Periodic Reporting for period 1 - AstroSignals (Spatiotemporal dynamics of subcellular energy metabolism in astrocytes) Reporting period: 2015-04-01 to 2017-03-31 Summary of the context and overall objectives of the project Subcellular compartmentalization of signal transduction is of critical importance for cellular function. Second messenger molecules within cells, such as calcium, activate or inhibit multiple targets, and it's only through the spatial and temporal compartmentalization of these messengers into microdomains that specificity and versatility of signal transduction is made possible. Thereby, instead of a global activation of all its subcellular targets, a second messenger activates only the targets that are within its microdomain. While it is well established that signal transduction inside cells in spatially and temporally compartmentalized, there remains a widespread assumption that the metabolic pathways providing energy, in the form of ATP, for sub-cellular signal transduction are not compartmentalized, but merely feed a global cellular pool of ATP. The overall objectives of this project are to compile and critically review the current evidence for sub-cellular compartmentalization of energy metabolism, and to establish experimentally whether we can find evidence for such compartmentalization in human astrocytes, the most abundant type of cell in the human brain. As a related issue, we developed a hypothesis of a novel pathway for subcellular glucose compartmentalization inside astrocytes. This pathway could explain the very efficient glucose uptake into these cells and facilitate astrocytic provision of metabolic support for neurons, one of the most important functions of astrocytes. An additional objective therefore is to experimentally test this hypothesis.Combining state-of-the-art techniques and expertise in the fields of cell signalling and metabolism, the results of this project enhance our understanding of metabolic regulation of signal transduction. A deeper understanding of the organization of subcellular energy metabolism and it's regulation of signalling may in the future open new possibilities for targeted treatments of brain diseases. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Using chemical, fluorescent indicators of cytosolic calcium in analyses of cell populations as well as single cells, in combination with an analysis of which genes are expressed in human astrocytes, we have characterised which subtypes of purinergic receptors are functionally expressed in human astrocytes. The results, indicating that only two types of receptors are functionally expressed, and that this expression profile differs from the one expected from previous analyses, are currently under review at an international peer-reviewed journal.Using chemical, fluorescent indicators of cytosolic calcium in analyses of astrocytic cell populations and specific inhibitors of different ATP-providing pathways, we determined that only two sources of cellular ATP provision appear to affect a certain type of calcium signals in human astrocytes, evoked by the receptor PAR1. This indicates that micro domains of cellular ATP might exists in these cells, locally supporting energy-demanding signalling processes. We are currently continuing this project to analyse which steps in the PAR1-evoked signalling cascade are dependent on local provision of ATP.We have obtained evidence that supports our hypothesis of a previously unknown pathway for subcellular glucose compartmentalization inside astrocytes. We have used advanced optical imaging techniques, such as total internal reflection fluorescence microscopy and confocal imaging, that allow visualization of sub-cellular processes with the aid of fluorescent biosensors. Using a sub-cellularly targeted fluorescent biosensor for glucose we found indications for a dynamic pathway for glucose uptake and glucose distribution inside astrocytes. We also establisehd a protocol that allows astrocytes to be grown inside microfluidic chambers, which permit fluidic isolation of cell body from cell extensions. This protocol enables to selectively change the chemical environment of cell body and cell extensions, mimicking conditions inside the body, where cell extensions will be exposed to different chemical signals than the cell body of brain cells. The results of this work are currently being finalized, and will be presented at the International Society for Neurochemistry Conference in Paris in August 2017. In addition the results will, upon completion within the next few months, be submitted to an international peer-reviewed journal. All publications from this project will be open access, allowing members of the public to access the articles. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) We have characterized the functional expression of purinergic receptors in human astrocytes. As these receptors are involved in a number of neruodegenerative processes, such as ischemia-induced injury and Alzheimer’s disease, our characterization will impact future research on these diseases.The protocol for fluidic isolation of astrocytic soma from astrocytic processes that was developed in the course of this project can have an impact on basic as well as applied brain research, allowing new experimental strategies by mimicking the heterogeneous environment of astrocytes inside the brain.The discovery of a new pathway for cellular glucose distribution inside astrocytes greatly enhances our understanding of the metabolic processes inside these cells, whose metabolic support of neurons is necessary for normal human brain function. These results will have an impact on our interpretation of past research on metabolism in astrocytes, and may open up a new research area for the neuroscience community. The results may also initiate research on whether analogous phenomena exists in other human cell types.
