Novel interactions of CLN3 protein link Batten disease to dysregulation of fodrin-Na+, K+ ATPase complex

Juvenile neuronal ceroid lipofuscinosis (JNCL, Batten disease) is the most common progressive neurodegenerative disorder of childhood. CLN3, the transmembrane protein underlying JNCL, is proposed to participate in multiple cellular events including membrane trafficking and cytoskeletal functions. We demonstrate here that CLN3 interacts with the plasma membrane-associated cytoskeletal and endocytic fodrin and the associated Na⁺, K⁺ ATPase. The ion pumping activity of Na⁺, K⁺ ATPase was unchanged in Cln3^−/− mouse primary neurons. However, the immunostaining pattern of fodrin appeared abnormal in JNCL fibroblasts and Cln3^−/− mouse brains suggesting disturbances in the fodrin cytoskeleton. Furthermore, the basal subcellular distribution as well as ouabain-induced endocytosis of neuron-specific Na⁺, K⁺ ATPase were remarkably affected in Cln3^−/− mouse primary neurons. These data suggest that CLN3 is involved in the regulation of plasma membrane fodrin cytoskeleton and consequently, the plasma membrane association of Na⁺, K⁺ ATPase. Most of the processes regulated by multifunctional fodrin and Na⁺, K⁺ ATPase are also affected in JNCL and Cln3-deficiency implicating that dysregulation of fodrin cytoskeleton and non-pumping functions of Na⁺, K⁺ ATPase may play a role in the neuronal degeneration in JNCL.     

Reversed Actrocytic GLT-1 during Ischemia is Crucial to Excitotoxic Death of Neurons, but Contributes to the Survival of Astrocytes themselves

During ischemia, the operation of astrocytic/neuronal glutamate transporters is reversed and glutamate and Na⁺ are co-transported to the extracellular space. This study aims to investigate whether this reversed operation of glutamate transporters has any functional meanings for astrocytes themselves. Oxygen/glucose deprivation (OGD) of neuron/astrocyte co-cultures resulted in the massive death of neurons, and the cell death was significantly reduced by treatment with either AP5 or DHK. In cultured astrocytes with little GLT-1 expression, OGD produced Na⁺ overload, resulting in the reversal of astrocytic Na⁺/Ca²⁺-exchanger (NCX). The reversed NCX then caused Ca²⁺ overload leading to the damage of astrocytes. In contrast, the OGD-induced Na⁺ overload and astrocytic damage were significantly attenuated in PACAP-treated astrocytes with increased GLT-1 expression, and the attenuation was antagonized by treatment with DHK. These results suggested that the OGD-induced reversal of GLT-1 contributed to the survival of astrocytes themselves by releasing Na⁺ with glutamate via reversed GLT-1.     

Computational Flux Balance Analysis Predicts that Stimulation of Energy Metabolism in Astrocytes and their Metabolic Interactions with Neurons Depend on Uptake of K<Superscript>+</Superscript> Rather than Glutamate

Brain activity involves essential functional and metabolic interactions between neurons and astrocytes. The importance of astrocytic functions to neuronal signaling is supported by many experiments reporting high rates of energy consumption and oxidative metabolism in these glial cells. In the brain, almost all energy is consumed by the Na⁺/K⁺ ATPase, which hydrolyzes 1 ATP to move 3 Na⁺ outside and 2 K⁺ inside the cells. Astrocytes are commonly thought to be primarily involved in transmitter glutamate cycling, a mechanism that however only accounts for few % of brain energy utilization. In order to examine the participation of astrocytic energy metabolism in brain ion homeostasis, here we attempted to devise a simple stoichiometric relation linking glutamatergic neurotransmission to Na⁺ and K⁺ ionic currents. To this end, we took into account ion pumps and voltage/ligand-gated channels using the stoichiometry derived from available energy budget for neocortical signaling and incorporated this stoichiometric relation into a computational metabolic model of neuron-astrocyte interactions. We aimed at reproducing the experimental observations about rates of metabolic pathways obtained by ¹³C-NMR spectroscopy in rodent brain. When simulated data matched experiments as well as biophysical calculations, the stoichiometry for voltage/ligand-gated Na⁺ and K⁺ fluxes generated by neuronal activity was close to a 1:1 relationship, and specifically 63/58 Na⁺/K⁺ ions per glutamate released. We found that astrocytes are stimulated by the extracellular K⁺ exiting neurons in excess of the 3/2 Na⁺/K⁺ ratio underlying Na⁺/K⁺ ATPase-catalyzed reaction. Analysis of correlations between neuronal and astrocytic processes indicated that astrocytic K⁺ uptake, but not astrocytic Na⁺-coupled glutamate uptake, is instrumental for the establishment of neuron-astrocytic metabolic partnership. Our results emphasize the importance of K⁺ in stimulating the activation of astrocytes, which is relevant to the understanding of brain activity and energy metabolism at the cellular level.     

Cell-Specific mRNA Alterations in Na+, K+-ATPase α and β Isoforms and FXYD in Mice Treated Chronically with Carbamazepine, an Anti-Bipolar Drug

Evidence accumulating during almost 50 years suggests Na⁺, K⁺-ATPase dysfunction in bipolar disorder, a disease treatable with chronic administration of lithium salts, carbamazepine or valproic acid. Three Na⁺, K⁺-ATPase α subunits (α1–3) and two β subunits (β1 and β2) are expressed in brain together with the auxiliary protein FXYD7. FXYD7 decreases K⁺ affinity, and thus contributes to stimulation of the enzyme at elevated extracellular K⁺ concentrations. Na⁺, K⁺-ATPase subtype and FXYD7 genes were determined by RT-PCR in mice co-expressing one fluorescent signal with an astrocytic marker or a different fluorescent signal with a neuronal marker and treated for 14 days with carbamazepine. Following fluorescence-activated cell sorting of neurons and astrocytes it was shown that α2 Expression was upregulated in astrocytes and neurons and α1 selectively in neurons, but α3 was unchanged. β1 was upregulated in astrocytes, but not in neurons. β2 was unaffected in astrocytes and absent in neurons. FXYD7 was downregulated specifically in neurons. According to cited literature data these changes should facilitate K⁺ uptake in neurons, without compromising preferential uptake in astrocytes at increased extracellular K⁺ concentrations. This process seems to be important for K⁺ homeostasis of the cellular level of the brain (Xu et al. Neurochem Res E-pub Dec. 12, 2012).     

MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation

MicroRNAs (miRNAs) are small RNAs with diverse regulatory roles. The miR-124 miRNA is expressed in neurons in the developing and adult nervous system. Here we show that overexpression of miR-124 in differentiating mouse P19 cells promotes neurite outgrowth, while blocking miR-124 function delays neurite outgrowth and decreases acetylated α-tubulin. Altered neurite outgrowth also was observed in mouse primary cortical neurons when miR-124 expression was increased, or when miR-124 function was blocked. In uncommitted P19 cells, miR-124 expression led to disruption of actin filaments and stabilization of microtubules. Expression of miR-124 also decreased Cdc42 protein and affected the subcellular localization of Rac1, suggesting that miR-124 may act in part via alterations to members of the Rho GTPase family. Furthermore, constitutively active Cdc42 or Rac1 attenuated neurite outgrowth promoted by miR-124. To obtain a broader perspective, we identified mRNAs downregulated by miR-124 in P19 cells using microarrays. mRNAs for proteins involved in cytoskeletal regulation were enriched among mRNAs downregulated by miR-124. A miR-124 variant with an additional 5′ base failed to promote neurite outgrowth and downregulated substantially different mRNAs. These results indicate that miR-124 contributes to the control of neurite outgrowth during neuronal differentiation, possibly by regulation of the cytoskeleton.     

Glutamate Induces Glutathione Efflux Mediated by Glutamate/Aspartate Transporter in Retinal Cell Cultures

This study was undertaken in order to characterize the role of the glutamate/aspartate transporter (GLAST) in the glutathione (GSH) efflux induced by glutamate. Our results demonstrated that retinal cell cultures exhibit two mechanisms of GSH release, one Na⁺-independent and other Na⁺-dependent. Glutamate and aspartate induced GSH efflux only in presence of Na⁺. Treatment with PCD (L-trans-Pyrrolidine-2,4-dicarboxylate), a transportable glutamate uptake blocker, increased GSH release indicating that GSH can be carried by glutamate transporters in retinal cell cultures. Added to this, treatment with zinc ion cultures, a recognized inhibitor of GLAST blocked GSH efflux evoked by glutamate. Treatment with NMDA antagonist (MK-801) did not have any effect on the GSH release induced by glutamate. These results suggest that glutamate induces GLAST-mediated release of GSH from retinal cell cultures and this could represent an important mechanism of cellular protection against glutamate toxicity in the CNS.     

TPEN,a Specific Zn<Superscript>2+</Superscript> Chelator,Inhibits Sodium Dithionite and Glucose Deprivation (SDGD)-Induced Neuronal Death by Modulating Apoptosis,Glutamate Signaling,and Voltage-Gated K<Superscript>+</Superscript> and Na<Superscript>+</Superscript> Channels

Hypoxia–ischemia-induced neuronal death is an important pathophysiological process that accompanies ischemic stroke and represents a major challenge in preventing ischemic stroke. To elucidate factors related to and a potential preventative mechanism of hypoxia–ischemia-induced neuronal death, primary neurons were exposed to sodium dithionite and glucose deprivation (SDGD) to mimic hypoxic–ischemic conditions. The effects of N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a specific Zn²⁺-chelating agent, on SDGD-induced neuronal death, glutamate signaling (including the free glutamate concentration and expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor (GluR2) and N-methyl-d-aspartate (NMDA) receptor subunits (NR2B), and voltage-dependent K⁺ and Na⁺ channel currents were also investigated. Our results demonstrated that TPEN significantly suppressed increases in cell death, apoptosis, neuronal glutamate release into the culture medium, NR2B protein expression, and I _K as well as decreased GluR2 protein expression and Na⁺ channel activity in primary cultured neurons exposed to SDGD. These results suggest that TPEN could inhibit SDGD-induced neuronal death by modulating apoptosis, glutamate signaling (via ligand-gated channels such as AMPA and NMDA receptors), and voltage-gated K⁺ and Na⁺ channels in neurons. Hence, Zn²⁺ chelation might be a promising approach for counteracting the neuronal loss caused by transient global ischemia. Moreover, TPEN could represent a potential cell-targeted therapy.     

How astrocytes feed hungry neurons

For years glucose was thought to constitute the sole energy substrate for neurons; it was believed to be directly provided to neurons via the extracellular space by the cerebral circulation. It was recently proposed that in addition to glucose, neurons might rely on lactate to sustain their activity. Therefore, it was demonstrated that lactate is a preferred oxidative substrate for neurons not only in vitro but also in vivo. Moreover, the presence of specific monocarboxylate transporters on neurons as well as on astrocytes is consistent with the hypothesis of a transfer of lactate from astrocytes to neurons. Evidence has been provided for a mechanism whereby astrocytes respond to glutamatergic activity by enhancing their glycolytic activity, resulting in increased lactate release. This is accomplished via the uptake of glutamate by glial glutamate transporters, leading to activation of the Na⁺/K⁺ ATPase and a stimulation of astrocytic glycolysis. Several recent observations obtained both in vitro and in vivo with different approaches have reinforced this view of brain energetics. Such an understanding might be critically important, not only because it forms the basis of some classical functional brain imaging techniques but also because several neurodegenerative diseases exhibit diverse alterations in energy metabolism.     

Regulation of neuronal excitability by release of proteins from glial cells

Effects of glial cells on electrical isolation and shaping of synaptic transmission between neurons have been extensively studied. Here we present evidence that the release of proteins from astrocytes as well as microglia may regulate voltage-activated Na⁺ currents in neurons, thereby increasing excitability and speed of transmission in neurons kept at distance from each other by specialized glial cells. As a first example, we show that basic fibroblast growth factor and neurotrophin-3, which are released from astrocytes by exposure to thyroid hormone, influence each other to enhance Na⁺ current density in cultured hippocampal neurons. As a second example, we show that the presence of microglia in hippocampal cultures can upregulate Na⁺ current density. The effect can be boosted by lipopolysaccharides, bacterial membrane-derived stimulators of microglial activation. Comparable effects are induced by the exposure of neuron-enriched hippocampal cultures to tumour necrosis factor-α, which is released from stimulated microglia. Taken together, our findings suggest that release of proteins from various types of glial cells can alter neuronal excitability over a time course of several days. This explains changes in neuronal excitability occurring in states of thyroid hormone imbalance and possibly also in seizures triggered by infectious diseases.     

Astrogliosis Is a Possible Player in Preventing Delayed Neuronal Death

Mitigating secondary delayed neuronal injury has been a therapeutic strategy for minimizing neurological symptoms after several types of brain injury. Interestingly, secondary neuronal loss appeared to be closely related to functional loss and/or death of astrocytes. In the brain damage induced by agonists of two glutamate receptors, N-ethyl-D-aspartic acid (NMDA) and kainic acid (KA), NMDA induced neuronal death within 3 h, but did not increase further thereafter. However, in the KA-injected brain, neuronal death was not obviously detectable even at injection sites at 3 h, but extensively increased to encompass the entire hemisphere at 7 days. Brain inflammation, a possible cause of secondary neuronal damage, showed little differences between the two models. Importantly, however, astrocyte behavior was completely different. In the NMDA-injected cortex, the loss of glial fibrillary acidic protein-expressing (GFAP⁺) astrocytes was confined to the injection site until 7 days after the injection, and astrocytes around the damage sites showed extensive gliosis and appeared to isolate the damage sites. In contrast, in the KA-injected brain, GFAP⁺ astrocytes, like neurons, slowly, but progressively, disappeared across the entire hemisphere. Other markers of astrocytes, including S100β, glutamate transporter EAAT2, the potassium channel Kir4.1 and glutamine synthase, showed patterns similar to that of GFAP in both NMDA- and KA-injected cortexes. More importantly, astrocyte disappearance and/or functional loss preceded neuronal death in the KA-injected brain. Taken together, these results suggest that loss of astrocyte support to neurons may be a critical cause of delayed neuronal death in the injured brain.     

Rottlerin Inhibits (Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>)-ATPase Activity in Brain Tissue and Alters <Emphasis Type="SmallCaps">d</Emphasis>-Aspartate Dependent Redistribution of Glutamate Transporter GLAST in Cultured Astrocytes

The naturally occurring toxin rottlerin has been used by other laboratories as a specific inhibitor of protein kinase C-delta (PKC-δ) to obtain evidence that the activity-dependent distribution of glutamate transporter GLAST is regulated by PKC-δ mediated phosphorylation. Using immunofluorescence labelling for GLAST and deconvolution microscopy we have observed that d-aspartate-induced redistribution of GLAST towards the plasma membranes of cultured astrocytes was abolished by rottlerin. In brain tissue in vitro, rottlerin reduced apparent activity of (Na⁺, K⁺)-dependent ATPase (Na⁺, K⁺-ATPase) and increased oxygen consumption in accordance with its known activity as an uncoupler of oxidative phosphorylation (“metabolic poison”). Rottlerin also inhibited Na⁺, K⁺-ATPase in cultured astrocytes. As the glutamate transport critically depends on energy metabolism and on the activity of Na⁺, K⁺-ATPase in particular, we suggest that the metabolic toxicity of rottlerin and/or the decreased activity of the Na⁺, K⁺-ATPase could explain both the glutamate transport inhibition and altered GLAST distribution caused by rottlerin even without any involvement of PKC-δ-catalysed phosphorylation in the process.     

Methylmercury alters the in vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells

In order to maintain normal functioning of the brain, glutamate homeostasis and extracellular levels of excitotoxic amino acids (EAA) must be tightly controlled. This is accomplished, in large measure, by the astroglial high-affinity Na⁺-dependent EAA transporters glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). Methylmercury (MeHg) is a potent neurotoxicant. Astrocytes are known targets for MeHg toxicity, representing a site for mercury localization. Mehg is known to cause astrocytic swelling, EAA release, and uptake inhibition in astrocytes, leading to increased extracellular glutamate levels and ensuing neuronal excitotoxicity and degeneration. However, the mechanisms and contribution of specific glutamate transporters to MeHg-induced glutamate dyshomeostasis remain unknown. Accordingly, the present study was carried out to investigate the effects of MeHg on the transport of [d-2, 3-³H]-d-aspartate, a nonmetabolizable glutamate analog in Chinese hamster ovary cells (CHO) transfected with the glutamate transporter subtypes GLAST or GLT-1. Additional studies examined the effects of MeHg on mRNA and protein levels of these transporters. Our results indicate the following (1) MeHg selectively affects glutamate transporter mRNA expression. MeHg treatment (6 h) led to no discernible changes in GLAST mRNA expression; however, GLT-1 mRNA expression significantly (p<0.001) increased following treatments with 5 or 10 μM MeHg. (2) Selective changes in the expression of glutamate transporter protein levels were also noted. GLAST transporter protein levels significantly (p<0.001, both at 5 and 10 μM MeHg) increased and GLT-1 transporter protein levels significantly (p<0.001) decreased followign MeHg exposure (5 μM). (3) MeHg exposure led to significant inhibition (p<0.05) of glutamate uptake by GLAST (both 5 and 10 μM MeHg), whereas GLT-1 transporter activity was significantly (p<0.01) increased following exposure to 5 and 10 μM MeHg. These studies suggest that MeHg contributes to the dysregulation of glutamate homeostasis and that its effects are distinct for GLAST and GLT-1.     

Neuroprotective properties of the excitatory amino acid carrier 1 (EAAC1)

Extracellular glutamate should be maintained at low levels to conserve optimal neurotransmission and prevent glutamate neurotoxicity in the brain. Excitatory amino acid transporters (EAATs) play a pivotal role in removing extracellular glutamate in the central nervous system (CNS). Excitatory amino acid carrier 1 (EAAC1) is a high-affinity Na⁺-dependent neuronal EAAT that is ubiquitously expressed in the brain. However, most glutamate released in the synapses is cleared by glial EAATs, but not by EAAC1 in vivo. In the CNS, EAAC1 is widely distributed in somata and dendrites but not in synaptic terminals. The contribution of EAAC1 to the control of extracellular glutamate levels seems to be negligible in the brain. However, EAAC1 can transport not only extracellular glutamate but also cysteine into the neurons. Cysteine is an important substrate for glutathione (GSH) synthesis in the brain. GSH has a variety of neuroprotective functions, while its depletion induces neurodegeneration. Therefore, EAAC1 might exert a critical role for neuroprotection in neuronal GSH metabolism rather than glutamatergic neurotransmission, while EAAC1 dysfunction would cause neurodegeneration. Despite the potential importance of EAAC1 in the brain, previous studies have mainly focused on the glutamate neurotoxicity induced by glial EAAT dysfunction. In recent years, however, several studies have revealed regulatory mechanisms of EAAC1 functions in the brain. This review will summarize the latest information on the EAAC1-regulated neuroprotective functions in the CNS.     

Primary cerebral and cerebellar astrocytes display differential sensitivity to extracellular sodium with significant effects on apoptosis

Central pontine myelinolysis is one of the idiopathic or iatrogenic brain dysfunction, and the most common cause is excessively rapid correction of chronic hyponatraemia. While myelin disruption is the main pathology, as the diagnostic name indicates, a previous study has reported that astrocyte death precedes the destruction of the myelin sheath after the rapid correction of chronic low Na⁺ levels, and interestingly, certain brain regions (cerebral cortex, hippocampus, etc.) are specifically damaged but not cerebellum. Here, using primary astrocyte cultures derived from rat cerebral cortex and cerebellum, we examined how extracellular Na⁺ alterations affect astrocyte death and whether the response is different between the two populations of astrocytes. Twice the amount of extracellular [Na⁺] and voltage‐gated Na⁺ channel opening induced substantial apoptosis in both populations of astrocytes, while, in contrast, one half [Na⁺] prevented apoptosis in cerebellar astrocytes, in which the Na⁺–Ca²⁺ exchanger, NCX2, was highly expressed but not in cerebral astrocytes. Strikingly, the rapid correction of chronic one half [Na⁺] exposure significantly increased apoptosis in cerebellar astrocytes but not in cerebral astrocytes. These results indicate that extracellular [Na⁺] affects astrocyte apoptosis, and the response to alterations in [Na⁺] is dependent on the brain region from which the astrocyte is derived. Copyright © 2014 John Wiley & Sons, Ltd.     

NOX activation in reactive astrocytes regulates astrocytic LCN2 expression and neurodegeneration

Reactive astrocytes (RA) secrete lipocalin-2 (LCN2) glycoprotein that regulates diverse cellular processes including cell death/survival, inflammation, iron delivery and cell differentiation. Elevated levels of LCN2 are considered as a biomarker of brain injury, however, the underlying regulatory mechanisms of its expression and release are not well understood. In this study, we investigated the role of astrocytic Na⁺/H⁺ exchanger 1 (NHE1) in regulating reactive astrocyte LCN2 secretion and neurodegeneration after stroke. Astrocyte specific deletion of Nhe1 in Gfap-Cre^ER+/−;Nhe1^f/f mice reduced astrogliosis and astrocytic LCN2 and GFAP expression, which was associated with reduced loss of NeuN⁺ and GRP78⁺ neurons in stroke brains. In vitro ischemia in astrocyte cultures triggered a significant increase of secreted LCN2 in astrocytic exosomes, which caused neuronal cell death and neurodegeneration. Inhibition of NHE1 activity during in vitro ischemia with its potent inhibitor HOE642 significantly reduced astrocytic LCN2⁺ exosome secretion. In elucidating the cellular mechanisms, we found that stroke triggered activation of NADPH oxidase (NOX)-NF-κB signaling and ROS-mediated LCN2 expression. Inhibition of astrocytic NHE1 activity attenuated NOX signaling and LCN2-mediated neuronal apoptosis and neurite degeneration. Our findings demonstrate for the first time that RA use NOX signaling to stimulate LCN2 expression and secretion. Blocking astrocytic NHE1 activity is beneficial to reduce LCN2-mediated neurotoxicity after stroke.Subject terms: Cell death in the nervous system, Astrocyte     

Cytoskeletal changes in diseases of the nervous system

The neuronal cytoskeleton not only provides the structural backbone of neurons, but also plays a fundamental role in maintaining neuronal functions. Dysregulation of neuronal architecture is evident in both injury and diseases of the central nervous system. These changes often result in the disruption of protein trafficking, loss of synapses and the death of neurons, ultimately impacting on signal transmission and manifesting in the disease phenotype. Furthermore, mutations in cytoskeletal proteins have been implicated in numerous diseases and, in some cases, identified as the cause of the disease, highlighting the critical role of the cytoskeleton in disease pathology. This review focuses on the role of cytoskeletal proteins in the pathology of mental disorders, neurodegenerative diseases and motor function deficits. In particular, we illustrate how cytoskeletal proteins can be directly linked to disease pathology and progression.     

Prostaglandin E2 modulates Na+,K+-ATPase activity in rat hippocampus: implications for neurological diseases

Prostaglandin E₂ (PGE₂) is quantitatively one of the major prostaglandins synthesized in mammalian brain, and there is evidence that it facilitates seizures and neuronal death. However, little is known about the molecular mechanisms involved in such excitatory effects. Na⁺,K⁺‐ATPase is a membrane protein which plays a key role in electrolyte homeostasis maintenance and, therefore, regulates neuronal excitability. In this study, we tested the hypothesis that PGE₂ decreases Na⁺,K⁺‐ATPase activity, in order to shed some light on the mechanisms underlying the excitatory action of PGE₂. Na⁺,K⁺‐ATPase activity was determined by assessing ouabain‐sensitive ATP hydrolysis. We found that incubation of adult rat hippocampal slices with PGE₂ (0.1–10 μM) for 30 min decreased Na⁺,K⁺‐ATPase activity in a concentration‐dependent manner. However, PGE₂ did not alter Na⁺,K⁺‐ATPase activity if added to hippocampal homogenates. The inhibitory effect of PGE₂ on Na⁺,K⁺‐ATPase activity was not related to a decrease in the total or plasma membrane immunocontent of the catalytic α subunit of Na⁺,K⁺‐ATPase. We found that the inhibitory effect of PGE₂ (1 μM) on Na⁺,K⁺‐ATPase activity was receptor‐mediated, as incubation with selective antagonists for EP1 (SC‐19220, 10 μM), EP3 (L‐826266, 1 μM) or EP4 (L‐161982, 1 μM) receptors prevented the PGE₂‐induced decrease of Na⁺,K⁺‐ATPase activity. On the other hand, incubation with the selective EP2 agonist (butaprost, 0.1–10 μM) increased enzyme activity per se in a concentration‐dependent manner, but did not prevent the inhibitory effect of PGE₂. Incubation with a protein kinase A (PKA) inhibitor (H‐89, 1 μM) and a protein kinase C (PKC) inhibitor (GF‐109203X, 300 nM) also prevented PGE₂‐induced decrease of Na⁺,K⁺‐ATPase activity. Accordingly, PGE₂ increased phosphorylation of Ser943 at the α subunit, a critical residue for regulation of enzyme activity. Importantly, we also found that PGE₂ decreases Na⁺,K⁺‐ATPase activity in vivo. The results presented here imply Na⁺,K⁺‐ATPase as a target for PGE₂‐mediated signaling, which may underlie PGE₂‐induced increase of brain excitability.     

A Novel Subtype of Astrocytes Expressing TRPV4 (Transient Receptor Potential Vanilloid 4) Regulates Neuronal Excitability via Release of Gliotransmitters

Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca²⁺ transients in astrocytes, and these Ca²⁺ transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4⁺ astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4⁺ astrocytes. After activation, both TRPV4⁺ and TRPV4⁻ astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4⁺ astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4⁺ astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands.     

The Na+/Ca2+exchanger in Alzheimer’s disease

As a pivotal player in regulating sodium (Na⁺) and calcium (Ca²⁺) homeostasis and signalling in excitable cells, the Na⁺/Ca²⁺ exchanger (NCX) is involved in many neurodegenerative disorders in which an imbalance of intracellular Ca²⁺ and/or Na⁺ concentrations occurs, including Alzheimer’s disease (AD). Although NCX has been mainly implicated in neuroprotective mechanisms counteracting Ca²⁺ dysregulation, several studies highlighted its role in the neuronal responses to intracellular Na⁺ elevation occurring in several pathophysiological conditions. Since the alteration of Na⁺ and Ca²⁺ homeostasis significantly contributes to synaptic dysfunction and neuronal loss in AD, it is of crucial importance to analyze the contribution of NCX isoforms in the homeostatic responses at neuronal and synaptic levels. Some studies found that an increase of NCX activity in brains of AD patients was correlated with neuronal survival, while other research groups found that protein levels of two NCX subtypes, NCX2 and NCX3, were modulated in parietal cortex of late stage AD brains. In particular, NCX2 positive synaptic terminals were increased in AD cohort while the number of NCX3 positive terminals were reduced. In addition, NCX1, NCX2 and NCX3 isoforms were up-regulated in those synaptic terminals accumulating amyloid-beta (Aβ), the neurotoxic peptide responsible for AD neurodegeneration. More recently, the hyperfunction of a specific NCX subtype, NCX3, has been shown to delay endoplasmic reticulum stress and apoptotic neuronal death in hippocampal neurons exposed to Aβ insult. Despite some issues about the functional role of NCX in synaptic failure and neuronal loss require further studies, these findings highlight the putative neuroprotective role of NCX in AD and open new strategies to develop new druggable targets for AD therapy.