Advancing Research in Metabolic Disorders through Integration of Model Systems and Metabolomics

The focus of this thesis is the development and application of different metabolomic(s) approaches to characterize alterations in model systems reflecting inborn errors of metabolism (IEM). IEM are diseases mostly caused by mutations in a single gene resulting in a change in the physiological processes that underly the functioning of a healthy individual. The impact of monogenetic perturbations depends on many different factors. In some cases, no effect is observed due to redundancy with similar enzymes or compensation via other metabolic routes. Alternatively, lack of the encoded gene product may result in the malfunctioning of a complete pathway or accumulation of a toxic intermediate. Both consequences can lead to severe clinical phenotypes. In addition, environmental factors like dietary intake, age and infections can influence clinical manifestations. By studying metabolic alterations, we aim to define the role of a gene in the context of the metabolic network with the ultimate goal of finding leads that can be used as a basis for therapeutic interventions.

In first part of this thesis we aimed to develop an untargeted fluxomics assay based on Direct Infusion High Resolution Mass Spectrometry (DI-HRMS) (chapter 2). This method, which we named DI-HRMS-BIT as an acronym for DI-HRMS based isotope tracing, is a metabolic approach that provides an overarching view of cellular metabolism. By combining isotope tracing approaches with DI-HRMS one can track the spread of labeled atoms over the metabolic network in time. Consequently, we can obtain insights into the fate of substrates and how their downstream products are orchestrated throughout metabolism.

In the second part of this thesis, we applied DI-HRMS-BIT in combination with targeted metabolomics approaches to examine the underlying pathophysiology of inherited disorders of NAD+ metabolism (chapter 3 and 4). NAD+ regulation is important for human health as it has a diverse range of biological functions both as a redox cofactor and as a substrate for important signaling enzymes like SIRTUINS (SIRTs) and Poly (ADP-ribose) polymerases (PARPs). This is evident from genetic disorders affecting NAD synthesis or redox status that have a severe impact on the patient’s health status.

In the third part of this thesis, we examine the consequences of enhanced mTORC1 signaling in different model systems. mTORC1 is considered one of the master regulators of metabolism and under favorable conditions stimulates anabolism and halts catabolism. Its activity is under the negative control of the tuberous sclerosis complex (TSC1/2). Loss of TSC1 or TSC2, or activating mutations in mTOR lead to pleiotropic phenotypes, including epilepsy. Epilepsy is one of the most common clinical features in patients suffering from tuberous sclerosis yet its link with the underlying physiological processes is poorly understood. In chapter 5 we therefore examined the metabolome of the cortex and hippocampus of mice with inducible neuronal specific deletion of Tsc1. To further examine the impact of enhanced mTORC1 activity we studied the metabolome of various cell model systems including lack of TSC2 and othergenetic alterations resulting in enhanced mTORC1 activity (chapter 6).