Persistent DNA methylation and down-regulation of homeostatic genes in astrocytes after pilocarpine-induced status epilepticus: implications for epileptogenesis.

Abstract

Epilepsy is a debilitating neurological disorder characterized by recurrent seizures, affecting millions of patients worldwide. Retrospective studies in temporal lobe epilepsy (TLE) patients have shown a high incidence of an initial precipitating event (IPE) in early childhood, followed by a silent period where epileptogenesis occurs to end up in chronic epilepsy. Epileptogenesis, the process through which a normal brain undergoes structural and functional changes leading to epilepsy, is not completely understood. We hypothesized that epigenetics may be involved in epileptogenesis, specifically affecting astrocytes through pathological remodeling. To study this process, we used three approaches: the lithium-pilocarpine model of TLE in rats, primary astroglial cultures exposed to epileptogenic DAMP named HMGB1, and brain tissue samples resected from TLE patients with drug-resistant epilepsy. We found that the IPE achieved by lithium-pilocarpine treatment (127/30 mg/kg IP) induced the DNA methylation of astrocytes at 7-, 21-, and 35-days post-IPE, indicating persistent epigenetic alterations in astrocytes during the epileptogenic period. In addition, we observed the down-regulation of homeostatic astroglial genes, including AQP4, glutamine synthase (GS), and Kir4.1, along with increased expression of proinflammatory genes (C3, MAFG) and DNA methyltransferases (DNMT). These alterations were mimicked in primary astrocyte cultures exposed to the epileptogenic HMGB1 (500 ng/ml; 18 h), which resulted in the hypermethylation of homeostatic astroglial genes and repression of homeostatic genes. HMGB1-induced repression of astroglial homeostatic genes was prevented by the treatment with DNMT inhibitor decitabine. Interestingly, astrocytes from TLE patients brains showed reactive astrogliosis, increased DNA methylation, and down-regulation of homeostatic genes Kir4.1 and GS. Taken together, these findings show that astrocytes are pathologically altered during the epileptogenic period by epigenetic modifications, combining the proinflammatory gain of function with the loss of homeostatic profile. This may contribute to the long-term alterations underlying epileptogenesis.