Authors:	Lerner, Raissa
Title:	Development of quantitative lipidomic strategies in neurological diseases for discovery and monitoring of tissue and blood markers -application to kainic acid mouse model of epilepsy
Online publication date:	21-Mar-2018
Year of first publication:	2018
Language:	english
Abstract:	Lipids are small hydrophobic or amphipathic biomolecules with a huge structural heterogeneity, which reflects the diversity of lipid function. Considering the broad spectrum of lipid functions, it becomes obvious that lipid related disturbances are implicated in several disorders. Consequently, a growing number of studies have evidenced the involvement of lipids in the development and progression of various diseases, such as epilepsy. This neurological disease, affecting about 1% of the human population, is characterized by recurrent seizures. A mouse model which mimics mainly seizures observed in temporal lobe epilepsy, a category of epilepsy which mainly occurs in the limbic system, can be obtained by injection of kainic acid, a ligand for a specific group of ionotropic glutamate receptors. A growing number of studies reveal alterations in lipid levels using such epileptic seizure models and thus evidence the implication of lipid signals and their potential role as biomarkers or therapeutic targets. In order to provide a tool for future lipid analysis under pathological conditions in clinical and research samples, we developed a method for lipidomic profiling and applied it in a first proof of concept study to a mouse model of epileptic seizures induced by systemic kainic acid injection. Mass spectrometry allows concurrent qualitative and quantitative profiling of multiple lipids without previous molecule identification and antibodies, thereby rendering the highest amount of structural data in short analysis time and at lower costs compared to other techniques. Moreover, mass spectrometry combined with liquid chromatography provides highest sensitivity for molecule quantification, thus allowing analysis of lowest sample amounts, which is often inevitable when investigating biological systems. Hence, mass spectrometry represents an adequate tool for comprehensive lipidomic research, most suitable for biological and clinical investigations and accordingly the technique of choice in this study. The protocol developed for lipidomic profiling was proved to be suitable for simultaneous analysis of phospholipids, endocannabinoids and endocannabinoid-related compounds from microgram amounts of tissue, e.g. brain punches, and revealed new insights in lipid plasticity associated with epileptic seizures. Since thorough elucidation of lipid pathways requires additional knowledge about other molecules involved, such as enzymes and receptors, in a second objective of this study, we developed a protocol for combined lipidomic and transcriptomic profiling. Therefore, we optimized the protocol for lipid extraction in order to allow simultaneous mRNA extraction and subsequent analysis. We demonstrated the established method as an excellent, versatile tool for combined lipidomic and transcriptomic profiling from microgram tissue amounts, and therefore benefiting analysis time and costs, as well as data reliability by circumventing pitfalls deriving from different animal batches and tissue heterogeneity. Applied to brain punches of the epileptic seizure mouse models, we gained new information about lipid-related processes within epilepsy at higher spatial resolution where distinct, less expected pathways of lipid metabolism are affected at acute epilepsy phase. Moreover, our data strongly support the need for subregional specificity when investigating neurological diseases, as for instance the dorsal and the ventral hippocampus showed contrary lipid changes, hence differentially regulated lipid metabolism, which might be masked when analyzing the whole hippocampus at once. This result was further underlined by mass spectrometry imaging experiments, applied in a third objective to the epileptic seizure mouse models, aiming at increased spatial resolution to target lipids across cell layers and populations. An additional outcome of these experiments revealed alterations in phospholipid adduct ions associated with epileptic seizures, pointing toward ion conversion in membrane lipids with acute epilepsy, which constitutes further potential target for disease treatment. In conclusion, the methods established in this thesis, represent valuable tools to increase our understanding about disturbances in lipid pathways associated with diseases, leverage new mechanistic knowledge on the diseases, enhance the predictability of diseases and thus allow reliable diagnosis and design of new therapeutic schemes for patients.
DDC:	570 Biowissenschaften 570 Life sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 04 Medizin
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-2643
URN:	urn:nbn:de:hebis:77-diss-1000019385
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	https://rightsstatements.org/vocab/InC/1.0/
Extent:	iv, 141 Seiten
Appears in collections:	JGU-Publikationen