Opposing effects of the purinergic P2X7 receptor on seizures in neurons and microglia in male mice.

BACKGROUND: The purinergic ATP-gated P2X7 receptor (P2X7R) is increasingly recognized to contribute to pathological neuroinflammation and brain hyperexcitability. P2X7R expression has been shown to be increased in the brain, including both microglia and neurons, in experimental models of epilepsy and patients. To date, the cell type-specific downstream effects of P2X7Rs during seizures remain, however, incompletely understood. METHODS: Effects of P2X7R signaling on seizures and epilepsy were analyzed in induced seizure models using male mice including the kainic acid model of status epilepticus and pentylenetetrazole model and in male and female mice in a genetic model of Dravet syndrome. RNA sequencing was used to analyze P2X7R downstream signaling during seizures. To investigate the cell type-specific role of the P2X7R during seizures and epilepsy, we generated mice lacking exon 2 of the P2rx7 gene in either microglia (P2rx7:Cx3cr1-Cre) or neurons (P2rx7:Thy-1-Cre). To investigate the protective potential of overexpressing P2X7R in GABAergic interneurons, P2X7Rs were overexpressed using adeno-associated virus transduction under the mDlx promoter. RESULTS: RNA sequencing of hippocampal tissue from wild-type and P2X7R knock-out mice identified both glial and neuronal genes, in particular genes involved in GABAergic signaling, under the control of the P2X7R following seizures. Mice with deleted P2rx7 in microglia displayed less severe acute seizures and developed a milder form of epilepsy, and microglia displayed an anti-inflammatory molecular profile. In contrast, mice lacking P2rx7 in neurons showed a more severe seizure phenotype when compared to epileptic wild-type mice. Analysis of single-cell expression data revealed that human P2RX7 expression is elevated in the hippocampus of patients with temporal lobe epilepsy in excitatory and inhibitory neurons. Functional studies determined that GABAergic interneurons display increased responses to P2X7R activation in experimental epilepsy. Finally, we show that viral transduction of P2X7R in GABAergic interneurons protects against evoked and spontaneous seizures in experimental temporal lobe epilepsy and in mice lacking Scn1a, a model of Dravet syndrome. CONCLUSIONS: Our results suggest a dual and opposing action of P2X7R in epilepsy and suggest P2X7R overexpression in GABAergic interneurons as a novel therapeutic strategy for acquired and, possibly, genetic forms of epilepsy.

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

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.

Epigenetic and transcriptomic reprogramming in monocytes of severe COVID-19 patients reflects alterations in myeloid differentiation and the influence of inflammatory cytokines.

BACKGROUND: COVID-19 manifests with a wide spectrum of clinical phenotypes, ranging from asymptomatic and mild to severe and critical. Severe and critical COVID-19 patients are characterized by marked changes in the myeloid compartment, especially monocytes. However, little is known about the epigenetic alterations that occur in these cells during hyperinflammatory responses in severe COVID-19 patients. METHODS: In this study, we obtained the DNA methylome and transcriptome of peripheral blood monocytes from severe COVID-19 patients. DNA samples extracted from CD14 + CD15- monocytes of 48 severe COVID-19 patients and 11 healthy controls were hybridized on MethylationEPIC BeadChip arrays. In parallel, single-cell transcriptomics of 10 severe COVID-19 patients were generated. CellPhoneDB was used to infer changes in the crosstalk between monocytes and other immune cell types. RESULTS: We observed DNA methylation changes in CpG sites associated with interferon-related genes and genes associated with antigen presentation, concordant with gene expression changes. These changes significantly overlapped with those occurring in bacterial sepsis, although specific DNA methylation alterations in genes specific to viral infection were also identified. We also found these alterations to comprise some of the DNA methylation changes occurring during myeloid differentiation and under the influence of inflammatory cytokines. A progression of DNA methylation alterations in relation to the Sequential Organ Failure Assessment (SOFA) score was found to be related to interferon-related genes and T-helper 1 cell cytokine production. CellPhoneDB analysis of the single-cell transcriptomes of other immune cell types suggested the existence of altered crosstalk between monocytes and other cell types like NK cells and regulatory T cells. CONCLUSION: Our findings show the occurrence of an epigenetic and transcriptional reprogramming of peripheral blood monocytes, which could be associated with the release of aberrant immature monocytes, increased systemic levels of pro-inflammatory cytokines, and changes in immune cell crosstalk in these patients.

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-22.

Objective: 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.

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

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.

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

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.

Microglial innate memory and epigenetic reprogramming in neurological disorders.

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.

The Degree of Cardiac Remodelling before Overload Relief Triggers Different Transcriptome and miRome Signatures during Reverse Remodelling (RR)-Molecular Signature Differ with the Extent of RR.

This study aims to provide new insights into transcriptome and miRome modifications occurring in cardiac reverse remodelling (RR) upon left ventricle pressure-overload relief in mice. Pressure-overload was established in seven-week-old C57BL/6J-mice by ascending aortic constriction. A debanding (DEB) surgery was performed seven weeks later in half of the banding group (BA). Two weeks later, cardiac function was evaluated through hemodynamics and echocardiography, and the hearts were collected for histology and small/bulk-RNA-sequencing. Pressure-overload relief was confirmed by the normalization of left-ventricle-end-systolic-pressure. DEB animals were separated into two subgroups according to the extent of cardiac remodelling at seven weeks and RR: DEB1 showed an incomplete RR phenotype confirmed by diastolic dysfunction persistence (E/e' ≥ 16 ms) and increased myocardial fibrosis. At the same time, DEB2 exhibited normal diastolic function and fibrosis, presenting a phenotype closer to myocardial recovery. Nevertheless, both subgroups showed the persistence of cardiomyocytes hypertrophy. Notably, the DEB1 subgroup presented a more severe diastolic dysfunction at the moment of debanding than the DEB2, suggesting a different degree of cardiac remodelling. Transcriptomic and miRomic data, as well as their integrated analysis, revealed significant downregulation in metabolic and hypertrophic related pathways in DEB1 when compared to DEB2 group, including fatty acid ß-oxidation, mitochondria L-carnitine shuttle, and nuclear factor of activated T-cells pathways. Moreover, extracellular matrix remodelling, glycan metabolism and inflammation-related pathways were up-regulated in DEB1. The presence of a more severe diastolic dysfunction at the moment of pressure overload-relief on top of cardiac hypertrophy was associated with an incomplete RR. Our transcriptomic approach suggests that a cardiac inflammation, fibrosis, and metabolic-related gene expression dysregulation underlies diastolic dysfunction persistence after pressure-overload relief, despite left ventricular mass regression, as echocardiographically confirmed.

Immunogenetic protective factors in Genetic Generalized Epilepsy.

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-1ß -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.

Apolipoprotein E isoforms and susceptibility to genetic generalized epilepsies.

Background: 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.