Memory formation shaped by astroglia

Astrocytes, the most heterogeneous glial cells in the central nervous system (CNS), execute a multitude of homeostatic functions and contribute to memory formation. Consolidation of synaptic and systemic memory is a prolonged process and hours are required to form long-term memory. In the past, neurons or their parts have been considered to be the exclusive cellular sites of these processes, however, it has now become evident that astrocytes provide an important and essential contribution to memory formation. Astrocytes participate in the morphological remodeling associated with synaptic plasticity, an energy-demanding process that requires mobilization of glycogen, which, in the CNS, is almost exclusively stored in astrocytes. Synaptic remodeling also involves bidirectional astroglial-neuronal communication supported by astroglial receptors and release of gliosignaling molecules. Astroglia exhibit cytoplasmic excitability that engages second messengers, such as Ca(2+), for phasic, and cyclic adenosine monophosphate (cAMP), for tonic signal coordination with neuronal processes. The detection of signals by astrocytes and the release of gliosignaling molecules, in particular by vesicle-based mechanisms, occurs with a significant delay after stimulation, orders of magnitude longer than that present in stimulus–secretion coupling in neurons. These particular arrangements position astrocytes as integrators ideally tuned to support time-dependent memory formation

Ca2+^{2+} as the prime trigger of aerobic glycolysis in astrocytes

Astroglial aerobic glycolysis, a process during which D-glucose is converted to L-lactate, a brain fuel and signal, is regulated by the plasmalemmal receptors, including adrenergic receptors (ARs) and purinergic receptors (PRs), modulating intracellular Ca$^{2+}$ and cAMP signals. However, the extent to which the two signals regulate astroglial aerobic glycolysis is poorly understood. By using agonists to stimulate intracellular α$_1$-/β-AR-mediated Ca$^{2+}$/cAMP signals, β-AR-mediated cAMP and P$_2$R-mediated Ca$^{2+}$ signals and genetically encoded fluorescence resonance energy transfer-based glucose and lactate nanosensors in combination with real-time microscopy, we show that intracellular Ca$^{2+}$, but not cAMP, initiates a robust increase in the concentration of intracellular free D-glucose ([glc]$_i$) and L-lactate ([lac]$_i$), both depending on extracellular D-glucose, suggesting Ca$^{2+}$-triggered glucose uptake and aerobic glycolysis in astrocytes. When the glycogen shunt, a process of glycogen remodelling, was inhibited, the α$_1$-/β-AR-mediated increases in [glc]$_i$ and [lac]$_i$ were reduced by ∼65 % and ∼30 %, respectively, indicating that at least ∼30 % of the utilization of D-glucose is linked to glycogen remodelling and aerobic glycolysis. Additional activation of β-AR/cAMP signals aided to α$_1$-/β-AR-triggered [lac]$_i$ increase, whereas the [glc]$_i$ increase was unaltered. Taken together, an increase in intracellular Ca$^{2+}$ is the prime mechanism of augmented aerobic glycolysis in astrocytes, while cAMP has only a moderate role. The results provide novel information on the signals regulating brain metabolism and open new avenues to explore whether astroglial Ca$^{2+}$ signals are dysregulated and contribute to neuropathologies with impaired brain metabolism

Astrocytes with TDP-43 inclusions exhibit reduced noradrenergic cAMP and Ca2^2+^+ signaling and dysregulated cell metabolism

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

Astrocyte arborization enhances Ca2+ but not cAMP signaling plasticity

The plasticity of astrocytes is fundamental for their principal function, maintaining homeostasis of the central nervous system throughout life, and is associated with diverse exposomal challenges. Here, we used cultured astrocytes to investigate at subcellular level basic cell processes under controlled environmental conditions. We compared astroglial functional and signaling plasticity in standard serum‐containing growth medium, a condition mimicking pathologic conditions, and in medium without serum, favoring the acquisition of arborized morphology. Using opto−/electrophysiologic techniques, we examined cell viability, expression of astroglial markers, vesicle dynamics, and cytosolic Ca(2+) and cAMP signaling. The results revealed altered vesicle dynamics in arborized astrocytes that was associated with increased resting [Ca(2+)](i) and increased subcellular heterogeneity in [Ca(2+)](i), whereas [cAMP](i) subcellular dynamics remained stable in both cultures, indicating that cAMP signaling is less prone to plastic remodeling than Ca(2+) signaling, possibly also in in vivo contexts

Astrocyte arborization enhances Ca2+^{2+} but not cAMP signaling plasticity

The plasticity of astrocytes is fundamental for their principal function, maintaining homeostasis of the central nervous system throughout life, and is associated with diverse exposomal challenges. Here, we used cultured astrocytes to investigate at subcellular level basic cell processes under controlled environmental conditions. We compared astroglial functional and signaling plasticity in standard serum-containing growth medium, a condition mimicking pathologic conditions, and in medium without serum, favoring the acquisition of arborized morphology. Using opto−/electrophysiologic techniques, we examined cell viability, expression of astroglial markers, vesicle dynamics, and cytosolic Ca$^{2+}$ and cAMP signaling. The results revealed altered vesicle dynamics in arborized astrocytes that was associated with increased resting [Ca$^{2+}$]$_i$ and increased subcellular heterogeneity in [Ca$^{2+}$]$_i$, whereas [cAMP]$_i$ subcellular dynamics remained stable in both cultures, indicating that cAMP signaling is less prone to plastic remodeling than Ca$^{2+}$ signaling, possibly also in in vivo contexts

Astrocyte specific remodeling of plasmalemmal cholesterol composition by ketamine indicates a new mechanism of antidepressant action

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]$_i$ in astrocytes. Membrane capacitance recordings, however, reveal fundamentally distinct mechanisms of effects of ketamine and [cAMP]$_i$ on vesicular secretion: a rise in [cAMP]$_i$ 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\u27s rapid antidepressant action

The activation of GPR27 increases cytosolic L-lactate in 3T3 embryonic cells and astrocytes

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]$_i$), 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]$_i$. 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]$_i$ was reduced compared with WT controls. Transfection of the GPR27-carrying plasmid into the 3T3KOGPR27 cells rescued the 8535-induced increase in [lactate]$_i$. 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]$_i$ was increased in comparison with controls, further supporting the view that GPR27 regulates L-lactate homeostasis

Astrocytes in stress accumulate lipid droplets

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 Ca$^{2+}$, 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

Enhancement of astroglial aerobic glycolysis by extracellular lactate-mediated increase in cAMP

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 (EC50 ≈ 5 mM) that is in fat cells part of an autocrine loop, in which the Gi-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-N-(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