, neurokinin B) will be investigated. Moreover the hypothesis will be tested that inhibition and/or over- expression of identified enzymes by infusion of respective viral vectors will significantly modify expression patterns of neuropeptides and epileptic seizure activity, ultimately leading to the identification of novel drug targets.

The consortial project included groups from Germany (2), Poland (1), Finland (1) and Austria (1) and two associated partners from France and Australia, respectively. It was focused on epigenetic mechanisms involved in the generation and manifestation of epilepsy. Epigenetic mechanisms include a great number of mechanisms controlling dynamic expression of genes. Most prominent mechanisms are modifications of histones, such as histone methylation, acetylation or phosphorylation, expression of transcription factors or small interfering RNAs. Expression of individual genes may be altered by life situations including states of disease such as epileptic seizures. Goal of the project was to investigate epigenetic changes in response to epileptic seizures and their involvement in the manifestation of chronic epilepsy (epileptogenesis). A main focus of the concerted project was put on alterations in histone methylation and acetylation in animal models of epilepsy with a focus on changes during epileptogenesis. The project was conducted using different animal models of epilepsy. The models used are mostly based on inducing an acute status epilepticus, which is followed by a phase of development of spontaneous seizures (epileptogenesis) and chronic epilepsy (recurrent unprovoked seizures).The Austrian partner has put his focus on characterization of changes in the expression of histone deacetylases (HDACs) during development of epilepsy. Whereas histone acetylation at individual histones promotes gene transcription, histone deacetylation reverses this process. Notably the potent anticonvulsive drug valproic acid may exert its action through inhibition of HDACs. Today four families of HDACs including 11 major enzymes are known. Using two different mouse models of epileptogenesis (intrahippocampal injection of the convulsive drug kainic acid and intraperitoneal application of pilocarpine) we observed highly specific temporal patterns of changes in mRNA expression of individual HDACs in different subfields of the hippocampus. Functionally these changes may be attributed to different states during epileptogenesis: 1) They included marked down-regulation of class I and IV (HDAC1, 2 and 11) during the acute status epilepticus (used to induce the process of epileptogenesis in the mice). This change may lead to increased expression of various genes including that of transcription factors and multiple proteins with pro- and anticonvulsive functions; 2) overexpression of certain class I and II HDACs (HDAC1, 2, 3 and 5) during epileptogenesis, possibly contributing to the manifestation of epilepsy and 3) overexpression of the class IIa HDACs 5 and 9, associated with granule cell dispersion, a pathological sign seen also in human temporal lobe epilepsy. Interestingly HDACs 5 and 9, beyond their function at acetylated histones, exert also a prominent action on cytoplasmatic proteins presumably involved in neuronal plasticity and intracellular migration.In conclusion, our study revealed prominent temporally and regionally different changes in the expression of individual HDACs during epileptogenesis. The findings suggest a role of HDACs during epileptogenesis and granule cell dispersion. Interference with these mechanisms may allow to interfere with the manifestation of epilepsy.