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Session 3: Imaging Brain Metabolism: Spatial and Temporal Domains

Session 3: Imaging Brain Metabolism: Spatial and Temporal Domains

Session 3: Imaging Brain Metabolism: Spatial and Temporal Domains

University Library - Seaview Area Level 2

Session 4: Novel Imaging Techniques and Modeling

Session 4: Novel Imaging Techniques and Modeling

Session 4: Novel Imaging Techniques and Modeling

Session 4: Novel Imaging Techniques and Modeling

Session 4: Novel Imaging Techniques and Modeling

Session 4: Novel Imaging Techniques and Modeling

Session 4: Novel Imaging Techniques and Modeling

Session 4: Novel Imaging Techniques and Modeling

Metabolites are the nodes of a complex network which is highly conserved throughout life. Understanding of local fluxes within the metabolic grid is constrained by technological limitations, mainly spatiotemporal resolution. For example, it has been known for a while that the first enzyme of glycolysis is attached to mitochondria, but we do not know what is the functional meaning of this location. Current models of cellular metabolism assume that all mitochondria in a cell behave equally. Yet mitochondria within a given cell differ in terms of morphology, age, location, connectivity, motility, and crosstalk with plasma membrane and organelles. Are they all similar in terms of their metabolism? Technologies are emerging that are capable of measuring metabolites with subcellular resolution in living cells. Genetically-encoded nanosensors are proteins build by the fusion of a ligand binding moiety with a fluorescent protein. The binding of the test molecule produces a conformational change that affect the spectroscopy properties of the fluorescent partner. Being genetically-encoded, they can be expressed in single-cells and targeted to subcellular localizations such as mitochondria. Here we introduce PyronicSF, a single-wavelength nanosensor for pyruvate. This sensor undergoes a maximal fluorescent change of 250% and detects pyruvate between 0.03 and 12 mM, spanning the physiological range in mammalian cells. PyronicSF was insensitive to pH and a panel of molecules structurally related to pyruvate. In combination with pharmacological inhibition of the mitochondrial pyruvate carrier (MPC) it was possible to obtain real-time, reversible measurements of the rate of pyruvate consumption in single mitochondria. Our results show that it is possible to characterize metabolic flux in individual organelles. We envisage that single-mitochondria pyruvate consumption measurements will allow to go deep into the understanding of how subcellular localization, morphology, aging and motility can modulate mitochondrial function.

High level cognitive tasks such as long-term memory formation require transcriptional regulations and de novo protein synthesis in the soma. Still, in this compartment, it is unknown if and how neuronal metabolism is regulated and sustained by glia, as it is well demonstrated at the level of the axon and the synapse (Bélanger et al., Cell Metabolism, 2011). In the model organism Drosophila melanogaster, five glial subtypes populate the central nervous system (Kremer et al., Glia, 2017). The brain is enclosed inside the equivalent of the brain-blood barrier, composed by perineurial and subperineurial glia. Axons and dendrites – the neuropil - are localized at the centre of the brain, while neuronal somas are segregated at the periphery. Astrocyte-like glia extend multiple processes deep into the neuropil, while ensheathing glia enwrap neuropil structures. In the periphery of the brain, cortex glial cells envelop each neuronal soma individually. Thanks to the diverse and precise genetic tools available in this model, we expressed the dominant thermosensitive allele of dynamin specifically in these different glial subtypes. By transferring flies at restrictive temperature, it allowed us to block exocytosis in the targeted glial cells in a spatiotemporally defined window. Using this approach, we showed that cortex glial cells were involved very early in olfactory memory formation, and specifically in long-term memory. Mushroom bodies are the integrative structures involved in associative memory in the fly brain. We now study if cortex glia could be activated by a signal derived from mushroom-body neurons, which are cholinergic (Barnstedt et al., Neuron, 2016). Data from feeding experiments indirectly suggest that, once activated, cortex glia would support neuronal cell bodies metabolism, but the pathways involved still need to be more precisely deciphered.

Mitochondrial Ca2+ plays a critical physiological role in cellular energy metabolism and signalling, and its overload contributes to various pathological conditions including neuronal apoptotic death in neurological diseases. It is known that the type-1 cannabinoid receptor (CB1) is present at the mitochondria (mtCB1) and the activation of these receptors decreases cellular respiration and dysregulates memory processes. Data from our lab now show that the CB1 receptor is also present in the mitochondria of astrocytes and can regulate energy production in this cell context. However, it is still not clear whether mtCB1 participates in the synaptic effect of CB1 in astrocytes and if this is mediated by the reported decrease in ATP production or by a regulation in the Ca2+ buffering capacity of mitochondria. Using confocal microscopy in isolated astrocytes transfected with a genetically encoded Ca2+ indicator (GCAMP6s) targeted to mitochondria and a cytosolic Ca2+ sensor (RCAMP2), we have shown that CB1 activation by the specific agonist WIN 55,212-2 leads to an increase of Ca2+ in both compartments. Conversely, a mutant CB1 that it is not addressed to mitochondria (DN22-CB1) only affects cytosolic Ca2+. These results demonstrate that mtCB1 receptor regulates Ca2+ levels in the mitochondria. The inhibitor of soluble adenylyl cyclase (sAC) KH7, that blocks mtCB1-dependent respiration, does not affect cannabinoid-induced increase of mitochondrial Ca2+ in astrocytes, suggesting that this effect of mtCB1 might be independent from cellular respiration. Moreover, the inhibitors of the IP3 receptor located in the endoplasmic reticulum (ER) 2-APB and Xestospongin C blocked the WIN-dependent mitochondrial Ca2+ increase, suggesting that the ER is the source responsible for the increase of Ca2+ in the mitochondria. To investigate if these “in vitro” experiments are reproducible in an “in vivo” model, the somatosensory cortex of WT mice was infected with AAV viruses expressing the mitochondrial GCAMP6s sensor under the astroglial promoter GFAP and the activity of mitochondrial Ca2+ monitored using a 2-photon microscopy in anesthetized animals. Preliminary results show that the intraperitoneal injection of the CB1 agonist THC increases the % of activated mitochondria particles. To conclude, this work shows that mtCB1 regulates Ca2+ levels in the mitochondria of astrocytes, representing a potential novel mechanism by which astroglial CB1 receptors could control astroglial functions and synaptic plasticity.

Session 5: Model Systems and Pathology

Session 5: Model Systems and Pathology

Session 5: Model Systems and Pathology

by Professor Pierre Magistretti