Villarreal, A. | Search bioRxiv

bioRxiv2025

Persistent DNA methylation and downregulation of homeostatic genes in astrocytes after pilocarpine-induced status epilepticus: Implications for epileptogenesis

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, remains an enigmatic phenomenon. We hypothesized that epigenetics may be involved in epileptogenesis and specifically astrocytes could be affected by 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 hypermethylation 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 downregulation of homeostatic astroglial genes AQP4; glutamine synthase (GS), and Kir4.1 with increased proinflammatory genes (C3, MAFG) and DNA methyl transferases (DNMT) expression. These alterations were mimicked in primary astrocyte cultures exposed to the epileptogenic HMGB1 (500 ng/ml; 18 hours) that also induced the hypermethylation of homeostatic astroglial genes. Astrocytes from TLE patients brains showed reactive astrogliosis, increased DNA methylation, and downregulation of homeostatic genes Kir4.1 and GS. These findings show that astrocytes are pathologically altered during the epileptogenic period, combining the proinflammatory gain of function with the loss of homeostatic profile. This may sustain the long-term alterations underlining epileptogenesis.

neuroscience↗

bioRxiv2021

Integrative multi-omics analyses reveal multi-modal FOXG1 functions acting on epigenetic processes and in concert with NEUROD1 to regulate synaptogenesis in the mouse hippocampus

BackgroundFOXG1 has important functions for neuronal differentiation and balances excitatory/inhibitory network activity. Mutations in the human FOXG1 gene cause a rare neurodevelopmental disorder, FOXG1-syndrome, which manifests differing phenotypes, including severe cognitive dysfunction, microencephaly, social withdrawal, and communication and memory deficits. Changes at the molecular level underlying these functional abnormalities upon FOXG1 haploinsufficiency are largely unexplored, in human patients as well as in animals modelling the debilitating disease. MethodsWe present multi-omics data and explore comprehensively how FOXG1 impacts neuronal maturation at the chromatin level in the adult mouse hippocampus. We used RNA-, ATAC- and ChIP-sequencing of primary hippocampal neurons and co-immunoprecipitation to explore various levels of epigenetic changes and transcription factor networks acting to alter neuronal differentiation upon reduction of FOXG1. ResultsWe provide the first comprehensive multi-omics data set exploring FOXG1 presence at the chromatin and identifying the consequences of reduced FOXG1 expression in primary hippocampal neurons. Analyzing the multi-omics data, our study reveals that FOXG1 uses various different ways to regulate transcription at the chromatin level. On a genome-wide level, FOXG1 (i) both represses and activates transcription, (ii) binds mainly to enhancer regions, and (iii) bidirectionally alters the epigenetic landscape in regard to levels of H3K27ac, H3K4me3, and chromatin accessibility. Genes affected by the chromatin alterations upon FOXG1 reduction impact synaptogenesis and axonogenesis. This finding emphasizes the importance of FOXG1 to integrate and coordinate transcription of genes necessary for proper neuronal function by acting on a genome-wide level. Interestingly, FOXG1 acts through histone deacetylases (HDACs) and inhibition of HDACs partly rescued transcriptional alterations observed upon FOXG1 reduction. On a more detailed level of analysis, we show that FOXG1 (iv) operates synergistically with NEUROD1. Interestingly, we could not detect a clear hierarchy of these two key transcription factors, but instead provide first evidence that they act in highly concerted and orchestrated manner to control neuronal differentiation. ConclusionsThis integrative and multi-omics view of changes upon FOXG1 reduction reveals an unprecedented multimodality of FOXG1 functions converging on neuronal maturation, fueling novel therapeutic options based on epigenetic drugs to alleviate, at least in part, neuronal dysfunctions.

neuroscience↗