Investigation of the Molecular Mechanisms Responsible for Absence Epilepsy in a Genetic Rat Model Using Spatiotemporal RNA Sequencing
ÖZDEMİR Ö. (Executive), ONAT F., ÇARÇAK YILMAZ N., ÖZBEK U.
TUBITAK Project, 1001 - Program for Supporting Scientific and Technological Research Projects, 2022 - 2025
- Project Type: TUBITAK Project
- Support Program: 1001 - Program for Supporting Scientific and Technological Research Projects
- Begin Date: November 2022
- End Date: November 2025
Project Abstract
Epilepsy is a health condition that affects approximately 1-3% of the population, with genetics playing a significant role in its etiology. Genetic generalized epilepsies (GGE), which include absence seizures as one of the classic seizure phenotypes and exhibit frequent genetic inheritance, affect approximately 0.3% of the population and account for 30-40% of all epilepsies. Despite the frequent observation of genetic inheritance, due to the complex nature of GGEs, previous studies have not provided high-resolution insights into the molecular etiopathogenesis of GGEs, and information on the epileptogenesis process remains insufficient. A subtype of GGEs, genetic absence epilepsies (GAE), which have a high prevalence particularly among children and adolescents, are complex disorders primarily caused by genetic factors. Among the most commonly used experimental models meeting the criteria for absence-type non-convulsive seizures are GAERS (Genetic Absence Epilepsy Rats from Strasbourg), a genetic animal model exhibiting spontaneous spike-wave discharges, which is one of the most widely used animal models in epilepsy research worldwide.
With recent advancements in genomic technologies, it has become possible to conduct genetic research at an unprecedented level of resolution. GAERS models, in particular, have the potential to provide highly detailed and novel insights into genetic epilepsies.
In the proposed project, electroencephalography (EEG) will be used to anatomically determine seizure foci in GAERS during the epileptogenesis phase on postnatal day 15 (P15) and postnatal day 30 (P30). EEG recordings will be taken, and these regions will be frozen for sectioning after stereotaxic surgery in GAERS and control rats at P15 and P30. From the frozen brain tissues, four series of sections will be obtained as 10μm, 10μm, 20μm, and 20μm. The first section will be used to determine tissue architecture, the second for spatiotemporal transcriptomic analysis using the 10X Genomics Visium platform, the third for laser microdissection of identified spatially differentiated cell groups, and the fourth for validation purposes. In this scope, epileptogenic cell groups, which are found to be distinct from their surrounding cells and healthy control cells in terms of spatiotemporal transcriptomic profile, will be dissected under a microscope using laser capture microdissection. Subsequently, RNA sequencing will be performed on the isolated "epileptogenic" cell RNA by adding unique molecular identifiers (UMIs). This will allow for the first-ever detailed transcriptomic profiling of epileptic cell groups specific to GAERS.
The data obtained from this study will facilitate the development of new drug targets and epileptogenic treatment approaches. Additionally, orthologous analysis conducted within the study will establish links between human genes and the rat transcriptomic profile, prioritizing genes based on their potential impact on epilepsy. All datasets generated within the project will be made available to researchers through a dedicated web platform. This will enable the re-evaluation of exome/genome sequencing data that initially yielded negative results for epilepsy phenotypes in human cases, potentially leading to the discovery of new human genes associated with epilepsy.