L. Felipe Barros

Modulation of the two-pore domain acid-sensitive K^+ channel TASK-2 (KCNK5) by changes in cell volume

The molecular identity of K+channels involved in Ehrlich cell volume regulation is unknown. A background K+ conductance is activated by cell swelling and is also modulated by extracellular pH. These characteristics are most similar to those of newly emerging TASK (TWIK-related acid-sensitive K+ channels)-type of two pore-domain K+ channels. mTASK-2, but not TASK-1 or -3, is present in Ehrlich cells and mouse kidney tissue from where the full coding sequences were obtained. Heterologous expression of mTASK-2 cDNA in HEK-293 cells generated K+ currents in the absence intracellular Ca2+. Exposure to hypotonicity enhanced mTASK-2 currents and osmotic cell shrinkage led to inhibition. This occurred without altering voltage dependence and with only slight decrease in pK a in hypotonicity but no change in hypertonicity. Replacement with other cations yields a permselectivity sequence for mTASK-2 of K+ > Rb+ ≫ Cs+ > NH 4 + > Na+ ≅ Li+, similar to that for the native conductance (I K, vol). Clofilium, a quaternary ammonium blocker of I K, vol, blocked the mTASK-2-mediated K+ current with an IC50 of 25 μm. The presence of mTASK-2 in Ehrlich cells, its functional similarities with I K, vol, and its modulation by changes in cell volume suggest that this two-pore domain K+ channel participates in the regulatory volume decrease phenomenon.

Metabolic signaling by lactate in the brain

High-resolution molecular and imaging techniques are shedding light on the mechanisms and functional significance of the transient rise in tissue lactate that accompanies synaptic activity. Despite high energy needs, neurons have a truncated glycolytic pathway that favors antioxidation over energy production, whereas astrocytes team up with oligodendrocytes to extract glucose from the blood, mobilize glycogen, and release lactate under neuronal command. Lactate energizes neurons but also diffuses beyond the active zone and modifies the activity of neurons and astrocytes in neighboring regions. Involved in a hierarchy of processes from neurovascular coupling to memory formation, lactate has a dual role as metabolic fuel and an intercellular messenger.

Antiribosomal-P autoantibodies from psychiatric lupus target a novel neuronal surface protein causing calcium influx and apoptosis

The interesting observation was made 20 years ago that psychotic manifestations in patients with systemic lupus erythematosus are associated with the production of antiribosomal-P protein (anti-P) autoantibodies. Since then, the pathogenic role of anti-P antibodies has attracted considerable attention, giving rise to long-term controversies as evidence has either contradicted or confirmed their clinical association with lupus psychosis. Furthermore, a plausible mechanism supporting an anti-P–mediated neuronal dysfunction is still lacking. We show that anti-P antibodies recognize a new integral membrane protein of the neuronal cell surface. In the brain, this neuronal surface P antigen (NSPA) is preferentially distributed in areas involved in memory, cognition, and emotion. When added to brain cellular cultures, anti-P antibodies caused a rapid and sustained increase in calcium influx in neurons, resulting in apoptotic cell death. In contrast, astrocytes, which do not express NSPA, were not affected. Injection of anti-P antibodies into the brain of living rats also triggered neuronal death by apoptosis. These results demonstrate a neuropathogenic potential of anti-P antibodies and contribute a mechanistic basis for psychiatric lupus. They also provide a molecular target for future exploration of this and other psychiatric diseases.

Glutamate Mediates Acute Glucose Transport Inhibition in Hippocampal Neurons

Although it is known that brain activity is fueled by glucose, the identity of the cell type that preferentially metabolizes the sugar remains elusive. To address this question, glucose uptake was studied simultaneously in cultured hippocampal neurons and neighboring astrocytes using a real-time assay based on confocal epifluorescence microscopy and fluorescent glucose analogs. Glutamate, although stimulating glucose transport in astrocytes, strongly inhibited glucose transport in neurons, producing in few seconds a 12-fold increase in the ratio of astrocytic-to-neuronal uptake rate. Neuronal transport inhibition was reversible on removal of the neurotransmitter and displayed an IC50 of 5 μm, suggesting its occurrence at physiological glutamate concentrations. The phenomenon was abolished by CNQX and mimicked by AMPA, demonstrating a role for the cognate subset of ionotropic glutamate receptors. Transport inhibition required extracellular sodium and calcium and was mimicked by veratridine but not by membrane depolarization with high K+ or by calcium overloading with ionomycin. Therefore, glutamate inhibits glucose transport via AMPA receptor-mediated sodium entry, whereas calcium entry plays a permissive role. This phenomenon suggests that glutamate redistributes glucose toward astrocytes and away from neurons and represents a novel molecular mechanism that may be important for functional imaging of the brain using positron emission tomography.

In Vivo Evidence for a Lactate Gradient from Astrocytes to Neurons

Investigating lactate dynamics in brain tissue is challenging, partly because in vivo data at cellular resolution are not available. We monitored lactate in cortical astrocytes and neurons of mice using the genetically encoded FRET sensor Laconic in combination with two-photon microscopy. An intravenous lactate injection rapidly increased the Laconic signal in both astrocytes and neurons, demonstrating high lactate permeability across tissue. The signal increase was significantly smaller in astrocytes, pointing to higher basal lactate levels in these cells, confirmed by a one-point calibration protocol. Trans-acceleration of the monocarboxylate transporter with pyruvate was able to reduce intracellular lactate in astrocytes but not in neurons. Collectively, these data provide in vivo evidence for a lactate gradient from astrocytes to neurons. This gradient is a prerequisite for a carrier-mediated lactate flux from astrocytes to neurons and thus supports the astrocyte-neuron lactate shuttle model, in which astrocyte-derived lactate acts as an energy substrate for neurons.

Glucose and lactate supply to the synapse.

The main source of energy for the mammalian brain is glucose, and the main sink of energy in the mammalian brain is the neuron, so the conventional view of brain energy metabolism is that glucose is consumed preferentially in neurons. But between glucose and the production of energy are several steps that do not necessarily take place in the same cell. An alternative model has been proposed that states that glucose preferentially taken by astrocytes, is degraded to lactate and then exported into neurons to be oxidized. Short of definitive data, opinions about the relative merits of these competing models are divided, making it a very exciting field of research. Furthermore, growing evidence suggests that lactate acts as a signaling molecule, involved in Na(+) sensing, glucosensing, and in coupling neuronal and glial activity to the modulation of vascular tone. In the present review, we discuss possible dynamics of glucose and lactate in excitatory synaptic regions, focusing on the transporters that catalyze the movement of these molecules.

Fast and Reversible Stimulation of Astrocytic Glycolysis by K+ and a Delayed and Persistent Effect of Glutamate

Synaptic activity is followed within seconds by a local surge in lactate concentration, a phenomenon that underlies functional magnetic resonance imaging and whose causal mechanisms are unclear, partly because of the limited spatiotemporal resolution of standard measurement techniques. Using a novel Förster resonance energy transfer-based method that allows real-time measurement of the glycolytic rate in single cells, we have studied mouse astrocytes in search for the mechanisms responsible for the lactate surge. Consistent with previous measurements with isotopic 2-deoxyglucose, glutamate was observed to stimulate glycolysis in cultured astrocytes, but the response appeared only after a lag period of several minutes. Na+ overloads elicited by engagement of the Na+-glutamate cotransporter with d-aspartate or application of the Na+ ionophore gramicidin also failed to stimulate glycolysis in the short term. In marked contrast, K+ stimulated astrocytic glycolysis by fourfold within seconds, an effect that was observed at low millimolar concentrations and was also present in organotypic hippocampal slices. After removal of the agonists, the stimulation by K+ ended immediately but the stimulation by glutamate persisted unabated for >20 min. Both stimulations required an active Na+/K+ ATPase pump. By showing that small rises in extracellular K+ mediate short-term, reversible modulation of astrocytic glycolysis and that glutamate plays a long-term effect and leaves a metabolic trace, these results support the view that astrocytes contribute to the lactate surge that accompanies synaptic activity and underscore the role of these cells in neurometabolic and neurovascular coupling.

High Resolution Measurement of the Glycolytic Rate

The glycolytic rate is sensitive to physiological activity, hormones, stress, aging, and malignant transformation. Standard techniques to measure the glycolytic rate are based on radioactive isotopes, are not able to resolve single cells and have poor temporal resolution, limitations that hamper the study of energy metabolism in the brain and other organs. A new method is described in this article, which makes use of a recently developed FRET glucose nanosensor to measure the rate of glycolysis in single cells with high temporal resolution. Used in cultured astrocytes, the method showed for the first time that glycolysis can be activated within seconds by a combination of glutamate and K+, supporting a role for astrocytes in neurometabolic and neurovascular coupling in the brain. It was also possible to make a direct comparison of metabolism in neurons and astrocytes lying in close proximity, paving the way to a high-resolution characterization of brain energy metabolism. Single-cell glycolytic rates were also measured in fibroblasts, adipocytes, myoblasts, and tumor cells, showing higher rates for undifferentiated cells and significant metabolic heterogeneity within cell types. This method should facilitate the investigation of tissue metabolism at the single-cell level and is readily adaptable for high-throughput analysis.

Higher Transport and Metabolism of Glucose in Astrocytes Compared with Neurons: A Multiphoton Study of Hippocampal and Cerebellar Tissue Slices

Glucose is the most important energy substrate for the brain, and its cellular distribution is a subject of great current interest. We have employed fluorescent glucose probes, the 2-deoxy-D-glucose derivates 6- and 2-([N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose) (2-NBDG), to measure transport and metabolism of glucose in acute slices of mouse hippocampus and cerebellum. In the hippocampus, 6-NBDG, which is not metabolized and hence indicates glucose transport, was taken up faster in astrocyte-rich layers (Stratum radiatum [S.r.], Stratum oriens [S.o.]) than in pyramidal cells. Metabolizable 2-NBDG showed larger signals in S.r. and S.o. than in Stratum pyramidale, suggesting faster glucose utilization rate in the astrocyte versus the neuronal compartment. Similarly, we found higher uptake and temperature-sensitive metabolism of 2-NBDG in Bergmann glia when compared with adjacent Purkinje neurons of cerebellar slices. A comparison between 6-NBDG transport and glucose transport in cultured cells using a fluorescence resonance energy transfer nanosensor showed that relative to glucose, 6-NBDG is transported better by neurons than by astrocytes. These results indicate that the preferential transport and metabolism of glucose by glial cells versus neurons proposed for the hippocampus and cerebellum by ourselves (in vitro) and for the barrel cortex by Chuquet et al. (in vivo) is more pronounced than anticipated.

Why glucose transport in the brain matters for PET

Neuronal activity is fueled by glucose metabolism, a phenomenon exploited in basic research and clinical diagnosis using fluorodeoxyglucose positron emission tomography (FDG-PET). According to the current view, glucose transport into the brain is not rate-limiting; thus, it cannot exert control over metabolism. This article challenges such a view by showing that basal transport hovers near its maximum, making metabolic activation unable to increase flux on its own. In the light of recent evidence on the identity of the cell type that preferentially breaks down glucose, we suggest that FDG-PET reports the synergistic activation of glucose transport and metabolism in astrocytes, rather than in neurons.