Project description:There are no blood-based molecular biomarkers of temporal lobe epilepsy (TLE) to support clinical diagnosis. MicroRNAs are short noncoding RNAs with strong biomarker potential due to their cell-specific expression, mechanistic links to brain excitability, and stable and reliable detection in biofluids. Altered expression of circulating microRNAs has been reported in human epilepsy, but most studies collected samples from one clinical site, relied on a single platform for profiling or conducted minimal validation. We collected plasma samples from video-electroencephalogram-monitored adult TLE patients at epilepsy specialist centers in two different countries, performed genome-wide PCR-based and RNA sequencing during the discovery phase and validated in a large cohort of samples (>300 samples) that included patients with psychogenic non-epileptic seizures. After profiling, validation of the discovery cohort and validation in the larger patient groups we identified miR-27a-3p, miR-328-3p and miR-654-3p with strong TLE biomarker potential. Plasma levels of these microRNAs were regulated in the same direction in plasma from epileptic mice, and furthermore were not different to healthy controls in patients with psychogenic non-epileptic seizures. The biomarker potential was extended by determining microRNA copy number in plasma and we demonstrate rapid detection of these microRNAs using an electrochemical RNA microfluidic disk as a prototype point-of-care device. Investigation of the molecular transport mechanism in plasma determined analysis of all three microRNAs within the exosome-enriched provided highest diagnostic accuracy while levels of Argonaute-bound miR-328-3p selectively increased in patient samples collected after seizures. In situ hybridization revealed the presence of miR-27a-3p and miR-328-3p within neurons in human brain and bioinformatics analysis predicted targets linked to growth factor signaling and apoptosis. Taken together, this study extends evidence for the biomarker potential of circulating microRNAs for epilepsy diagnosis and mechanistic links to underlying pathomechanisms. microRNA expression in the plasma of 16 patients with TLE, before and after seizure, and 16 controls was measured by TaqMan OpenArray Human MicroRNA Panel.

Project description:Aims: To identify transcriptome signatures underlying epileptogenesis in temporal lobe epilepsy (TLE).Methods: Robust rank aggregation analysis was used to integrate multiple microarrays in rodent models of TLE and determine differentially expressed genes (DEGs) in acute, latent, and chronic stages. Functional annotation and protein-protein interaction analysis were performed to explore the potential functions of the DEGs and identify hub genes with the highest intramodular connectivity. The association between hub genes and hippocampal sclerosis/seizure frequency was analyzed using publicly available RNA-sequencing datasets from TLE patients. We subsequently established a pilocarpine-induced status epilepticus (SE) model in rats and validated mRNA expression of hub genes by quantitative reverse transcription PCR (qRT-PCR).Results: The DEGs in the acute, latent, and chronic phases of TLE in animal models were prominently enriched in inflammatory response. Hub genes identified in the acute phase mainly participated in biological processes including inflammation, blood-brain barrier damage, and cell adhesion. The hub genes in the latent phase were related to microglia/macrophage activation (Emr1 and Aif1) and phagocytosis (Cd68, Tyrobp, and Lyz). In the chronic phase, the hub genes were associated with activation of complements and microglia/macrophages. We further found that some hub genes identified in human TLE, such as Tlr2, Lgals3, and Stat3, were positively correlated with seizure frequency. Other hub genes, including Lgals3 and Serpine1, were associated with hippocampus sclerosis. qRT-PCR analysis confirmed that the mRNA levels of hub genes in rat hippocampus were significantly up-regulated after SE induction.Conclusions: Our integrated analysis identified hub genes in different stages of epilepsy. The functional annotations suggest that the activation and phagocytic activities of microglia/macrophages may play critical roles in epileptogenesis of TLE.

Project description:There are no blood-based molecular biomarkers of temporal lobe epilepsy (TLE) to support clinical diagnosis. MicroRNAs are short noncoding RNAs with strong biomarker potential due to their cell-specific expression, mechanistic links to brain excitability, and stable and reliable detection in biofluids. Altered expression of circulating microRNAs has been reported in human epilepsy, but most studies collected samples from one clinical site, relied on a single platform for profiling or conducted minimal validation. We collected plasma samples from video-electroencephalogram-monitored adult TLE patients at epilepsy specialist centers in two different countries, performed genome-wide PCR-based and RNA sequencing during the discovery phase and validated in a large cohort of samples (>300 samples) that included patients with psychogenic non-epileptic seizures. After profiling, validation of the discovery cohort and validation in the larger patient groups we identified miR-27a-3p, miR-328-3p and miR-654-3p with strong TLE biomarker potential. Plasma levels of these microRNAs were regulated in the same direction in plasma from epileptic mice, and furthermore were not different to healthy controls in patients with psychogenic non-epileptic seizures. The biomarker potential was extended by determining microRNA copy number in plasma and we demonstrate rapid detection of these microRNAs using an electrochemical RNA microfluidic disk as a prototype point-of-care device. Investigation of the molecular transport mechanism in plasma determined analysis of all three microRNAs within the exosome-enriched provided highest diagnostic accuracy while levels of Argonaute-bound miR-328-3p selectively increased in patient samples collected after seizures. In situ hybridization revealed the presence of miR-27a-3p and miR-328-3p within neurons in human brain and bioinformatics analysis predicted targets linked to growth factor signaling and apoptosis. Taken together, this study extends evidence for the biomarker potential of circulating microRNAs for epilepsy diagnosis and mechanistic links to underlying pathomechanisms.

Dataset Information

Genome-wide microRNA profiling of human temporal lobe epilepsy identifies modulators of the immune response.

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Genome-wide microRNA profiling of human temporal lobe epilepsy identifies modulators of the immune response.

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