Glycolytically impaired Drosophila glial cells fuel neural metabolism via ß-oxidation.

Neuronal function is highly energy demanding and thus requires efficient and constant metabolite delivery by glia. Drosophila glia are highly glycolytic and provide lactate to fuel neuronal metabolism. Flies are able to survive for several weeks in the absence of glial glycolysis. Here, we study how Drosophila glial cells maintain sufficient nutrient supply to neurons under conditions of impaired glycolysis. We show that glycolytically impaired glia rely on mitochondrial fatty acid breakdown and ketone body production to nourish neurons, suggesting that ketone bodies serve as an alternate neuronal fuel to prevent neurodegeneration. We show that in times of long-term starvation, glial degradation of absorbed fatty acids is essential to ensure survival of the fly. Further, we show that Drosophila glial cells act as a metabolic sensor and can induce mobilization of peripheral lipid stores to preserve brain metabolic homeostasis. Our study gives evidence of the importance of glial fatty acid degradation for brain function, and survival, under adverse conditions in Drosophila.

Single-Fluorophore Indicator to Explore Cellular and Sub-cellular Lactate Dynamics.

Lactate is an energy substrate and an intercellular signal, which can be monitored in intact cells with the genetically encoded FRET indicator Laconic. However, the structural complexity, need for sophisticated equipment, and relatively small fluorescent change limit the use of FRET indicators for subcellular targeting and development of high-throughput screening methodologies. Using the bacterial periplasmic binding protein TTHA0766 from Thermus thermophilus, we have now developed a single-fluorophore indicator for lactate, CanlonicSF. This indicator exhibits a maximal fluorescence change of 200% and a KD of ∼300 µM. The fluorescence is not affected by other monocarboxylates. The lactate indicator was not significantly affected by Ca2+ at the physiological concentrations prevailing in the cytosol, endoplasmic reticulum, and extracellular space, but was affected by Ca2+ in the low micromolar range. Targeting the indicator to the endoplasmic reticulum revealed for the first time sub-cellular lactate dynamics. Its improved lactate-induced fluorescence response permitted the development of a multiwell plate assay to screen for inhibitors of the monocarboxylate transporters MCTs, a pharmaceutical target for cancer and inflammation. The functionality of the indicator in living tissue was demonstrated in the brain of Drosophila melanogaster larvae. CanlonicSF is well suited to explore lactate dynamics with sub-cellular resolution in intact systems.

Genetically Encoded Sensors to Study Metabolism in Drosophila.

The rather recent development of genetically encoded metabolite sensors has changed the way we can study metabolism in living cells, ex vivo tissues, and in vivo immensely. In recent years, these sensors have also been adapted for use in Drosophila tissues. Here, we describe a standard protocol to image such sensors in ex vivo Drosophila larval brains using the glucose sensor FLII12Pglu-700µÎ´6. The protocol, however, can be adapted for the use of other sensors, tissues, and can even be used in vivo.

Starvation-induced regulation of carbohydrate transport at the blood-brain barrier is TGF-ß-signaling dependent.

During hunger or malnutrition, animals prioritize alimentation of the brain over other organs to ensure its function and, thus, their survival. This protection, also-called brain sparing, is described from Drosophila to humans. However, little is known about the molecular mechanisms adapting carbohydrate transport. Here, we used Drosophila genetics to unravel the mechanisms operating at the blood-brain barrier (BBB) under nutrient restriction. During starvation, expression of the carbohydrate transporter Tret1-1 is increased to provide more efficient carbohydrate uptake. Two mechanisms are responsible for this increase. Similar to the regulation of mammalian GLUT4, Rab-dependent intracellular shuttling is needed for Tret1-1 integration into the plasma membrane; even though Tret1-1 regulation is independent of insulin signaling. In addition, starvation induces transcriptional upregulation that is controlled by TGF-ß signaling. Considering TGF-ß-dependent regulation of the glucose transporter GLUT1 in murine chondrocytes, our study reveals an evolutionarily conserved regulatory paradigm adapting the expression of sugar transporters at the BBB.

ATF4-Induced Warburg Metabolism Drives Over-Proliferation in Drosophila.

The mitochondrial electron transport chain (ETC) enables essential metabolic reactions; nonetheless, the cellular responses to defects in mitochondria and the modulation of signaling pathway outputs are not understood. We show that Notch signaling and ETC attenuation via knockdown of COX7a induces massive over-proliferation. The tumor-like growth is caused by a transcriptional response through the eIF2α-kinase PERK and ATF4, which activates the expression of metabolic enzymes, nutrient transporters, and mitochondrial chaperones. We find this stress adaptation to be beneficial for progenitor cell fitness, as it renders cells sensitive to proliferation induced by the Notch signaling pathway. Intriguingly, over-proliferation is not caused by transcriptional cooperation of Notch and ATF4, but it is mediated in part by pH changes resulting from the Warburg metabolism induced by ETC attenuation. Our results suggest that ETC function is monitored by the PERK-ATF4 pathway, which can be hijacked by growth-promoting signaling pathways, leading to oncogenic pathway activity.

Metabolite transport across the mammalian and insect brain diffusion barriers.

The nervous system in higher vertebrates is separated from the circulation by a layer of specialized endothelial cells. It protects the sensitive neurons from harmful blood-derived substances, high and fluctuating ion concentrations, xenobiotics or even pathogens. To this end, the brain endothelial cells and their interlinking tight junctions build an efficient diffusion barrier. A structurally analogous diffusion barrier exists in insects, where glial cell layers separate the hemolymph from the neural cells. Both types of diffusion barriers, of course, also prevent influx of metabolites from the circulation. Because neuronal function consumes vast amounts of energy and necessitates influx of diverse substrates and metabolites, tightly regulated transport systems must ensure a constant metabolite supply. Here, we review the current knowledge about transport systems that carry key metabolites, amino acids, lipids and carbohydrates into the vertebrate and Drosophila brain and how this transport is regulated. Blood-brain and hemolymph-brain transport functions are conserved and we can thus use a simple, genetically accessible model system to learn more about features and dynamics of metabolite transport into the brain.