Objectives
GDI1
gene encodes for αGDI, a protein controlling the cycling of small GTPases, reputed to orchestrate vesicle trafficking. Mutations in human
GDI1
are responsible for intellectual disability (ID). In mice with ablated
Gdi1
, a model of ID, impaired working and associative short-term memory was recorded. This cognitive phenotype worsens if the deletion of αGDI expression is restricted to neurons. However, whether astrocytes, key homeostasis providing neuroglial cells, supporting neurons via aerobic glycolysis, contribute to this cognitive impairment is unclear.
Methods
We carried out proteomic analysis and monitored [
18
F]-fluoro-2-deoxy-
d
-glucose uptake into brain slices of
Gdi1
knockout and wild type control mice.
d
-Glucose utilization at single astrocyte level was measured by the Förster Resonance Energy Transfer (FRET)-based measurements of cytosolic cyclic AMP,
d
-glucose and L-lactate, evoked by agonists selective for noradrenaline and L-lactate receptors. To test the role of astrocyte-resident processes in disease phenotype, we generated an inducible
Gdi1
knockout mouse carrying the
Gdi1
deletion only in adult astrocytes and conducted behavioural tests.
Results
Proteomic analysis revealed significant changes in astrocyte-resident glycolytic enzymes. Imaging [
18
F]-fluoro-2-deoxy-
d
-glucose revealed an increased
d
-glucose uptake in
Gdi1
knockout tissue versus wild type control mice, consistent with the facilitated
d
-glucose uptake determined by FRET measurements. In mice with
Gdi1
deletion restricted to astrocytes, a selective and significant impairment in working memory was recorded, which was rescued by inhibiting glycolysis by 2-deoxy-
d
-glucose injection.
Conclusions
These results reveal a new astrocyte-based mechanism in neurodevelopmental disorders and open a novel therapeutic opportunity of targeting aerobic glycolysis, advocating a change in clinical practice.
The mammalian brain critically depends on a sustained delivery of energy, which mostly derives from d-glucose (Bittner et al., 2010). To ensure an energy supply that is constantly available for fluctuating neural activity, the brain must be capable of rapid nutrient uptake from the blood (Harris et al., 2012; Machler et al., 2016). Neurons are highly differentiated and specialized cells, which are not directly
Glycogen synthase kinase 3 (GSK-3) plays an important role in metabolic regulation in skeletal muscles, and both insulin and adrenaline stimulate GKS-3 phosphorylation. The aim of the present study was to study the effect of insulin and adrenaline on GSK-3 localisation in skeletal muscles.We characterized subcellular localization of (GSK-3) signal protein in fully differentiated muscle fibre by immunofluorescence and confocal microscopy. We stimulated muscle fibres with insulin and/or adrenaline. Images were analysed by segmentation of single central optical section of the muscle.We found GSK-3 to be localised in clusters. The number of GSK-3 clusters and their average size were increased after stimulation with insulin and/or adrenaline. Average GSK-3 particle size is linearly related to their quantity.We conclude that subcellular GSK-3 in isolated skeletal muscle fibres is localized in clusters and clustering increased after stimulation with insulin and/or adrenaline.
A single sub-anesthetic dose of ketamine evokes rapid and long-lasting beneficial effects in patients with a major depressive disorder. However, the mechanisms underlying this effect are unknown. It has been proposed that astrocyte dysregulation of extracellular K+ concentration ([K+]o) alters neuronal excitability, thus contributing to depression. We examined how ketamine affects inwardly rectifying K+ channel Kir4.1, the principal regulator of K+ buffering and neuronal excitability in the brain. Cultured rat cortical astrocytes were transfected with plasmid-encoding fluorescently tagged Kir4.1 (Kir4.1-EGFP) to monitor the mobility of Kir4.1-EGFP vesicles at rest and after ketamine treatment (2.5 or 25 µM). Short-term (30 min) ketamine treatment reduced the mobility of Kir4.1-EGFP vesicles compared with the vehicle-treated controls (p < 0.05). Astrocyte treatment (24 h) with dbcAMP (dibutyryl cyclic adenosine 5′-monophosphate, 1 mM) or [K+]o (15 mM), which increases intracellular cAMP, mimicked the ketamine-evoked reduction of mobility. Live cell immunolabelling and patch-clamp measurements in cultured mouse astrocytes revealed that short-term ketamine treatment reduced the surface density of Kir4.1 and inhibited voltage-activated currents similar to Ba2+ (300 µM), a Kir4.1 blocker. Thus, ketamine attenuates Kir4.1 vesicle mobility, likely via a cAMP-dependent mechanism, reduces Kir4.1 surface density, and inhibits voltage-activated currents similar to Ba2+, known to block Kir4.1 channels.
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