Abstract
Epileptogenesis is a common consequence of brain insults, however, the prevention or delay of the epileptogenic process remains an important unmet medical challenge. Overexpression of glycine transporter 1 (GlyT1) is proposed as a pathological hallmark in the hippocampus of patients with temporal lobe epilepsy (TLE), and we previously demonstrated in rodent epilepsy models that augmentation of glycine suppressed chronic seizures and altered acute seizure thresholds. In the present study we evaluated the effect of the GlyT1 inhibitor, sarcosine (aka N-methylglycine), on epileptogenesis and also investigated possible mechanisms. We developed a modified rapid kindling model of epileptogenesis in rats combined with seizure score monitoring to evaluate the antiepileptogenic effect of sarcosine. We used immunohistochemistry and Western blot analysis for the evaluation of GlyT1 expression and epigenetic changes of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in the epileptogenic hippocampi of rats, and further evaluated expression changes in enzymes involved in the regulation of DNA methylation, ten-eleven translocation methylcytosine dioxygenase 1 (TET1), DNA-methyltransferase 1 (DNMT1), and DNMT3a. Our results demonstrated: (i) experimental evidence that sarcosine (3 g/kg, i.p. daily) suppressed kindling epileptogenesis in rats; (ii) the sarcosine-induced antiepileptogenic effect was accompanied by a suppressed hippocampal GlyT1 expression as well as a reduction of hippocampal 5mC levels and a corresponding increase in 5hmC; and (iii) sarcosine treatment caused differential expression changes of TET1 and DNMTs. Together, these findings suggest that sarcosine has unprecedented disease-modifying properties in a kindling model of epileptogenesis in rats, which was associated with altered hippocampal DNA methylation. Thus, manipulation of the glycine system is a potential therapeutic approach to attenuate the development of epilepsy.
Highlights
Prevention of epileptogenesis remains an unmet medical challenge because underlying mechanisms involved in this process remain unclear (Pitkanen and Lukasiuk, 2011; Terrone et al, 2016)
The overexpressed glycine transporter 1 (GlyT1) was predominantly located at the dentate inner molecular layer, whereas a lesser increase was seen in the dentate outer molecular layer (Figure 1D)
Quantitative analysis from dentate gyrus (DG)-focused IF staining (Figure 1E) demonstrated that neuronal nuclei (NeuN)-positive staining in the DG was not significantly different between fully kindled rats and the non-kindled naive controls (p = 0.6476, unpaired t-test, n = 3–4 per group) (Figure 1F); this is in line with Nissl observations and strongly indicates that neuronal cell death is not a contributing pathophysiological feature of our rapid kindling model of epileptogenesis
Summary
Prevention of epileptogenesis remains an unmet medical challenge because underlying mechanisms involved in this process remain unclear (Pitkanen and Lukasiuk, 2011; Terrone et al, 2016). Epigenetic modification may play an important role in epileptogenesis (Hsieh and Gage, 2005; Kobow and Blumcke, 2012) and in particular global hypermethylation was identified in the epileptic hippocampus (Zhu et al, 2012). Such epigenetic changes can modify pathophysiological events in epileptogenesis and is linked to the resistance to antiepileptic drugs (AEDs) (Kobow et al, 2013). These works support a role for methylation in the development and maintenance of epilepsy. Whether sarcosine’s actions through transmethylation have any relationship to seizure outcomes and epileptogenesis has not been previously evaluated
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