Project description:Etiology matters - Comparing Genomic DNA Methylation Patterns in Three Rat Models of Acquired Epilepsy (Amygdala stimulation – MBD-seq)
Project description:Etiology matters - Comparing Genomic DNA Methylation Patterns in Three Rat Models of Acquired Epilepsy (Amygdala stimulation – mRNA-seq)
Project description:Purpose: Traumatic brain injury (TBI) causes 10%–20% of structural epilepsies and 5% of all epilepsies. The lack of prognostic biomarkers for post-traumatic epilepsy (PTE) is a major obstacle to the development of anti-epileptogenic treatments. In this study, we conducted high throughput small RNA-Seq analysis from tail-vein plasma samples collected from a rat model of PTE to discover miRNA biomarker candidates that could serve as prognostic biomarkers for brain damage severity and the development of PTE. Methods: Epileptogenesis was induced in adult male Sprague-Dawley rats by the lateral fluid-percussion-induced TBI. Epilepsy was defined as the occurrence of at least 1 unprovoked seizure during continuous 1-month video-electroencephalography monitoring in the sixth post-TBI month. Small RNA-seq was performed from tail-vein plasma samples collected from a subset of 20 animals (4 sham-operated controls, 7 TBI rats with epilepsy, 9 TBI rats without epilepsy) at 2 days and 9 days post-TBI. RNA was extracted from 200 μl plasma using an miRNeasy Mini Kit. Small RNA-Seq was conducted by GenomeScan (Leiden, the Netherlands). Small RNA library preparation was performed using the Illumina TruSeq Small RNA Sample Prep Kit. Single-end sequencing was performed on the Illumina HiSeq 4000.
Project description:TBI was induced with lateral fluid-percussion injury in adult male rats. MBD-seq of the perilesional cortex, ipsilateral thalamus and ipsilateral hippocampus was performed at 3 months post-TBI. The data was used to identify differential methylation of gene promoter, gene body, and exons in the perilesional cortex , thalamus and hippocampus.
Project description:Although it is well known that stroke and head trauma are one of the high risk factors for the development of acquired epilepsy, the cellular mechanisms underlying the epileptogenesis is not well understood. Using rodent models of ischemic stroke and head trauma (partial cortical isolation, undercut), we comparatively analyzed transcription profiles between two different models to explore the commonality. Despite well-known risk factors such as stroke and head trauma, it has not been clinically effective to prevent acquired epilepsy. To do so, it is crucial to understand what commonly drives neural hyperexcitability. Using comparative transcriptome analyses with different models of acquired epilepsy including stroke and head trauma (the present GEO case), as well as blood-brain barrier (BBB) disruption, albumin, and TGFβ application (GEO accession number: GSE12304), we show that TGFβ signaling activation following BBB disruption commonly occurs regardless of brain region and insult types, accompanied by the strong upregulation of genes relevant to inflammation and extracellular matrix (ECM) modulation.
Project description:DNA methylation and hydroxymethylation have been implicated in normal development and differentiation, but our knowledge about the genome-wide distribution of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) during cellular differentiation remains limited. Using in vitro model system of gradual differentiation of human embryonic stem (hES) cells into ventral midbrain-type neural precursor (NP) cells and terminally into dopamine (DA) neurons, we explored changes in 5mC or 5hmC patterns during lineage commitment. We used three techniques, 450K DNA methylation array, MBD-seq, and hMeDIP-seq, and found combination of these methods can provide comprehensive information on the genome-wide 5mC or 5hmC patterns. We observed dramatic changes of 5mC patterns during differentiation of hES cells into NP cells. Although genome-wide 5hmC distribution was more stable than 5mC, coding exons, CpG islands and shores showed dynamic 5hmC patterns during differentiation. In addition to the role of DNA methylation as a mechanism to initiating gene silencing, we also found DNA methylation as a locking system to maintain gene silencing. More than 1,000 genes including mesoderm development related genes acquired promoter methylation during neuronal differentiation even though they were already silenced in hES cells. Finally, we found that activated genes lost 5mC in transcription start site (TSS) but acquired 5hmC around TSS and gene body during differentiation. Our findings may provide clues for elucidating the molecular mechanisms underlying lineage specific differentiation of pluripotent stem cells during human embryonic development. Examination of hMeDIP-Seq and MBD-Seq in 3 cell types (human embryonic stem, neural precursor, and dopamine neuron cells)
Project description:Traumatic brain injury (TBI) is a prevalent and debilitating condition, which often leads to the development of post-traumatic epilepsy (PTE), a condition that yet lacks preventive strategies. Biperiden, an anticholinergic drug, is a promising candidate that has shown efficacy in murine models of PTE. MicroRNAs (miRNAs), small regulatory RNAs, can help in understanding the biological basis of PTE, and act as TBI- and PTE-relevant biomarkers that can be detected peripherally, as they are present in extracellular vesicles (EVs) that cross the blood brain barrier. This study aimed to investigate miRNAs in serum EVs from patients with TBI, and their association with biperiden treatment and PTE. Blood samples of 37 TBI patients were collected 10 days after trauma and treatment initiation in a double-blind clinical trial. A total of 18 patients received biperiden, with 3 subjects developing PTE, and 19 received placebo, with 2 developing PTE. Serum EVs were characterized by size distribution and protein profiling, followed by high-throughput sequencing of the EV miRNome. Differential expression analysis revealed no significant differences in miRNA expression between TBI patients with and without PTE. Interestingly, miR-9-5p displayed decreased expression in biperiden-treated patients compared to the placebo group. This miRNA regulates genes enriched in stress response pathways, including axonogenesis and neuronal death, relevant to both PTE and TBI. These findings suggest serum miR-9-5p as a possible marker for biperiden treatment, which may play a role in TBI resolution.