Browsing by Author "Martins-Ferreira, Ricardo"
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- Apolipoprotein E isoforms and susceptibility to genetic generalized epilepsiesPublication . Chaves, João; Martins-Ferreira, Ricardo; Carvalho, Cláudia; Bettencourt, Andreia; Brás, Sandra; Chorão, Rui; Freitas, Joel; Samões, Raquel; Lopes, João; Ramalheira, João; Silva, Berta; Costa, Paulo; Martins Da Silva, António; Leal, BárbaraBackground: Apolipoprotein E (ApoE) is the main lipoprotein secreted in brain. It has a critical immunomodulatory function, influences neurotransmission and it is involved in repairing damaged neurons. ApoE e4 is an isoform of ApoE with altered function, and was previously associated with early onset epilepsy and refractoriness, both in animal models and in patients with focal epilepsies. There is a limited knowledge on ApoE’s role in Genetic Generalized Epilepsies (GGE). Aim: To determine if ApoE isoforms are risk factors for GGE development. Methods: A group of 337 GGE patients (193 F, 144 M, 33.6 ± 14.2 years) was compared with a group of 342 healthy individuals in a case-control genetic association study. ApoE genotyping was performed using PCR-RFLP. Results: The genotypic frequency of ApoE e3/e2 was lower in GGE patients relative to controls (6.5% in GGE vs. 11.7% in controls, p = 0.019, OR (95% CI) = 0.53 (0.305–0.905). No associations with other clinical data such as photosensitivity or age at disease onset were observed. Conclusion: Our results show that ApoE e3/e2 genotype may be a protective factor for GGE development. There is evidence that this genotype could be neuroprotective, preventing oxidative damage and promoting neuronal survival. Although replication studies are warranted, our data suggest that ApoE isoforms have a role in epileptogenic mechanisms regardless of the specific epileptic manifestations.
- Circulating cell-free DNA methylation mirrors alterations in cerebral patterns in epilepsyPublication . Martins-Ferreira, Ricardo; Leal, Bárbara; Chaves, João; Ciudad, Laura; Samões, Raquel; Martins da Silva, António; Pinho Costa, Paulo; Ballestar, EstebanBackground: 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.
- Circulating miR-134 in mesial temporal lobe epilepsy: implications in hippocampal sclerosis development and drug resistancePublication . Guerra Leal, Bárbara; Carvalho, Cláudia; Santos, Cristina; Samões, Raquel; Martins-Ferreira, Ricardo; Teixeira, Catarina; Rodrigues, Diana; Freitas, Joel; Lemos, Carolina; Chorão, Rui; Ramalheira, João; Lopes, João; Martins da Silva, António; Pinho E Costa, Paulo; Chaves, JoãoAim: miR-134 has been widely reported as upregulated in experimental and human studies of Mesial Temporal Lobe Epilepsy the most common drug-resistant epilepsy (DRE). Studies have shown that the use of antagomirs, anti-miR-134, may be a promising therapeutic approach to these epilepsies. However, data on miR-134 in other epileptic syndromes is scarce. In this study, we aimed to quantify serum levels of miR-134 in a cohort of patients with Mesial Temporal Lobe Epilepsy-Hippocampal Sclerosis (MTLE-HS) and with Genetic Generalized Epilepsies (GGE). Additionally, we explored the correlation between miR-134 serum levels and clinical parameters, such as age at onset or febrile seizures antecedents, to evaluate its potential as a biomarker and therapeutic target in epilepsy. Methods: miR-134 levels were evaluated in cell-free serum of 131 patients with epilepsy (75 women, 56 men; age 41.10 ± 13.12 years; 72 with DRE) and 42 healthy individuals (25 women, 17 men; age 42.40 ± 9.80 years). The epilepsy cohort included 77 MTLE-HS patients and 54 GGE patients. Results: Patients with elevated miR-134 circulating levels were at higher risk of drug-resistant epilepsy (OR [95% CI] = 2.246 [1.111–4.539], p = 0.021). Other risk factors included an older age (OR [95% CI] = 1.032 [1.004–1.061], p = 0.025), history of febrile seizures (OR [95% CI] = 2.994 [1.385–6.471], p = 0.005) and higher disease duration (OR [95% CI] = 1.038 [1.011–1.066], p = 0.006). The strongest predictor of DRE was hippocampal sclerosis (OR [95% CI] = 10.338 [4.566–23.404], p < 0.001). Circulating miR-134 levels were significantly higher in MTLE-HS patients compared to controls (p < 0.05) and GGE patients (p < 0.05). However, the clinical utility of miR-134 in discriminating MTLE-HS patients from controls was only moderated (AUC = 0.651 ± 0.051 95% CI 0.551–0.751, p = 0.007). Conclusion: We show that miR-134 circulating levels are associated with DRE, especially in MTLE-HS, a syndrome characterized by severe hippocampal damage, consistent with activity-regulated miR-134 expression. This overexpression likely contributes to disease progression and our results support the potential of targeting miR-134 as a novel therapeutic approach for refractory epilepsy.
- Epilepsy progression is associated with cumulative DNA methylation changes in inflammatory genesPublication . Martins-Ferreira, Ricardo; Leal, Bárbara; Chaves, João; Li, Tianlu; Ciudad, Laura; Rangel, Rui; Santos, Agostinho; Martins da Silva, António; Pinho Costa, Paulo; Ballestar, EstebanMesial 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.
- Immunogenetic protective factors in Genetic Generalized EpilepsyPublication . 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, BárbaraBackground: 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.
- Mesial Temporal Lobe Epilepsy (MTLE) Drug-Refractoriness Is Associated With P2X7 Receptors Overexpression in the Human Hippocampus and Temporal Neocortex and May Be Predicted by Low Circulating Levels of miR-22Publication . Guerra Leal, Bárbara; Barros-Barbosa, Aurora; Ferreirinha, Fátima; Chaves, João; Rangel, Rui; Santos, Agostinho; Carvalho, Cláudia; Martins-Ferreira, Ricardo; Samões, Raquel; Freitas, Joel; Lopes, João; Ramalheira, João; Lobo, Maria Graça; Martins da Silva, António; Costa, Paulo P.; Correia-de-Sá, PauloObjective: ATP-gated ionotropic P2X7 receptors (P2X7R) actively participate in epilepsy and other neurological disorders. Neocortical nerve terminals of patients with Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis (MTLE-HS) express higher P2X7R amounts. Overexpression of P2X7R bolsters ATP signals during seizures resulting in glial cell activation, cytokines production, and GABAergic rundown with unrestrained glutamatergic excitation. In a mouse model of status epilepticus, increased expression of P2X7R has been associated with the down-modulation of the non-coding micro RNA, miR-22. MiR levels are stable in biological fluids and normally reflect remote tissue production making them ideal disease biomarkers. Here, we compared P2X7R and miR-22 expression in epileptic brains and in the serum of patients with MTLE-HS, respectively. Methods: Quantitative RT-PCR was used to evaluate the expression of P2X7R in the hippocampus and anterior temporal lobe of 23 patients with MTLE-HS and 10 cadaveric controls. Confocal microscopy and Western blot analysis were performed to assess P2X7R protein amounts. MiR-22 expression was evaluated in cell-free sera of 40 MTLE-HS patients and 48 healthy controls. Results: Nerve terminals of the hippocampus and neocortical temporal lobe of MTLE-HS patients overexpress (p < 0.05) an 85 kDa P2X7R protein whereas the normally occurring 67 kDa receptor protein dominates in the brain of the cadaveric controls. Contrariwise, miR-22 serum levels are diminished (p < 0.001) in MTLE-HS patients compared to age-matched control blood donors, a situation that is more evident in patients requiring multiple (>3) anti-epileptic drug (AED) regimens. Conclusion: Data show that there is an inverse relationship between miR-22 serum levels and P2X7R expression in the hippocampus and neocortex of MTLE-HS patients, which implies that measuring serum miR-22 may be a clinical surrogate of P2X7R brain expression in the MTLE-HS. Moreover, the high area under the ROC curve (0.777; 95% CI 0.629-0.925; p = 0.001) suggests that low miR-22 serum levels may be a sensitive predictor of poor response to AEDs among MTLE-HS patients. Results also anticipate that targeting the miR-22/P2X7R axis may be a good strategy to develop newer AEDs.
- Microglial innate memory and epigenetic reprogramming in neurological disordersPublication . Martins-Ferreira, Ricardo; Leal, Bárbara; Costa, Paulo; Ballestar, EstebanMicroglia 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.
- Purinergic exposure induces epigenomic and transcriptomic-mediated preconditioning resembling epilepsy-associated microglial statesPublication . Martins-Ferreira, Ricardo; Calafell-Segura, Josep; Chaves, João; Ciudad, Laura; Martins da Silva, António; Pinho E Costa, Paulo; Leal, Bárbara; Ballestar, EstebanMicroglia 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.
- The Potential of Circulating Cell-Free DNA Methylation as an Epilepsy BiomarkerPublication . Martins-Ferreira, Ricardo; Leal, Bárbara Guerra; Costa, Paulo PinhoCirculating 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.