Unit for Multidisciplinary Research in Biomedicine

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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

Exercise training counteracts the cardiac metabolic remodelling induced by experimental pulmonary arterial hypertension

Publication . Morais, Filipe; Nogueira-Ferreira, Rita; Rocha, Hugo; Duarte, José A.; Vilarinho, Laura; Silva, Ana F.; Leite-Moreira, Adelino; Santos, Mário; Ferreira, Rita; Moreira-Gonçalves, Daniel

Exercise training provides several cardiovascular benefits in both physiological and pathological conditions; however, its use as a therapeutic tool for pulmonary arterial hypertension (PAH) has been poorly explored. This study aimed to extend the comprehension of the cardioprotective effects of exercise training in the set of PAH focusing on the metabolic changes promoted by exercise in the right ventricle (RV). The monocrotaline animal model of PAH was used and male Wistar rats were submitted to two weeks of treadmill exercise training (5 days/week, 60 min/day, 25 m/min) following disease establishment. Trained rats showed an improved diastolic function (lower end-diastolic pressure and tau) despite the presence of cardiac overload (increased peak systolic pressure, end-diastolic pressure and arterial elastance). This enhanced hemodynamic response was paralleled by an increased uptake of glucose to cardiomyocytes through glucose transporter type 4 (GLUT4) followed by increased lactate dehydrogenase (LDH) activity. Exercise did not reverse the decrease of fatty acid oxidation related to PAH but increased the content of the transcription factors peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and peroxisome proliferator-activated receptor gamma (PPAR-γ). Two weeks of exercise did not modulate the changes in amino acid metabolism secondary to PAH. Our work suggests that continuous aerobic exercise of moderate intensity, despite its short-term duration and application in a late stage of the disease, supports the RV response to PAH by promoting a shift in the cardiac metabolic phenotype.

2022-11-15Journal article

Restricted access

Epilepsy progression is associated with cumulative DNA methylation changes in inflammatory genes

Publication . Martins-Ferreira, Ricardo; Leal, Bárbara; Chaves, João; Li, Tianlu; Ciudad, Laura; Rangel, Rui; Santos, Agostinho; Martins da Silva, António; Pinho Costa, Paulo; Ballestar, Esteban

Mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) is the most common focal epilepsy in adults. It is characterized by alarming rates of pharmacoresistance. Epileptogenesis is associated with the occurrence of epigenetic alterations, and the few epigenetic studies carried out in MTLE-HS have mainly focused on the hippocampus. In this study, we obtained the DNA methylation profiles from both the hippocampus and anterior temporal neocortex of MTLE-HS patients subjected to resective epilepsy surgery and autopsied non-epileptic controls. We assessed the progressive nature of DNA methylation changes in relation to epilepsy duration. We identified significantly altered hippocampal DNA methylation patterns encompassing multiple pathways known to be involved in epileptogenesis. DNA methylation changes were even more striking in the neocortex, wherein pathogenic pathways and genes were common to both tissues. Most importantly, DNA methylation changes at many genomic sites varied significantly with epilepsy duration. Such progressive changes were associated with inflammation-related genes in the hippocampus. Our results suggest that the neocortex, relatively spared of extensive histopathological damage, may also be involved in epilepsy development. These results also open the possibility that the observed neocortical impairment could represent a preliminary stage of epileptogenesis before the establishment of chronic lesions or a consequence of prolonged seizure exposure. Our two-tissue multi-level characterization of the MTLE-HS DNA methylome suggests the occurrence of a self-propagating inflammatory wave of epigenetic dysregulation.

2022-02Journal article

Restricted 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