Common Metabolites, Distinct Pathways: The Use of High-field NMR Spectroscopy Metabolomics in Neurology and Immunology

Abstract

While current metabolic imaging approaches, such as positron emission tomography, hyperpolarized magnetic resonance (MR) imaging and localized MR spectroscopy, provide information on localization and visualization of metabolically-active tissues and metabolites in vivo, additional ex vivo validation and investigations are used for a deeper molecular elucidation of biological events. Metabolomics provides an insight into ongoing cellular processes in a living organism by analyzing and investigating alterations of small molecule polar compounds and lipids. Many of these alterations are directly affected by environmental and genetic factors, such as RNA expression. In recent years, besides mass spectrometry-based approaches, nuclear magnetic resonance (NMR) spectroscopy has been successfully established as an important analytical technique in metabolomics due to its non-destructive nature, high reproducibility and generation of absolute concentration values. NMR, furthermore, is one of the closest approaches to established in vivo MR technologies allowing a direct link from in vivo MR spectra to ex vivo NMR. NMR spectroscopy is, therefore, a perfect tool to quantitatively analyze small polar molecules, such as amino acids, short-chain fatty acids, energy, growth and redox metabolism-related compounds as well as metabolites of gut microbiota and lipids. The broad variety of NMR applications has led to the development of increased sensitivity probes and fully-automated commercial assays. Inventions, such as the ultrasensitive 1.7 mm microprobe, have made it possible to analyze smaller tissue or biofluid amounts of precious samples, including small preclinical organs and tissues or patient tumor biopsies, with appropriate precision and minimal tissue metabolite dilution. In this thesis project, we aimed to investigate and characterize specific metabolic alterations to clinically-relevant immunological and neurological conditions by employing NMR spectroscopy-based metabolomics. In line with the previous preclinical imaging work, two preclinical models – i) acute and chronic inflammation progression and ii) neurological gut-brain axis – were selected as core examples for a comprehensive ex vivo metabolome characterization. In the first project i), the delayed-type hypersensitivity reaction (DTHR) mouse model was subjected to a dynamic immunometabolism and inflammation characterization with the hypothesizing that the inflammation progression is related to dynamic systemic alterations for which metabolomics readout can elucidate the ongoing bio-molecular events. The inflammation was induced by repeated contact tissue challenges on mouse ear tissue. Different metabolic patterns were identified arising from either acute or chronic DTHR that correlated with the resident immune cell response and further active cell infiltration to the inflamed location. Distinct metabolic events, including switches between the scavenging of reactive oxygen and nitrogen species, facilitated the detailed characterization of the detrimental effect of prolonged inflammation and the emergency state of the system. Continuous inflammation led to limited access to substrates for energy metabolism. Chronic DTHR further required alternative anabolic pathways to sustain the cellular growth and repair process. In the second project ii), a gastric bypass surgery rat model was used for the metabolomic characterization of gut microbiota metabolites and potential gut-brain axis communication. We hypothesized that gut-brain axis communication could be responsible for the beneficial lasting effects after surgical intervention. Rats were fed a liquid sucrose diet to induce obesity since high-sugar beverages and liquid caloric consumption have become a pandemic in the adolescent population hindered by the general concept of the Western diet. Plasma and feces were studied as gut metabolism readout and further analyzed in the context of fecal microbiome, hepatic lipid profiles, and brain activity imaging to obtain a holistic overview of the systemic effects of the Roux-En-Y gastric bypass (RYGB) surgery. The gut metabolite γ-aminobutyrate (GABA) was increased in surgery animal feces together with GABA-producing microbiota species abundance compared to sham controls. RYGB surgery animals showed greater neuronal activation in midbrain regions that are known to be rich in GABAergic cells, pointing towards an activated gut-brain communication resulting from the surgery. Two main projects demonstrate how the usage of optimized preanalytical procedures, together with harmonized analytical NMR workflows provide reproducible data with comprehensive insight into the metabolism of an organism ex vivo. Further outlook includes metabolomics result integration in the context of in vivo imaging data, as the combined result evaluation can facilitate the understanding of health and disease progression, and help to streamline diagnostic, such as novel radiotracer development, and therapeutic approaches.