Epigenetic regulation of signalling pathways in MTLE-HS and its impact on epileptogenesis

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Circulating cell-free DNA methylation mirrors alterations in cerebral patterns in epilepsy

Publication . Martins-Ferreira, Ricardo; Leal, Bárbara; Chaves, João; Ciudad, Laura; Samões, Raquel; Martins da Silva, António; Pinho Costa, Paulo; Ballestar, Esteban

Background: DNA methylation profiling of circulating cell-free DNA (cfDNA) has rapidly become a promising strategy for biomarker identification and development. The cell-type-specific nature of DNA methylation patterns and the direct relationship between cfDNA and apoptosis can potentially be used non-invasively to predict local alterations. In addition, direct detection of altered DNA methylation patterns performs well as a biomarker. In a previous study, we demonstrated marked DNA methylation alterations in brain tissue from patients with mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS). Results: We performed DNA methylation profiling in cfDNA isolated from the serum of MTLE patients and healthy controls using BeadChip arrays followed by systematic bioinformatic analysis including deconvolution analysis and integration with DNase accessibility data sets. Differential cfDNA methylation analysis showed an overrepresentation of gene ontology terms and transcription factors related to central nervous system function and regulation. Deconvolution analysis of the DNA methylation data sets ruled out the possibility that the observed differences were due to changes in the proportional contribution of cortical neurons in cfDNA. Moreover, we found no overrepresentation of neuron- or glia-specific patterns in the described cfDNA methylation patterns. However, the MTLE-HS cfDNA methylation patterns featured a significant overrepresentation of the epileptic DNA methylation alterations previously observed in the hippocampus. Conclusions: Our results support the use of cfDNA methylation profiling as a rational approach to seeking non-invasive and reproducible epilepsy biomarkers.

2022-12-28Journal article

Open access

The Potential of Circulating Cell-Free DNA Methylation as an Epilepsy Biomarker

Publication . Martins-Ferreira, Ricardo; Leal, Bárbara Guerra; Costa, Paulo Pinho

Circulating cell-free DNA (cfDNA) are highly degraded DNA fragments shed into the bloodstream. Apoptosis is likely to be the main source of cfDNA due to the matching sizes of cfDNA and apoptotic DNA cleavage fragments. The study of cfDNA in liquid biopsies has served clinical research greatly. Genetic analysis of these circulating fragments has been used in non-invasive prenatal testing, detection of graft rejection in organ transplants, and cancer detection and monitoring. cfDNA sequencing is, however, of limited value in settings in which genetic association is not well-established, such as most neurodegenerative diseases.Recent studies have taken advantage of the cell-type specificity of DNA methylation to determine the tissue of origin, thus detecting ongoing cell death taking place in specific body compartments. Such an approach is yet to be developed in the context of epilepsy research. In this article, we review the different approaches that have been used to monitor cell-type specific death through DNA methylation analysis, and recent data detecting neuronal death in neuropathological settings. We focus on the potential relevance of these tools in focal epilepsies, like Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis (MTLE-HS), characterized by severe neuronal loss. We speculate on the potential relevance of cfDNA methylation screening for the detection of neuronal cell death in individuals with high risk of epileptogenesis that would benefit from early diagnosis and consequent early treatment.

2022-03-24Journal article

Open access

Immunogenetic protective factors in Genetic Generalized Epilepsy

Publication . Chaves, João; Martins-Ferreira, Ricardo; Ferreira, Ana Marta; Brás, Sandra; Carvalho, Cláudia; Bettencourt, Andreia; Samões, Raquel; Monteiro, Fábio; Freitas, Joel; Chorão, Rui; Lopes, João; Ramalheira, João; Silva, Berta; Costa, Paulo; Martins Da Silva, António; Leal, Bárbara

Background: Genetic Generalized Epilepsies (GGEs) are a heterogeneous group of syndromes characterized by generalized seizure activity that affects both hemispheres, with mainly genetic causes. Neuroinflammation has been established as an important mechanism in epileptogenesis. The ability to develop an appropriated immune response is strongly determined by immunogenetic factors. In this setting, our aim was to evaluate potential associations between GGEs and immunogenetic factors. Methods: The rs16944 (IL-1β -511 T > C) polymorphism and the HLA-DRB1 locus were genotyped in a Portuguese GGE population. Association with two clinicopathological features, photosensitivity and refractoriness, was investigated. This case-control study included 323 GGE patients (187 F, 136 M, 34.0 ± 13.9 years of age), 145 of which with JME diagnosis (88 F, 57 M, 34.1 ± 14.0 years), and 282 healthy controls (174 F, 108 M, 37.7 ± 11.6 years). Results: Decreased frequencies of the HLA-DRB1*09 and DRB1*13 alleles were observed in the GGE population. HLA-DRB1*07 frequency was increased in JME. Rs16944 allelic frequencies were similar between patients and controls. Conclusions: These results, not entirely consistent with previous reports, suggest that HLA molecules may have a complex role in epileptogenesis.

2020-10Journal article

Restricted access

Microglial innate memory and epigenetic reprogramming in neurological disorders

Publication . Martins-Ferreira, Ricardo; Leal, Bárbara; Costa, Paulo; Ballestar, Esteban

Microglia are myeloid-derived cells recognized as brain-resident macrophages. They act as the first and main line of immune defense in the central nervous system (CNS). Microglia have high phenotypic plasticity and are essential for regulating healthy brain homeostasis, and their dysregulation underlies the onset and progression of several CNS pathologies through impaired inflammatory responses. Aberrant microglial activation, following an inflammatory insult, is associated with epigenetic dysregulation in various CNS pathologies. Emerging data suggest that certain stimuli to myeloid cells determine enhanced or attenuated responses to subsequent stimuli. These phenomena, generally termed innate immune memory (IIM), are highly dependent on epigenetic reprogramming. Microglial priming has been reported in several neurological diseases and corresponds to a state of increased permissiveness or exacerbated response, promoted by continuous exposure to a chronic pro-inflammatory environment. In this article, we provide extensive evidence of these epigenetic-mediated phenomena under neurological conditions and discuss their contribution to pathogenesis and their clinical implications, including those concerning potential novel therapeutic approaches.

2020-12-09Journal article

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Purinergic exposure induces epigenomic and transcriptomic-mediated preconditioning resembling epilepsy-associated microglial states

Publication . Martins-Ferreira, Ricardo; Calafell-Segura, Josep; Chaves, João; Ciudad, Laura; Martins da Silva, António; Pinho E Costa, Paulo; Leal, Bárbara; Ballestar, Esteban

Microglia play a crucial role in a range of neuropathologies through exacerbated activation. Microglial inflammatory responses can be influenced by prior exposures to noxious stimuli, like increased levels of extracellular adenosine and ATP. These are characteristic of brain insults like epileptic seizures and could potentially shape subsequent responses through epigenetic regulation. We investigated DNA methylation and expression changes in human microglia-like cells differentiated from monocytes following ATP-mediated preconditioning. We demonstrate that microglia-like cells display homeostatic microglial features, shown by surface markers, transcriptome, and DNA methylome. After exposure to ATP, TLR-mediated activation leads to an exacerbated pro-inflammatory response. These changes are accompanied by methylation and transcriptional reprogramming associated with enhanced immune-related functions. The reprogramming associated with ATP-mediated preconditioning leads to profiles found in microglial subsets linked to epilepsy. Purine-driven microglia immune preconditioning drives epigenetic and transcriptional changes that could contribute to altered functions of microglia during seizure development and progression.

2024-08-16Research article

Open access