Emerging Roles of Astrocyte Kir4.1 Channels in the Pathogenesis and Treatment of Brain Diseases

Inwardly rectifying Kir4.1 channels in astrocytes mediate spatial potassium (K+) buffering, a clearance mechanism for excessive extracellular K+, in tripartite synapses. In addition to K+ homeostasis, astrocytic Kir4.1 channels also play an essential role in regulating extracellular glutamate levels via coupling with glutamate transporters. Moreover, Kir4.1 channels act as novel modulators of the expression of brain-derived neurotrophic factor (BDNF) in astrocytes. Specifically, inhibition of astrocytic Kir4.1 channels elevates extracellular K+ and glutamate levels at synapses and facilitates BDNF expression in astrocytes. These changes elevate neural excitability, which may facilitate synaptic plasticity and connectivity. In this article, we summarize the functions and pharmacological features of Kir4.1 channels in astrocytes and highlight the importance of these channels in the treatment of brain diseases. Although further validation in animal models and human patients is required, astrocytic Kir4.1 channel could potentially serve as a novel therapeutic target for the treatment of depressive disorders and epilepsy.

Paraquat-induced intracellular Zn2+ dysregulation causes dopaminergic degeneration in the substantia nigra, but not in the striatum

Parkinson's disease (PD) is characterized by a selective death of nigrostriatal dopaminergic neurons, while the difference in the vulnerability to the death between the substantia nigra pars compacta (SNpc) and the striatum is poorly understood. Here we tested the difference focused on paraquat (PQ)-induced intracellular Zn2+ toxicity via extracellular glutamate accumulation. When PQ was locally injected into the SNpc and the striatum, dopaminergic degeneration was observed in the SNpc, but not in the striatum. Intracellular hydrogen peroxide (H2O2) produced by PQ was increased in both the SNpc and the striatum. In contrast, extracellular glutamate accumulation was observed only in the SNpc and rescued in the presence of N-(p-amylcinnamoyl)anthranilic acid (ACA), a blocker of the transient receptor potential melastatin 2 (TRPM2) cation channels. PQ increased intracellular Zn2+ level in the SNpc, but not in the striatum. The increase was rescued by 1-naphthyl acetyl spermine (NASPM), a selective blocker of Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptors. PQ-induced dopaminergic degeneration in the SNpc was rescued by ACA, NASPM, and GBR, a dopamine reuptake inhibitor. The present study indicates intracellular H2O2 produced by PQ, which is taken up through dopamine transporters, is retrogradely transported to presynaptic glutamatergic terminals, activates TRPM2 channels, accumulates glutamate in the extracellular compartment, and induces intracellular Zn2+ dysregulation via Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptor activation, resulting in dopaminergic degeneration in the SNpc. However, H2O2 signaling is not the case in the striatum. Paraquat-induced Zn2+ dysregulation plays a key role for neurodegeneration in the SNpc, but not in the striatum.

The Use of Ketamine as Therapy for Depression

Depressive disorder is still a significant problem in several developed countries and is morbidity caused by mental disorders. With the development of science, now discovered the unique pharmacodynamic properties of ketamine, which is used as an antidepressant. As we know in clinical practice, ketamine is used for anaesthesia, analgesia, sedation, and chronic pain management. Rapid-onset antidepressants resulted from increased levels of BDNF in the hippocampus. Extracellular glutamate agents are not new for the treatment of depression. According to the neurobiology view, depression is a monoaminergic phenomenon, so this is the impetus for discovering a new generation of antidepressants. Ketamine can be given intravenously in subanesthetic doses. Still, monitoring must be carrying in therapy administration because of the possible side effects such as hypersalivation, tachycardia, increased systemic arterial pressure, and intracranial pressure.

Malignant astrocyte swelling and impaired glutamate clearance drive the expansion of injurious spreading depolarization foci

Spreading depolarizations (SDs) indicate injury progression and predict worse clinical outcome in acute brain injury. We demonstrate in rodents that acute brain swelling upon cerebral ischemia impairs astroglial glutamate clearance and increases the tissue area invaded by SD. The cytotoxic extracellular glutamate accumulation (>15 µM) predisposes an extensive bulk of tissue (4–5 mm2) for a yet undescribed simultaneous depolarization (SiD). We confirm in rat brain slices exposed to osmotic stress that SiD is the pathological expansion of prior punctual SD foci (0.5–1 mm2), is associated with astrocyte swelling, and triggers oncotic neuron death. The blockade of astrocytic aquaporin-4 channels and Na+/K+/Cl− co-transporters, or volume-regulated anion channels mitigated slice edema, extracellular glutamate accumulation (<10 µM) and SiD occurrence. Reversal of slice swelling by hyperosmotic mannitol counteracted glutamate accumulation and prevented SiD. In contrast, inhibition of glial metabolism or inhibition of astrocyte glutamate transporters reproduced the SiD phenotype. Finally, we show in the rodent water intoxication model of cytotoxic edema that astrocyte swelling and altered astrocyte calcium waves are central in the evolution of SiD. We discuss our results in the light of evidence for SiD in the human cortex. Our results emphasize the need of preventive osmotherapy in acute brain injury.

Ketamine decreases neuronally released glutamate via retrograde stimulation of presynaptic adenosine A1 receptors

Ketamine produces a rapid antidepressant response in patients with major depressive disorder (MDD), but the underlying mechanisms appear multifaceted. One hypothesis, proposes that by antagonizing NMDA receptors on GABAergic interneurons, ketamine disinhibits afferens to glutamatergic principal neurons and increases extracellular glutamate levels. However, ketamine seems also to reduce rapid glutamate release at some synapses. Therefore, clinical studies in MDD patients have stressed the need to identify mechanisms whereby ketamine decreases presynaptic activity and glutamate release. In the present study, the effect of ketamine and its antidepressant metabolite, (2R,6R)-HNK, on neuronally derived glutamate release was examined in rodents. We used FAST methodology to measure depolarization-evoked extracellular glutamate levels in vivo in freely moving or anesthetized animals, synaptosomes to detect synaptic recycling ex vivo and primary cortical neurons to perform functional imaging and to examine intracellular signaling in vitro. In all these versatile approaches, ketamine and (2R,6R)-HNK reduced glutamate release in a manner which could be blocked by AMPA receptor antagonism. Antagonism of adenosine A1 receptors, which are almost exclusively expressed at nerve terminals, also counteracted ketamine’s effect on glutamate release and presynaptic activity. Signal transduction studies in primary neuronal cultures demonstrated that ketamine reduced P-T286-CamKII and P-S9-Synapsin, which correlated with decreased synaptic vesicle recycling. Moreover, systemic administration of A1R antagonist counteracted the antidepressant-like actions of ketamine and (2R,6R)-HNK in the forced swim test. To conclude, by studying neuronally released glutamate, we identified a novel retrograde adenosinergic feedback mechanism that mediate inhibitory actions of ketamine on glutamate release that may contribute to its rapid antidepressant action.

CB1R activation in nucleus accumbens core promotes stress-induced reinstatement of cocaine seeking by elevating extracellular glutamate in a drug-paired context

Preclinical models of stress-induced relapse to drug use have shown that the dysregulation of glutamatergic transmission within the nucleus accumbens (NA) contributes notably to the reinstatement of cocaine-seeking behavior in rodents. In this sense, there has been increasing interest in the cannabinoid type-1 receptor (CB1R), due to its crucial role in modulating glutamatergic neurotransmission within brain areas involved in drug-related behaviors. This study explored the involvement of CB1R within the NA subregions in the restraint stress-induced reinstatement of cocaine-conditioned place preference (CPP), as well as in the regulation of glutamatergic transmission, by using a pharmacological approach and the in vivo microdialysis sampling technique in freely moving rats. CB1R blockade by the antagonist/inverse agonist AM251 (5 nmol/0.5 μl/side) or CB1R activation by the agonist ACEA (0.01 fmol/0.5 μl/side), prevented or potentiated restraint stress-induced reinstatement of cocaine-CPP, respectively, after local administration into NAcore, but not NAshell. In addition, microdialysis experiments demonstrated that restraint stress elicited a significant increase in extracellular glutamate in NAcore under reinstatement conditions, with the local administration of AM251 or ACEA inhibiting or potentiating this, respectively. Interestingly, this rise specifically corresponded to the cocaine-associated CPP compartment. We also showed that this context-dependent change in glutamate paralleled the expression of cocaine-CPP, and disappeared after the extinction of this response. Taken together, these findings demonstrated the key role played by CB1R in mediating reinstatement of cocaine-CPP after restraint stress, through modulation of the context-specific glutamate release within NAcore. Additionally, CB1R regulation of basal extracellular glutamate was demonstrated and proposed as the underlying mechanism.

Abstract P812: Characterizing NMDA Receptor Subunits on B Cells

Background: N-methyl-D-aspartate (NMDARs) play a critical role in neuronal excitotoxicity after stroke. The actions of NMDARs have been shown mostly in obligatory GluN1 subunits on neurons and not GluN2A/B subunits. In B cells, these subunits have not been highly characterized though the presence of NMDARs has been shown. The function of the GluN2A/B subunits can be neuroprotective or pro-death in neurons, respectively. We hypothesized that GluN2A and GluN2B subunit presence on B cells would be affected by exposure to extracellular glutamate. Methods: Splenic B cells were isolated from 3-4mo-old C57BL/6 male mice via magnetic separation and treated with physiologic levels of L-glutamate (glu; 1uM) in the presence or absence of 5ug/mL LPS. B cell cytospins were stained for B220, GluN2A, and GluN2B, imaged using confocal microscopy, and quantified in FIJI. An average of 10.7 B cells were quantified per image at 80-157x magnification. RGB channels of the z-stacks were quantified to identify positive B220 expression. The z-stacks were split into 2D images and quantified plane-by-plane to identify GluN2A/B subunit clusters. Each cluster of subunits was recorded per cell in view across all planes of the original z-stack to yield total subunit count. Groups included 14-43 B cells quantified, and the number of subunits per cell were analyzed via ordinary two-way ANOVA, Sidak post-hoc test (Graphpad Prism). Significance was p<0.05. Results: There was an average of 19.3±7.2 GluN2A subunits and 19.0±5.0 GluN2B subunits per cell for unstimulated, untreated B cells. Neither glu treatment (p=0.23) nor LPS stimulation (p= 0.10) impacted the number of GluN2A subunits per B cell. LPS decreased GluN2B subunits when compared to unstimulated B cells (11.1±5.1 subunits; p=0.02). Glu treatment normalized GluN2B subunits per B cell near untreated baseline levels (18.2±11 subunits per cell; p=0.01), resulting in an interaction between LPS stimulation and glu treatment in B cells (F (1, 86) =6.180, P=0.015). Conclusions: Our data suggests activated B cells downregulate GluN2B-containing NMDARs following LPS stimulation. This downregulation mimics that of NMDAR activity on neurons upon excitoxicity (PMID: 24361499) but future studies should confirm GluN2B internalization.

Disruption of Glutamate Transport and Homeostasis by Acute Metabolic Stress

High-affinity, Na+-dependent glutamate transporters are the primary means by which synaptically released glutamate is removed from the extracellular space. They restrict the spread of glutamate from the synaptic cleft into the perisynaptic space and reduce its spillover to neighboring synapses. Thereby, glutamate uptake increases the spatial precision of synaptic communication. Its dysfunction and the entailing rise of the extracellular glutamate concentration accompanied by an increased spread of glutamate result in a loss of precision and in enhanced excitation, which can eventually lead to neuronal death via excitotoxicity. Efficient glutamate uptake depends on a negative resting membrane potential as well as on the transmembrane gradients of the co-transported ions (Na+, K+, and H+) and thus on the proper functioning of the Na+/K+-ATPase. Consequently, numerous studies have documented the impact of an energy shortage, as occurring for instance during an ischemic stroke, on glutamate clearance and homeostasis. The observations range from rapid changes in the transport activity to altered expression of glutamate transporters. Notably, while astrocytes account for the majority of glutamate uptake under physiological conditions, they may also become a source of extracellular glutamate elevation during metabolic stress. However, the mechanisms of the latter phenomenon are still under debate. Here, we review the recent literature addressing changes of glutamate uptake and homeostasis triggered by acute metabolic stress, i.e., on a timescale of seconds to minutes.

Role of EphrinA3 in HIV-1 Neuropathogenesis

Glial cells perform important supporting functions for neurons through a dynamic crosstalk. Neuron–glia communication is the major phenomenon to sustain homeostatic functioning of the brain. Several interactive pathways between neurons and astrocytes are critical for the optimal functioning of neurons, and one such pathway is the ephrinA3–ephA4 signaling. The role of this pathway is essential in maintaining the levels of extracellular glutamate by regulating the excitatory amino acid transporters, EAAT1 and EAAT2 on astrocytes. Human immunodeficiency virus-1 (HIV-1) and its proteins cause glutamate excitotoxicity due to excess glutamate levels at sites of high synaptic activity. This study unravels the effects of HIV-1 transactivator of transcription (Tat) from clade B on ephrinA3 and its role in regulating glutamate levels in astrocyte–neuron co-cultures of human origin. It was observed that the expression of ephrinA3 increases in the presence of HIV-1 Tat B, while the expression of EAAT1 and EAAT2 was attenuated. This led to reduced glutamate uptake and therefore high neuronal death due to glutamate excitotoxicity. Knockdown of ephrinA3 using small interfering RNA, in the presence of HIV-1 Tat B reversed the neurotoxic effects of HIV-1 Tat B via increased expression of glutamate transporters that reduced the levels of extracellular glutamate. The in vitro findings were validated in autopsy brain sections from acquired immunodeficiency syndrome patients and we found ephrinA3 to be upregulated in the case of HIV-1-infected patients. This study offers valuable insights into astrocyte-mediated neuronal damage in HIV-1 neuropathogenesis.