Astrocytes, heterogeneous neuroglial cells, contribute to metabolic homeostasis in the brain by providing energy substrates to neurons. In contrast to predominantly oxidative neurons, astrocytes are ...considered primarily as glycolytic cells. They take up glucose from the circulation and in the process of aerobic glycolysis (despite the normal oxygen levels) produce
L
-lactate, which is then released into the extracellular space
via
lactate transporters and possibly channels. Astroglial
L
-lactate can enter neurons, where it is used as a metabolic substrate, or exit the brain
via
the circulation. Recently,
L
-lactate has also been considered to be a signaling molecule in the brain, but the mechanisms of
L
-lactate signaling and how it contributes to the brain function remain to be fully elucidated. Here, we provide an overview of
L
-lactate signaling mechanisms in the brain and present novel insights into the mechanisms of
L
-lactate signaling
via
G-protein coupled receptors (GPCRs) with the focus on astrocytes. We discuss how increased extracellular
L
-lactate upregulates cAMP production in astrocytes, most likely
via
L
-lactate-sensitive G
s
-protein coupled GPCRs. This activates aerobic glycolysis, enhancing
L
-lactate production and accumulation of lipid droplets, suggesting that
L
-lactate augments its own production in astrocytes (i.e., metabolic excitability) to provide more
L
-lactate for neurons and that astrocytes in conditions of increased extracellular
L
-lactate switch to lipid metabolism.
Abstract
Most cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in neurons and non-neuronal cells, ...including astrocytes, which metabolically support neurons with nutrients. Neuronal metabolism largely depends on the activation of the noradrenergic system releasing noradrenaline. Activation of astroglial adrenergic receptors with noradrenaline triggers cAMP and Ca
2+
signaling and augments aerobic glycolysis with production of lactate, an important neuronal energy fuel. Astrocytes with cytoplasmic TDP-43 inclusions can cause motor neuron death, however, whether astroglial metabolism and metabolic support of neurons is altered in astrocytes with TDP-43 inclusions, is unclear. We measured lipid droplet and glucose metabolisms in astrocytes expressing the inclusion-forming C-terminal fragment of TDP-43 or the wild-type TDP-43 using fluorescent dyes or genetically encoded nanosensors. Astrocytes with TDP-43 inclusions exhibited a 3-fold increase in the accumulation of lipid droplets versus astrocytes expressing wild-type TDP-43, indicating altered lipid droplet metabolism. In these cells the noradrenaline-triggered increases in intracellular cAMP and Ca
2+
levels were reduced by 35% and 31%, respectively, likely due to the downregulation of β
2
-adrenergic receptors. Although noradrenaline triggered a similar increase in intracellular lactate levels in astrocytes with and without TDP-43 inclusions, the probability of activating aerobic glycolysis was facilitated by 1.6-fold in astrocytes with TDP-43 inclusions and lactate MCT1 transporters were downregulated. Thus, while in astrocytes with TDP-43 inclusions noradrenergic signaling is reduced, aerobic glycolysis and lipid droplet accumulation are facilitated, suggesting dysregulated astroglial metabolism and metabolic support of neurons in TDP-43-associated ALS and FTD.
When the brain is in a pathological state, the content of lipid droplets (LDs), the lipid storage organelles, is increased, particularly in glial cells, but rarely in neurons. The biology and ...mechanisms leading to LD accumulation in astrocytes, glial cells with key homeostatic functions, are poorly understood. We imaged fluorescently labeled LDs by microscopy in isolated and brain tissue rat astrocytes and in glia‐like cells in Drosophila brain to determine the (sub)cellular localization, mobility, and content of LDs under various stress conditions characteristic for brain pathologies. LDs exhibited confined mobility proximal to mitochondria and endoplasmic reticulum that was attenuated by metabolic stress and by increased intracellular Ca2+, likely to enhance the LD–organelle interaction imaged by electron microscopy. When de novo biogenesis of LDs was attenuated by inhibition of DGAT1 and DGAT2 enzymes, the astrocyte cell number was reduced by ~40%, suggesting that in astrocytes LD turnover is important for cell survival and/or proliferative cycle. Exposure to noradrenaline, a brain stress response system neuromodulator, and metabolic and hypoxic stress strongly facilitated LD accumulation in astrocytes. The observed response of stressed astrocytes may be viewed as a support for energy provision, but also to be neuroprotective against the stress‐induced lipotoxicity.
Main points
Astroglial lipid droplets are dynamic organelles with confined mobility.
Lipid droplet turnover is important for astrocyte survival/proliferation.
Stressed astrocytes accumulate lipid droplets likely to provide energy and reduce lipotoxicity.
Ketamine is an antidepressant with rapid therapeutic onset and long-lasting effect, although the underlying mechanism(s) remain unknown. Using FRET-based nanosensors we found that ketamine increases ...cAMP
in astrocytes. Membrane capacitance recordings, however, reveal fundamentally distinct mechanisms of effects of ketamine and cAMP
on vesicular secretion: a rise in cAMP
facilitated, whereas ketamine inhibited exocytosis. By directly monitoring cholesterol-rich membrane domains with a fluorescently tagged cholesterol-specific membrane binding domain (D4) of toxin perfringolysin O, we demonstrated that ketamine induced cholesterol redistribution in the plasmalemma in astrocytes, but neither in fibroblasts nor in PC 12 cells. This novel mechanism posits that ketamine affects density and distribution of cholesterol in the astrocytic plasmalemma, consequently modulating a host of processes that may contribute to ketamine's rapid antidepressant action.
Edema in the central nervous system can rapidly result in life‐threatening complications. Vasogenic edema is clinically manageable, but there is no established medical treatment for cytotoxic edema, ...which affects astrocytes and is a primary trigger of acute post‐traumatic neuronal death. To test the hypothesis that adrenergic receptor agonists, including the stress stimulus epinephrine protects neural parenchyma from damage, we characterized its effects on hypotonicity‐induced cellular edema in cortical astrocytes by in vivo and in vitro imaging. After epinephrine administration, hypotonicity‐induced swelling of astrocytes was markedly reduced and cytosolic 3'‐5'‐cyclic adenosine monophosphate (cAMP) was increased, as shown by a fluorescence resonance energy transfer nanosensor. Although, the kinetics of epinephrine‐induced cAMP signaling was slowed in primary cortical astrocytes exposed to hypotonicity, the swelling reduction by epinephrine was associated with an attenuated hypotonicity‐induced cytosolic Ca2+ excitability, which may be the key to prevent astrocyte swelling. Furthermore, in a rat model of spinal cord injury, epinephrine applied locally markedly reduced neural edema around the contusion epicenter. These findings reveal new targets for the treatment of cellular edema in the central nervous system. GLIA 2016;64:1034–1049
Main Points
Epinephrine reduces hypotonic astrocyte swelling in vitro and in vivo.
Cell swelling reduction involves cAMP‐mediated Ca2+ hypoexcitability.
Epinephrine reduces trauma‐induced astrocyte swelling; a new possibility for the treatment of cellular edema.
Besides being a neuronal fuel, L-lactate is also a signal in the brain. Whether extracellular L-lactate affects brain metabolism, in particular astrocytes, abundant neuroglial cells, which produce ...L-lactate in aerobic glycolysis, is unclear. Recent studies suggested that astrocytes express low levels of the L-lactate GPR81 receptor (EC
≈ 5 mM) that is in fat cells part of an autocrine loop, in which the G
-protein mediates reduction of cytosolic cyclic adenosine monophosphate (cAMP). To study whether a similar signaling loop is present in astrocytes, affecting aerobic glycolysis, we measured the cytosolic levels of cAMP, D-glucose and L-lactate in single astrocytes using fluorescence resonance energy transfer (FRET)-based nanosensors. In contrast to the situation in fat cells, stimulation by extracellular L-lactate and the selective GPR81 agonists, 3-chloro-5-hydroxybenzoic acid (3Cl-5OH-BA) or 4-methyl-
-(5-(2-(4-methylpiperazin-1-yl)-2-oxoethyl)-4-(2-thienyl)-1,3-thiazol-2-yl)cyclohexanecarboxamide (Compound 2), like adrenergic stimulation, elevated intracellular cAMP and L-lactate in astrocytes, which was reduced by the inhibition of adenylate cyclase. Surprisingly, 3Cl-5OH-BA and Compound 2 increased cytosolic cAMP also in GPR81-knock out astrocytes, indicating that the effect is GPR81-independent and mediated by a novel, yet unidentified, excitatory L-lactate receptor-like mechanism in astrocytes that enhances aerobic glycolysis and L-lactate production via a positive feedback mechanism.
•Genetically encoded fluorescent cAMP sensors have enabled the first measurements of the temporal dynamics of cAMP signalling in living astrocytes.•Compared with Ca2+ signalling with fast phasic ...dynamics, cAMP signalling in astrocytes -exhibits >4-fold slower tonic dynamics.•Ca2+ potentiates cAMP signalling -in astrocytes, indicating cross-talk between the Ca2+- and cAMP-signalling pathways.•Astroglial cAMP signalling in subcellular compartments needs to be evaluated in the future.
To maintain a high level of specificity and normal cell function, the cyclic adenosine monophosphate (cAMP) pathway is tightly regulated in space and time. Recent advances in cAMP reporter technology have provided insights into spatio-temporal characteristics of cAMP signalling in individual living cells, including astrocytes. Astrocytes are glial cells in the central nervous system with many homeostatic functions. In contrast to neurons, astrocytes are electrically silent, but, in response to extracellular stimuli through activation of surface receptors, they can increase intracellular levels of secondary messengers, e.g. Ca2+ and cAMP. This enables them to communicate with neighbouring cells, such as neurons and endothelial cells of blood vessels. The dynamics of receptor-mediated Ca2+ signalling in astrocytes has been extensively studied in the past in contrast to cAMP signalling. Here, we present the first insights into the temporal dynamics of cAMP signalling in living astrocytes, which revealed that cAMP signals in astrocytes exhibit tonic dynamics and are slower than Ca2+ signals with phasic dynamics. We debate on the heterogeneity of basal cAMP levels in astrocytes and how hypotonicity-induced astrocyte swelling affects temporal dynamics of cAMP signalling. Understanding the spatio-temporal characteristics of cAMP signalling in astrocytes is of extreme importance because cAMP governs many important cellular processes and any malfunctions may lead to pathology.
G-protein-coupled receptors (GPCRs) represent a family with over 800 members in humans, and one-third of these are targets for approved drugs. A large number of GPCRs have unknown physiologic roles. ...Here, we investigated GPR27, an orphan GPCR belonging to the family of super conserved receptor expressed in the brain, with unknown functions. Cytosolic levels of L-lactate (lactate
), the end product of aerobic glycolysis, were measured with the Laconic fluorescence resonance energy transfer nanosensor. In single 3T3 wild-type (WT) embryonic cells, the application of 8535 (1 µM), a surrogate agonist known to activate GPR27, resulted in an increase in lactate
. Similarly, an increase was recorded in primary rat astrocytes, a type of neuroglial cell abundant in the brain, which contain glycogen and express enzymes of aerobic glycolysis. In CRISPR-Cas9 GPR27 knocked out 3T3 cells, the 8535-induced increase in lactate
was reduced compared with WT controls. Transfection of the GPR27-carrying plasmid into the 3T3KOGPR27 cells rescued the 8535-induced increase in lactate
. These results indicate that stimulation of GPR27 enhances aerobic glycolysis and L-lactate production in 3T3 cells and astrocytes. Interestingly, in the absence of GPR27 in 3T3 cells, resting lactate
was increased in comparison with controls, further supporting the view that GPR27 regulates L-lactate homeostasis.
During the arousal and startle response, locus coeruleus neurons, innervating practically all brain regions, release catecholamine noradrenaline, which reaches neural brain cells, including ...astrocytes. These glial cells respond to noradrenergic stimulation by simultaneous activation of the α- and β-adrenergic receptors (ARs) in the plasma membrane with increasing cytosolic levels of Ca(2+) and cAMP, respectively. AR-activation controls a myriad of processes in astrocytes including glucose metabolism, gliosignal vesicle homeostasis, gene transcription, cell morphology and antigen-presenting functions, all of which have distinct temporal characteristics. It is known from biochemical studies that Ca(2+) and cAMP signals in astrocytes can interact, however it is presently unclear whether the temporal properties of the two second messengers are time associated upon AR-activation. We used confocal microscopy to study AR agonist-induced intracellular changes in Ca(2+) and cAMP in single cultured cortical rat astrocytes by real-time monitoring of the Ca(2+) indicator Fluo4-AM and the fluorescence resonance energy transfer-based nanosensor A-kinase activity reporter 2 (AKAR2), which reports the activity of cAMP via its downstream effector protein kinase A (PKA). The results revealed that the activation of α1-ARs by phenylephrine triggers periodic (phasic) Ca(2+) oscillations within 10s, while the activation of β-ARs by isoprenaline leads to a ∼10-fold slower tonic rise to a plateau in cAMP/PKA activity devoid of oscillations. Thus the concomitant activation of α- and β-ARs triggers the Ca(2+) and cAMP second messenger systems in astrocytes with distinct temporal properties, which appears to be tailored to regulate downstream effectors in different time domains.
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