Modelling astrocytic metabolism in actual cell morphologies

The human brain is the most structurally and biochemically complex organ, and its broad spectrum of diverse functions is accompanied by high energy demand. In order to address this high energy demand, brain cells of the central nervous system are organised in a complex and balanced ecosystem, and perturbation of brain energy metabolism is known to be associated with neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's disease. Among all cells composing this ecosystem, astrocytes contribute metabolically to produce the primary energy substrate of life, \ATP, and lactate, which can be exported to neurons to support their metabolism. Astrocytes have a star-shaped morphology, allowing them to connect on the one side with blood vessels to uptake glucose and on the other side with neurons to provide lactate. Astrocytes may also exhibit metabolic dysfunctions and modify their morphology in response to diseases. A mechanistic understanding of the morphology-dysfunction relation is still elusive. This thesis developed and applied a mechanistic multiscale modelling approach to investigate astrocytic metabolism in physiological morphologies in healthy and diseased human subjects. The complexity of cellular systems is a significant obstacle in investigating cellular behaviour. Systems biology tackles biological unknowns by combining computational and biological investigations. In order to address the elusive connection between metabolism and morphology in astrocytes, we developed a computational model of central energy metabolism in realistic morphologies. The underlying processes are described by a reaction-diffusion system that can represent cells more realistically by considering the actual three-dimensional shape than classical ordinary differential equation models where the cells are assumed to be spatially punctual, i.e. have no spatial dimension. Thus, the computational model we developed integrates high-resolution microscopy images of astrocytes from human post-mortem brain samples and simulates glucose metabolism in different physiological astrocytic human morphologies associated with AD and healthy conditions. The first part of the thesis is dedicated to presenting a numerical approach that includes complex morphologies. We investigate the classical finite element method (FEM) and cut finite element method (\cutfem{}) for simplified metabolic models in complex geometries. Establishing our image-driven numerical method leads to the second part of this thesis, where we investigate the crucial role played by the locations of reaction sites. We demonstrate that spatial organisation and chemical diffusivity play a pivotal role in the system output. Based on these new findings, we subsequently use microscopy images of healthy and Alzheimer's diseased human astrocytes to build simulations and investigate cell metabolism. In the last part of the thesis, we consider another critical process for astrocytic functionality: calcium signalling. The energy produced in metabolism is also partially used for calcium exchange between cell compartments and mainly can drive mitochondrial activity as a main ATP generating entity. Thus, the active cross-talk between glucose metabolism and calcium signalling can significantly impact the metabolic functionality of cells and requires deeper investigation. For this purpose, we extend our established metabolic model by a calcium signalling module and investigate the coupled system in two-dimensional geometries. Overall, the investigations showed the importance of spatially organised metabolic modelling and paved the way for a new direction of image-driven-meshless modelling of metabolism. Moreover, we show that complex morphologies play a crucial role in metabolic robustness and how astrocytes' morphological changes to AD conditions lead to impaired energy metabolism

Degradation and degeneration :synergistic impact of autophagy and mitochondrial dysfunction in Parkinson's disease

PhD ThesisThe single greatest risk factor for the development of idiopathic Parkinson’s Disease is advancing age. The differences at the cellular level that cause some individuals to develop this highly debilitating disease over healthy ageing are not fully understood. Mitochondrial dysfunction has been implicated in the pathogenesis of Parkinson’s disease (PD) since the drug MPTP, known to cause Parkinson’s like symptoms, was shown to invoke its deleterious effect through inhibition of Complex I (CI) of the mitochondrial electron transport chain. Since this discovery in the 1980s, several causative genes in the much rarer familial forms of PD have been shown to encode proteins which function within, or in association with mitochondria. Through inherited cases of the disorder the process through which mitochondria are removed, mitophagy, a specialized form of autophagy has also been associated with the pathogenesis that leads to en masse cell death in this disorder. This work explores the interplay between mitochondrial deficiencies, through complex I dysfunction, and changes to autophagic processes. The methodologies to enable these observations are also described in detail with the development of novel and specialized techniques necessary to answer many of the specific research questions. The mechanisms behind complex I deficiency’s impact upon cellular processes is also explored as part of this thesis. Mitochondria and autophagy are irrevocably linked through mitochondrial dynamics, to this end an exploration of the greater impact complex I dysfunction has upon mitochondrial motility, fission and fusion was investigated. As the most prevalent neurodegenerative movement disorder of old age, understanding the molecular changes that result in Parkinson's Disease is vital to increase knowledge and offer novel therapeutic targets. Parallel studies in human upper midbrain tissue and cybrid cell lines within this work have revealed significant changes to both autophagy and mitochondrial dynamics in response to complex I deficiency. Given that mitochondrial ‘health’ and autophagic regulation directly impact upon one another identifying how exactly these may contribute to neuronal loss will hopefully allow therapeutic modulation at a point of PD pathogenesis where cells can still be retained

Mitochondrial Fission After Traumatic Brain Injury

Mitochondrial dysfunction is a central feature in the pathophysiology of Traumatic Brain Injury (TBI). Loss of mitochondrial function disrupts normal cellular processes in the brain, as well as impedes the ability for repair and recovery, creating a vicious cycle that perpetuates damage after injury. To maintain metabolic homeostasis and cellular health, mitochondria constantly undergo regulated processes of fusion and fission and functionally adapt to changes in the cellular environment. An imbalance of these processes can disrupt the ability for mitochondria to functionally meet the metabolic needs of the cell, therefore resulting in mitochondrial damage and eventual cell death. Excessive fission, in particular, has been identified as a key pathological event in neuronal damage and death in many neurodegenerative disease models. Specifically, dysregulation of the primary protein regulator of mitochondrial fission, Dynamin-related Protein 1 (Drp1), has been implicated as an underlying mechanism associated with excessive fission and neurodegeneration; however, whether dysregulation of Drp1 and excessive fission occur after TBI and contribute to neuropathological outcome is not well known. The studies described in this dissertation investigate the following hypothesis: TBI causes dysregulation of Drp1 and increases mitochondrial fission in the hippocampus, and inhibiting Drp1 will reduce mitochondrial dysfunction, reduce neuronal damage, and improve cognitive function after injury. Results from these studies revealed four key findings: 1) Experimental TBI increases Drp1 association with mitochondria, and 2) causes acute changes in Drp1-mediated mitochondrial morphology that persists post-injury, indicating increased mitochondrial fission acutely after injury. Additionally, 3) post-injury treatment with a pharmacological inhibitor of Drp1, Mdivi-1, improved survival of newly born neurons in the injured hippocampus, and 4) improved hippocampal-dependent cognitive function after experimental TBI. Taken together, results from these studies reveal that TBI causes excessive Drp1-mediated mitochondrial fission and that this pathological fission state may play a key role in hippocampal neuronal death and cognitive deficits after TBI. Furthermore, these findings indicate inhibition of Drp1 and mitochondrial fission as a potential therapeutic strategy to improve neuronal recovery and cognitive function after injury

Dissecting tumor cell heterogeneity in 3D cell culture systems by combining imaging and next generation sequencing technologies

Three-dimensional (3D) in vitro cell culture systems have advanced the modeling of cellular processes in health and disease by reflecting physiological characteristics and architectural features of in vivo tissues. As a result, representative patient-derived 3D culture systems are emerging as advanced pre-clinical tumor models to support individualized therapy decisions. Beside the additional progress that has been achieved in molecular and pathological analyses towards personalized treatments, a remaining problem in both primary lesions and in vitro cultures is our limited understanding of functional tumor cell heterogeneity. This phenomenon is increasingly recognized as key driver of tumor progression and treatment resistance. Recent technological advances in next generation sequencing (NGS) have enabled unbiased identification of gene expression in low-input samples and single cells (scRNA-seq), thereby providing the basis to reveal cellular subtypes and drivers of cell state transitions. However, these methods generally require dissociation of tissues into single cell suspensions, which consequently leads to the loss of multicellular context. Thus, a direct or indirect combination of gene expression profiling with in situ microscopy is necessary for single cell analyses to precisely understand the association between complex cellular phenotypes and their underlying genetic programs. In this thesis, I will present two complementing strategies based on combinations of NGS and microscopy to dissect tumor cell heterogeneity in 3D culture systems. First, I will describe the development and application of the new method ‘pheno-seq’ for integrated high-throughput imaging and transcriptomic profiling of clonal tumor spheroids derived from models of breast and colorectal cancer (CRC). By this approach, we revealed characteristic gene expression that is associated with heterogeneous invasive and proliferative behavior, identified transcriptional regulators that are missed by scRNA-seq, linked visual phenotypes and associated transcriptional signatures to inhibitor response and inferred single-cell regulatory states by deconvolution. Second, by applying scRNA-seq to 12 patient-derived CRC spheroid cultures, we identified shared expression programs that relate to intestinal lineages and revealed metabolic signatures that are linked to cancer cell differentiation. In addition, we validated and complemented sequencing results by quantitative microscopy using live-dyes and multiplexed RNA fluorescence in situ hybridization, thereby revealing metabolic compartmentalization and potential cell-cell interactions. Taken together, we believe that our approaches provide a framework for translational research to dissect heterogeneous transcriptional programs in 3D cell culture systems which will pave the way for a deeper understanding of functional tumor cell heterogeneity

Molecular support for temporal dynamics of induced anti-herbivory defenses in the brown seaweed Fucus Vesiculosus

Grazing by the isopod Idotea baltica induces chemical defenses in the brown seaweed Fucus vesiculosus. A combination of a 33 day induction experiment, feeding choice assays and functional genomic analyses was used to investigate temporal defense patterns and to correlate changes in palatability to changes in gene expression. Despite permanent grazing, seaweed palatability varied over time. Controls were significantly more consumed than grazed pieces only after 18 and 27 days of grazing. Relative to controls, 562/402 genes were up-/down-regulated in seaweed pieces that were grazed for 18 days, i.e. when defense induction was detected. Reprogramming of the regulative expression orchestra (translation, transcription), up-regulation of genes involved in lipid and carbohydrate metabolism, intracellular trafficking, defense and stress response, as well as downregulation of photosynthesis was found in grazed seaweed. These findings indicate short-term temporal variation in defenses and that modified gene expression patterns arise at the same time when grazed seaweed pieces show reduced palatability. Several genes with putative defensive functions and cellular processes potentially involved in defence, such as reallocation of resources from primary to secondary metabolism, were reveale