Dysregulated Transcript Expression but Not Function of the Glutamate Transporter EAAT2 in the Dorsolateral Prefrontal Cortex in Schizophrenia.

BACKGROUND: Schizophrenia (SCZ) is a serious mental illness with complex pathology, including abnormalities in the glutamate system. Glutamate is rapidly removed from the synapse by excitatory amino acid transporters (EAATs). Changes in the expression and localization of the primary glutamate transporter EAAT2 are found in the brain in central nervous system (CNS) disorders including SCZ. We hypothesize that neuronal expression and function of EAAT2 are increased in the frontal cortex in subjects diagnosed with SCZ. STUDY DESIGN: EAAT2 protein expression and glutamate transporter function were assayed in synaptosome preparations from the dorsolateral prefrontal cortex (DLPFC) of SCZ subjects and age- and sex-matched nonpsychiatrically ill controls. EAAT2 splice variant transcript expression was assayed in enriched populations of neurons and astrocytes from the DLPFC. Pathway analysis of publicly available transcriptomic datasets was carried out to identify biological changes associated with EAAT2 perturbation in different cell types. RESULTS: We found no significant changes in EAAT2 protein expression or glutamate uptake in the DLPFC in SCZ subjects compared with controls (n = 10/group). Transcript expression of EAAT2 and signaling molecules associated with EAAT2b trafficking (CaMKIIa and DLG1) were significantly altered in enriched populations of astrocytes and pyramidal neurons (P < .05) in SCZ (n = 16/group). These changes were not associated with antipsychotic medications. Pathway analysis also identified cell-type-specific enrichment of biological pathways associated with perturbation of astrocyte (immune pathways) and neuronal (metabolic pathways) EAAT2 expression. CONCLUSIONS: Overall, these data support the growing body of evidence for the role of dysregulation of the glutamate system in the pathophysiology of SCZ.

Hyperglycemia impairs EAAT2 glutamate transporter trafficking and glutamate clearance in islets of Langerhans: implications for type 2 diabetes pathogenesis and treatment.

Pancreatic endocrine cells employ a sophisticated system of paracrine and autocrine signals to synchronize their activities, including glutamate, which controls hormone release and ß-cell viability by acting on glutamate receptors expressed by endocrine cells. We here investigate whether alteration of the excitatory amino acid transporter 2 (EAAT2), the major glutamate clearance system in the islet, may occur in type 2 diabetes mellitus and contribute to ß-cell dysfunction. Increased EAAT2 intracellular localization was evident in islets of Langerhans from T2DM subjects as compared with healthy control subjects, despite similar expression levels. Chronic treatment of islets from healthy donors with high-glucose concentrations led to the transporter internalization in vesicular compartments and reduced [H3]-d-glutamate uptake (65 ± 5% inhibition), phenocopying the findings in T2DM pancreatic sections. The transporter relocalization was associated with decreased Akt phosphorylation protein levels, suggesting an involvement of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the process. In line with this, PI3K inhibition by a 100-µM LY294002 treatment in human and clonal ß-cells caused the transporter relocalization in intracellular compartments and significantly reduced the glutamate uptake compared to control conditions, suggesting that hyperglycemia changes the trafficking of the transporter to the plasma membrane. Upregulation of the glutamate transporter upon treatment with the antibiotic ceftriaxone rescued hyperglycemia-induced ß-cells dysfunction and death. Our data underscore the significance of EAAT2 in regulating islet physiology and provide a rationale for potential therapeutic targeting of this transporter to preserve ß-cell survival and function in diabetes.NEW & NOTEWORTHY The glutamate transporter SLC1A2/excitatory amino acid transporter 2 (EAAT2) is expressed on the plasma membrane of pancreatic ß-cells and controls islet glutamate clearance and ß-cells survival. We found that the EAAT2 membrane expression is lost in the islets of Langerhans from type 2 diabetes mellitus (T2DM) patients due to hyperglycemia-induced downregulation of the phosphoinositide 3-kinase/Akt pathway and modification of its intracellular trafficking. Pharmacological rescue of EAAT2 expression prevents ß-cell dysfunction and death, suggesting EAAT2 as a new potential target of intervention in T2DM.

The Conventional and Breakthrough Tool for the Study of L-Glutamate Transporters.

In our recent report, we clarified the direct interaction between the excitatory amino acid transporter (EAAT) 1/2 and polyunsaturated fatty acids (PUFAs) by applying electrophysiological and molecular biological techniques to Xenopus oocytes. Xenopus oocytes have a long history of use in the scientific field, but they are still attractive experimental systems for neuropharmacological studies. We will therefore summarize the pharmacological significance, advantages (especially in the study of EAAT2), and experimental techniques that can be applied to Xenopus oocytes; our new findings concerning L-glutamate (L-Glu) transporters and PUFAs; and the significant outcomes of our data. The data obtained from electrophysiological and molecular biological studies of Xenopus oocytes have provided us with further important questions, such as whether or not some PUFAs can modulate EAATs as allosteric modulators and to what extent docosahexaenoic acid (DHA) affects neurotransmission and thereby affects brain functions. Xenopus oocytes have great advantages in the studies about the interactions between molecules and functional proteins, especially in the case when the expression levels of the proteins are small in cell culture systems without transfections. These are also proper to study the mechanisms underlying the interactions. Based on the data collected in Xenopus oocyte experiments, we can proceed to the next step, i.e., the physiological roles of the compounds and their significances. In the case of EAAT2, the effects on the neurotransmission should be examined by electrophysiological approach using acute brain slices. For new drug development, pharmacokinetics pharmacodynamics (PKPD) data and blood brain barrier (BBB) penetration data are also necessary. In order not to miss the promising candidate compounds at the primary stages of drug development, we should reconsider using Xenopus oocytes in the early phase of drug development.

KYNA Ameliorates Glutamate Toxicity of HAND by Enhancing Glutamate Uptake in A2 Astrocytes.

Reactive astrocytes are key players in HIV-associated neurocognitive disorders (HAND), and different types of reactive astrocytes play opposing roles in the neuropathologic progression of HAND. A recent study by our group found that gp120 mediates A1 astrocytes (neurotoxicity), which secrete proinflammatory factors and promote HAND disease progression. Here, by comparing the expression of A2 astrocyte (neuroprotective) markers in the brains of gp120 tgm mice and gp120+/α7nAChR-/- mice, we found that inhibition of alpha 7 nicotinic acetylcholine receptor (α7nAChR) promotes A2 astrocyte generation. Notably, kynurenine acid (KYNA) is an antagonist of α7nAChR, and is able to promote the formation of A2 astrocytes, the secretion of neurotrophic factors, and the enhancement of glutamate uptake through blocking the activation of α7nAChR/NF-κB signaling. In addition, learning, memory and mood disorders were significantly improved in gp120 tgm mice by intraperitoneal injection of kynurenine (KYN) and probenecid (PROB). Meanwhile, the number of A2 astrocytes in the mouse brain was significantly increased and glutamate toxicity was reduced. Taken together, KYNA was able to promote A2 astrocyte production and neurotrophic factor secretion, reduce glutamate toxicity, and ameliorate gp120-induced neuropathological deficits. These findings contribute to our understanding of the role that reactive astrocytes play in the development of HAND pathology and provide new evidence for the treatment of HAND via the tryptophan pathway.

Still in Search for an EAAT Activator: GT949 Does Not Activate EAAT2, nor EAAT3 in Impedance and Radioligand Uptake Assays.

Excitatory amino acid transporters (EAATs) are important regulators of amino acid transport and in particular glutamate. Recently, more interest has arisen in these transporters in the context of neurodegenerative diseases. This calls for ways to modulate these targets to drive glutamate transport, EAAT2 and EAAT3 in particular. Several inhibitors (competitive and noncompetitive) exist to block glutamate transport; however, activators remain scarce. Recently, GT949 was proposed as a selective activator of EAAT2, as tested in a radioligand uptake assay. In the presented research, we aimed to validate the use of GT949 to activate EAAT2-driven glutamate transport by applying an innovative, impedance-based, whole-cell assay (xCELLigence). A broad range of GT949 concentrations in a variety of cellular environments were tested in this assay. As expected, no activation of EAAT3 could be detected. Yet, surprisingly, no biological activation of GT949 on EAAT2 could be observed in this assay either. To validate whether the impedance-based assay was not suited to pick up increased glutamate uptake or if the compound might not induce activation in this setup, we performed radioligand uptake assays. Two setups were utilized; a novel method compared to previously published research, and in a reproducible fashion copying the methods used in the existing literature. Nonetheless, activation of neither EAAT2 nor EAAT3 could be observed in these assays. Furthermore, no evidence of GT949 binding or stabilization of purified EAAT2 could be observed in a thermal shift assay. To conclude, based on experimental evidence in the present study GT949 requires specific assay conditions, which are difficult to reproduce, and the compound cannot simply be classified as an activator of EAAT2 based on the presented evidence. Hence, further research is required to develop the tools needed to identify new EAAT modulators and use their potential as a therapeutic target.

Leat-associated seizures the possible role of EAAT2, pyruvate carboxylase and glutamine synthetase.

BACKGROUND: Drug-resistant epilepsy is a common condition in patients with brain neoplasms. The pathogenesis of tumor-associated seizures is poorly understood. Among the possible pathogenetic mechanisms, the increase in glutamate concentration has been proposed. Glutamate transporters, glutamine synthetase and pyruvate carboxylase are involved in maintaining the physiological concentration of glutamate in the intersynaptic spaces. In our previous research on angiocentric gliomas, we demonstrated that all tumors lacked the expression of the main glutamate transporter EAAT2, while the expression of glutamine synthetase and pyruvate carboxylase was mostly preserved. METHODS: In the present study, we evaluated the immunohistochemical expression of EAAT2, glutamine synthetase and pyruvate carboxylase in a heterogeneous series of 25 long-term epilepsy-associated tumors (10 dysembryoplastic neuroepithelial tumors, 7 gangliogliomas, 3 subependymal giant cell astrocytomas, 3 rosette forming glioneuronal tumors, 1 diffuse astrocytoma MYB- or MYBL1-altered and 1 angiocentric glioma). In order to evaluate the incidence of variants in the SLC1A2 gene, encoding EAAT2, in a large number of central nervous system tumors we also queried the PedcBioPortal. RESULTS: EAAT2 protein expression was lost in 9 tumors (36 %: 3 dysembryoplastic neuroepithelial tumors, 1 ganglioglioma, 3 subependymal giant cell astrocytomas, 1 diffuse astrocytoma MYB- or MYBL1-altered and 1 angiocentric glioma). Glutamine synthetase protein expression was completely lost in 2 tumors (8 %; 1 ganglioglioma and 1 diffuse astrocytoma MYB- or MYBL1-altered). All tumors of our series but rosette forming glioneuronal tumors (in which neurocytic cells were negative) were diffusely positive for pyruvate carboxylase. Consultation of the PedcBioPortal revealed that of 2307 pediatric brain tumors of different histotype and grade, 20 (< 1%) had variants in the SLC1A2 gene. Among the SLC1A2-mutated tumors, there were no angiocentric gliomas or other LEATs CONCLUSIONS: In conclusion, unlike angiocentric gliomas where the EAAT2 loss is typical and constant, the current study shows the loss of EAAT2 expression only in a fraction of the LEATs. In these cases, we may hypothesize some possible epileptogenic role of the EAAT2 loss. The retained expression of pyruvate carboxylase may contribute to determining a pathological glutamate excess unopposed by glutamine synthetase that resulted expressed to a variable extent in the majority of the tumors. Furthermore, we can assume that the EAAT2 loss in brain tumors in general and in LEATs in particular is more conceivably epigenetic.

HSP90ß regulates EAAT2 expression and participates in ischemia­reperfusion injury in rats.

Cerebrovascular diseases (CVDs) have become a global public health problem and ischemia­reperfusion injury, the major cause of neurological impairment exacerbation, is closely related to excitotoxicity. The present study aimed to investigate the effects of changes in heat shock protein (HSP)90ß expression and verify whether HSP90ß regulates EAAT2 expression in a cerebral ischemia­reperfusion injury model. Healthy adult Sprague­Dawley (SD) male rats were used to establish a control group, sham­operated group, middle cerebral artery occlusion (MCAO) group, empty virus group and lentivirus group. A model of cerebral ischemia­reperfusion was established using the MCAO method. Lentivirus construction and injection were used to interfere with the expression of HSP90ß. The modified neurological severity score was used to assess neurological deficits. Triphenyltetrazolium chloride staining was used to detect infarct areas. Immunofluorescence was used to detect HSP90ß expression localization and the expression levels of HSP90ß and EAAT2 were determined using western blotting and reverse transcription­quantitative PCR. An MCAO model was successfully established and it was found that HSP90ß, but not HSP90α, was upregulated after MCAO. HSP90ß expression coincided with astrocyte markers in the ischemic penumbra area, while no expression was observed in microglia. Inhibition of HSP90ß expression improved neurological deficits and alleviated brain injury by increasing EAAT2 expression. These results suggested that HSP90ß is involved in the process of cerebral ischemia­reperfusion injury in rats and that inhibition of HSP90ß expression increases EAAT2 levels, conferring a neuroprotective effect in MCAO model rats.

PS1/gamma-secretase acts as rogue chaperone of glutamate transporter EAAT2/GLT-1 in Alzheimer's disease.

The recently discovered interaction between presenilin 1 (PS1), a catalytic subunit of γ-secretase responsible for the generation of amyloid-ß(Aß) peptides, and GLT-1, the major glutamate transporter in the brain (EAAT2 in the human) may provide a mechanistic link between two important pathological aspects of Alzheimer's disease (AD): abnormal Aßoccurrence and neuronal network hyperactivity. In the current study, we employed a FRET-based approach, fluorescence lifetime imaging microscopy (FLIM), to characterize the PS1/GLT-1 interaction in its native environment in the brain tissue of sporadic AD (sAD) patients. There was significantly less interaction between PS1 and GLT-1 in sAD brains, compared to tissue from patients with frontotemporal lobar degeneration (FTLD), or non-demented age-matched controls. Since PS1 has been shown to adopt pathogenic "closed" conformation in sAD but not in FTLD, we assessed the impact of changes in PS1 conformation on the interaction. Familial AD (fAD) PS1 mutations which induce a "closed" PS1 conformation similar to that in sAD brain and gamma-secretase modulators (GSMs) which induce a "relaxed" conformation, reduced and increased the interaction, respectively. This indicates that PS1 conformation seems to have a direct effect on the interaction with GLT-1. Furthermore, using biotinylation/streptavidin pull-down, western blotting, and cycloheximide chase assays, we determined that the presence of PS1 increased GLT-1 cell surface expression and GLT-1 homomultimer formation, but did not impact GLT-1 protein stability. Together, the current findings suggest that the newly described PS1/GLT-1 interaction endows PS1 with chaperone activity, modulating GLT-1 transport to the cell surface and stabilizing the dimeric-trimeric states of the protein. The diminished PS1/GLT-1 interaction suggests that these functions of the interaction may not work properly in AD.

Neuroprotective Potential of L-Glutamate Transporters in Human Induced Pluripotent Stem Cell-Derived Neural Cells against Excitotoxicity.

Human induced pluripotent stem cell (hiPSC)-derived neural cells have started to be used in safety/toxicity tests at the preclinical stage of drug development. As previously reported, hiPSC-derived neurons exhibit greater tolerance to excitotoxicity than those of primary cultures of rodent neurons; however, the underlying mechanisms remain unknown. We here investigated the functions of L-glutamate (L-Glu) transporters, the most important machinery to maintain low extracellular L-Glu concentrations, in hiPSC-derived neural cells. We also clarified the contribution of respective L-Glu transporter subtypes. At 63 days in vitro (DIV), we detected neuronal circuit functions in hiPSC-derived neural cells by a microelectrode array system (MEA). At 63 DIV, exposure to 100 µM L-Glu for 24 h did not affect the viability of neural cells. 100 µM L-Glu in the medium decreased to almost 0 µM in 60 min. Pharmacological inhibition of excitatory amino acid transporter 1 (EAAT1) and EAAT2 suppressed almost 100% of L-Glu decrease. In the presence of this inhibitor, 100 µM L-Glu dramatically decreased cell viability. These results suggest that in hiPSC-derived neural cells, EAAT1 and EAAT2 are the predominant L-Glu transporters, and their uptake potentials are the reasons for the tolerance of hiPSC-derived neurons to excitotoxicity.

ß_lactam antibiotics against drug addiction: A novel therapeutic option.

Drug addiction as a problem for the health of the individual and the society is the result of a complex process in which there is an interaction between brain nuclei and neurotransmitters (such as glutamate). ß-lactam antibiotics, due to their enhancing properties on the glutamate transporter glutamate transporter-1, can affect and counteract the addictive mechanisms of drugs through the regulation of extracellular glutamate. Since glutamate is a key neurotransmitter in the development of drug addiction, it seems that ß-lactams can be considered as a promising treatment for addiction. However, more research in this field is necessary to identify other mechanisms involved in their effectiveness. This article is a review of the studies conducted on the effect of ß-lactam administration in preventing the development of drug addiction, as well as their possible cellular and molecular mechanisms. This review suggests the clinical use of ß-lactam antibiotics that have weak antimicrobial properties (such as clavulanic acid) in the treatment of drug dependence.

Activation of Gq-Coupled Receptors in Astrocytes Restores Cognitive Function in Alzheimer's Disease Mice Model.

Alzheimer's disease (AD) is one of the most widespread neurodegenerative diseases. Most of the current AD therapeutic developments are directed towards improving neuronal cell function or facilitating Aß amyloid clearance from the brain. However, some recent evidence suggests that astrocytes may play a significant role in the pathogenesis of AD. In this paper, we evaluated the effects of the optogenetic activation of Gq-coupled exogenous receptors expressed in astrocytes as a possible way of restoring brain function in the AD mouse model. We evaluated the effects of the optogenetic activation of astrocytes on long-term potentiation, spinal morphology and behavioral readouts in 5xFAD mouse model of AD. We determined that in vivo chronic activation of astrocytes resulted in the preservation of spine density, increased mushroom spine survival, and improved performance in cognitive behavioral tests. Furthermore, chronic optogenetic stimulation of astrocytes resulted in the elevation of EAAT-2 glutamate uptake transporter expression, which could be a possible explanation for the observed in vivo neuroprotective effects. The obtained results suggest that the persistent activation of astrocytes may be considered a potential therapeutic approach for the treatment of AD and possibly other neurodegenerative disorders.

Novel EAAT2 activators improve motor and cognitive impairment in a transgenic model of Huntington's disease.

Introduction: Glutamate excitotoxicity is causal in striatal neurodegeneration underlying motor dysfunction and cognitive deficits in Huntington's disease (HD). Excitatory amino acid transporter 2 (EAAT2), the predominant glutamate transporter accounting for >90% of glutamate transport, plays a key role in preventing excitotoxicity by clearing excess glutamate from the intrasynaptic cleft. Accordingly, EAAT2 has emerged as a promising therapeutic target for prevention of neuronal excitotoxicity underlying HD and other neurodegenerative diseases. Methods: We have previously designed novel EAAT2 positive allosteric modulator GT951, GTS467, and GTS551, with low nanomolar efficacy in glutamate uptake and favorable pharmacokinetic properties. In this study, we test the neuroprotective abilities of these novel EAAT2 activators in vivo using the robust Drosophila HD transgenic model expressing human huntingtin gene with expanded repeats (Htt128Q). Results: All three compounds significantly restored motor function impaired under HD pathology over a wide dose range. Additionally, treatment with all three compounds significantly improved HD-associated olfactory associative learning and short-term memory defects, while GT951 and GTS551 also improved middle-term memory in low-performing group. Similarly, treatment with GT951 and GTS551 partially protected against early mortality observed in our HD model. Further, treatment with all three EAAT2 activators induced epigenetic expression of EAAT2 Drosophila homolog and several cognition-associated genes. Conclusion: Together, these results highlight the efficacy of GT951, GTS467 and GTS551 in treating motor and cognitive impairments under HD pathology and support their development for treatment of HD.

Riluzole treatment modulates KCC2 and EAAT-2 receptor expression and Ca2+ accumulation following ventral root avulsion injury.

Avulsion injury results in motoneuron death due to the increased excitotoxicity developing in the affected spinal segments. This study focused on possible short and long term molecular and receptor expression alterations which are thought to be linked to the excitotoxic events in the ventral horn with or without the anti-excitotoxic riluzole treatment. In our experimental model the left lumbar 4 and 5 (L4, 5) ventral roots of the spinal cord were avulsed. Treated animals received riluzole for 2 weeks. Riluzole is a compound that acts to block voltage-activated Na+ and Ca2+ channels. In control animals the L4, 5 ventral roots were avulsed without riluzole treatment. Expression of astrocytic EAAT-2 and that of KCC2 in motoneurons on the affected side of the L4 spinal segment were detected after the injury by confocal and dSTORM imaging, intracellular Ca2+ levels in motoneurons were quantified by electron microscopy. The KCC2 labeling in the lateral and ventrolateral parts of the L4 ventral horn was weaker compared with the medial part of L4 ventral horn in both groups. Riluzole treatment dramatically enhanced motoneuron survival but was not able to prevent the down-regulation of KCC2 expression in injured motoneurons. In contrast, riluzole successfully obviated the increase of intracellular calcium level and the decrease of EAAT-2 expression in astrocytes compared with untreated injured animals. We conclude that KCC2 may not be an essential component for survival of injured motoneurons and riluzole is able to modulate the intracellular level of calcium and expression of EAAT-2.

The Amyloid Cascade Hypothesis in Alzheimer's Disease: Should We Change Our Thinking?

Old age increases the risk of Alzheimer's disease (AD), the most common neurodegenerative disease, a devastating disorder of the human mind and the leading cause of dementia. Worldwide, 50 million people have the disease, and it is estimated that there will be 150 million by 2050. Today, healthcare for AD patients consumes 1% of the global economy. According to the amyloid cascade hypothesis, AD begins in the brain by accumulating and aggregating Aß peptides and forming ß-amyloid fibrils (Aß42). However, in clinical trials, reducing Aß peptide production and amyloid formation in the brain did not slow cognitive decline or improve daily life in AD patients. Prevention studies in cognitively unimpaired people at high risk or genetically destined to develop AD also have not slowed cognitive decline. These observations argue against the amyloid hypothesis of AD etiology, its development, and disease mechanisms. Here, we look at other avenues in the research of AD, such as the presenilin hypothesis, synaptic glutamate signaling, and the role of astrocytes and the glutamate transporter EAAT2 in the development of AD.

Yes-associated protein regulates glutamate homeostasis through promoting the expression of excitatory amino acid transporter-2 in astrocytes via ß-catenin signaling.

The homeostasis of glutamate is mainly regulated by the excitatory amino acid transporters (EAATs), especially by EAAT2 in astrocytes. Excessive glutamate in the synaptic cleft caused by dysfunction or dysregulation of EAAT2 can lead to excitotoxicity, neuronal death and cognitive dysfunction. However, it remains unclear about the detailed regulation mechanism of expression and function of astrocytic EAAT2. In this study, first, we found increased neuronal death and impairment of cognitive function in YAPGFAP -CKO mice (conditionally knock out Yes-associated protein [YAP] in astrocytes), and identified EAAT2 as a downstream target of YAP through RNA sequencing. Second, the expression of EAAT2 was decreased in cultured YAP-/- astrocytes and the hippocampus of YAPGFAP -CKO mice, and glutamate uptake was reduced in YAP-/- astrocytes, but increased in YAP-upregulated astrocytes. Third, further investigation of the mechanism showed that the mRNA and protein levels of ß-catenin were decreased in YAP-/- astrocytes and increased in YAP-upregulated astrocytes. Wnt3a activated YAP signaling and up-regulated EAAT2 through ß-catenin. Furthermore, over-expression or activation of ß-catenin partially restored the downregulation of EAAT2, the impairment of glutamate uptake, neuronal death and cognitive decline that caused by YAP deletion. Finally, activation of EAAT2 also rescued neuronal death and cognitive decline in YAPGFAP -CKO mice. Taken together, our study identifies an unrecognized role of YAP signaling in the regulation of glutamate homeostasis through the ß-catenin/EAAT2 pathway in astrocytes, which may provide novel insights into the pathogenesis of brain diseases that closely related to the dysfunction or dysregulation of EAAT2, and promote the development of clinical strategy.

Diminished motor neuron activity driven by abnormal astrocytic EAAT1 glutamate transporter activity in spinal muscular atrophy is not fully restored after lentiviral SMN delivery.

Spinal muscular atrophy (SMA) is characterized by the loss of the lower spinal motor neurons due to survival motor neuron (SMN) deficiency. The motor neuron cell autonomous and non-cell autonomous disease mechanisms driving early glutamatergic dysfunction, a therapeutically targetable phenotype prior to motor neuron cell loss, remain unclear. Using microelectrode array analysis, we demonstrate that the secretome and cell surface proteins needed for proper synaptic modulation are likely disrupted in human SMA astrocytes and lead to diminished motor neuron activity. While healthy astrocyte conditioned media did not improve SMA motor neuron activity, SMA motor neurons robustly responded to healthy astrocyte neuromodulation in direct contact cultures. This suggests an important role of astrocyte synaptic-associated plasma membrane proteins and contact-mediated cellular interactions for proper motor neuron function in SMA. Specifically, we identified a significant reduction of the glutamate Na+ dependent excitatory amino acid transporter EAAT1 within human SMA astrocytes and SMA lumbar spinal cord tissue. The selective inhibition of EAAT1 in healthy co-cultures phenocopied the diminished neural activity observed in SMA astrocyte co-cultures. Caveolin-1, an SMN-interacting protein previously associated with local translation at the plasma membrane, was abnormally elevated in human SMA astrocytes. Although lentiviral SMN delivery to SMA astrocytes partially rescued EAAT1 expression, limited activity of healthy motor neurons was still observed in SMN-transduced SMA astrocyte co-cultures. Together, these data highlight the detrimental impact of astrocyte-mediated disease mechanisms on motor neuron function in SMA and that SMN delivery may be insufficient to fully restore astrocyte function at the synapse.

Ceftriaxone induces glial EAAT-2 promotor region via NF-kB conformational changes: An interaction analysis using HADDOCK.

Excitotoxicity, depletion of energy metabolites, and ionic imbalance are the major factors involved in neurodegeneration mediated through excitatory amino acid transporter-2 (EAAT-2) dysfunction in ischemic insult. Recent studies have revealed that ceftriaxone expresses EAAT-2 via nuclear transcription factor kappa-B (NF-kB) signaling pathway, stimulation of EAAT-2 expression in the ischemic, and excitotoxic conditions that could provide potential benefits to control neurodegeneration. In this study, we have predicted the in silico model for interaction between NF-kB and EAAT-2 promoter region to rule out the conformational changes for the expression of EAAT-2 protein. Using homology-built model of NF-kB, we identified ceftriaxone-induced conformational changes in gene locus -272 of DNA where NF-kB binding with EAAT-2 promoter region through protein-DNA docking calculation. The interaction profile and conformational dynamics occurred between ceftriaxone predocked and postdocked conformations of NF-kB with DNA employing HADDOCK 2.2 web server followed by 250 ns long all atom explicit solvent molecular dynamics simulations. Both the protein and DNA exhibited modest conformational changes with respect to HADDOCK score, energy terms (desolvation energy [Edesolv ]), van der waal energy (Evdw ), electrostatic energy (Eelec ), restraints energy (Eair ), buried surface area, root mean square deviation, RMSF, radius of gyration, total hydrogen bonds when ceftriaxone pre- and postdocked NF-kB conformations were bound to DNA. Hence, the conformational changes in the C-terminal domain could be the reason for EAAT-2 expression through ceftriaxone specific binding pocket of -272 of DNA.

The role of excitatory amino acid transporter 2 (EAAT2) in epilepsy and other neurological disorders.

Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS). Excitatory amino acid transporters (EAATs) have important roles in the uptake of glutamate and termination of glutamatergic transmission. Up to now, five EAAT isoforms (EAAT1-5) have been identified in mammals. The main focus of this review is EAAT2. This protein has an important role in the pathoetiology of epilepsy. De novo dominant mutations, as well as inherited recessive mutation in this gene, have been associated with epilepsy. Moreover, dysregulation of this protein is implicated in a range of neurological diseases, namely amyotrophic lateral sclerosis, alzheimer's disease, parkinson's disease, schizophrenia, epilepsy, and autism. In this review, we summarize the role of EAAT2 in epilepsy and other neurological disorders, then provide an overview of the therapeutic modulation of this protein.

Formulation of tizanidine hydrochloride-loaded provesicular system for improved oral delivery and therapeutic activity employing a 23 full factorial design.

Tizanidine hydrochloride (TZN) is one of the most effective centrally acting skeletal muscle relaxants. The objective of this study is to prepare TZN-loaded proniosomes (TZN-PN) aiming at enhanced oral delivery and therapeutic activity. TZN-PN were prepared by coacervation phase separation method. The developed vesicles were characterized via entrapment efficiency percentage (EE%), vesicular size (VS), and zeta potential (ZP). A 23 full factorial design was employed to attain an optimized TZN-PN formulation. The optimized TZN-PN were further characterized via in vitro release study and transmission electron microscopy (TEM). In vivo rotarod test was employed for determination of the muscle relaxant activities of rats and levels of GABA and EAAT2 were detected. The developed TZN-PN exhibited relatively high EE% (75.78-85.45%), a VS ranging between (348-559 nm), and a ZP (-26.47 to -59.64). In vitro release profiles revealed sustained release of TZN from the optimized TZN-PN, compared to free drug up to 24 h. In vivo rotarod study revealed that the elevation in coordination was in the following order: normal control < free TZN < market product < TZN-PN (F6). Moreover, the optimized TZN-PN exhibited significant elevated coordination activity by 39% and 26% compared to control group and market product group, respectively. This was accompanied with an elevation in both GABA and EAAT2 serum levels. Thus, it could be concluded that encapsulation of TZN in the provesicular nanosystem proniosomes has enhanced the anti-nociceptive effect of the drug and consequently its therapeutic activity.

Neuromyelitis optica spectrum disorder: pathophysiological approach.

Aim: To review the main pathological findings of Neuromyelitis Optica Spectrum Disorder (NMOSD) associated with the presence of autoantibodies to aquaporin-4 (AQP4) as well as the mechanisms of astrocyte dysfunction and demyelination. Methods: An comprehensive search of the literature in the field was carried out using the database of The National Center for Biotechnology Information from . Systematic searches were performed until July 2022. Results: NMOSD is an inflammatory and demyelinating disease of the central nervous system mainly in the areas of the optic nerves and spinal cord, thus explaining mostly the clinical findings. Other areas affected in NMOSD are the brainstem, hypothalamus, and periventricular regions. Relapses in NMOSD are generally severe and patients only partially recover. NMOSD includes clinical conditions where autoantibodies to aquaporin-4 (AQP4-IgG) of astrocytes are detected as well as similar clinical conditions where such antibodies are not detected. AQP4 are channel-forming integral membrane proteins of which AQ4 isoforms are able to aggregate in supramolecular assemblies termed orthogonal arrays of particles (OAP) and are essential in the regulation of water homeostasis and the adequate modulation of neuronal activity and circuitry. AQP4 assembly in orthogonal arrays of particles is essential for AQP4-IgG pathogenicity since AQP4 autoantibodies bind to OAPs with higher affinity than for AQP4 tetramers. NMOSD has a complex background with prominent roles for genes encoding cytokines and cytokine receptors. AQP4 autoantibodies activate the complement-mediated inflammatory demyelination and the ensuing damage to AQP4 water channels, leading to water influx, necrosis and axonal loss. Conclusions: NMOSD as an astrocytopathy is a nosological entity different from multiple sclerosis with its own serological marker: immunoglobulin G-type autoantibodies against the AQP4 protein which elicits a complement-dependent cytotoxicity and neuroinflammation. Some patients with typical manifestations of NMSOD are AQP4 seronegative and myelin oligodendrocyte glycoprotein positive. Thus, the detection of autoantibodies against AQP4 or other autoantibodies is crucial for the correct treatment of the disease and immunosuppressant therapy is the first choice.