Melatonin prevents EAAC1 deletion-induced retinal ganglion cell degeneration by inhibiting apoptosis and senescence.

Normal tension glaucoma (NTG) is referred to as a progressive degenerative disorder of the retinal ganglion cells (RGCs), resulting in nonreversible visual defects, despite intraocular pressure levels within the statistically normal range. Current therapeutic strategies for NTG yield limited benefits. Excitatory amino acid carrier 1 (EAAC1) knockout (EAAC1-/- ) in mice has been shown to induce RGC degeneration without elevating intraocular pressure, mimicking pathological characteristics of NTG. In this study, we explored whether daily oral administration of melatonin could block RGCs loss and prevent retinal morphology and function defects associated with EAAC1 deletion. We also explored the molecular mechanisms underlying EAAC1 deletion-induced RGC degeneration and the neuroprotective effects of melatonin. Our RNA sequencing and in vivo data indicated EAAC1 deletion caused elevated oxidative stress, activation of apoptosis and cellular senescence pathways, and neuroinflammation in RGCs. However, melatonin administration efficiently prevented these detrimental effects. Furthermore, we investigated the potential role of apoptosis- and senescence-related redox-sensitive factors in EAAC1 deletion-induced RGCs degeneration and the neuroprotective effects of melatonin administration. We observed remarkable upregulation of p53, whereas NRF2 and Sirt1 expression were significantly decreased in EAAC1-/- mice, which were prevented by melatonin treatment, suggesting that melatonin exerted its neuroprotective effects possibly through modulating NRF2/p53/Sirt1 redox-sensitive signaling pathways. Overall, our study provided a solid foundation for the application of melatonin in the management of NTG.

Rapid Regulation of Glutamate Transport: Where Do We Go from Here?

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). A family of five Na+-dependent transporters maintain low levels of extracellular glutamate and shape excitatory signaling. Shortly after the research group of the person being honored in this special issue (Dr. Baruch Kanner) cloned one of these transporters, his group and several others showed that their activity can be acutely (within minutes to hours) regulated. Since this time, several different signals and post-translational modifications have been implicated in the regulation of these transporters. In this review, we will provide a brief introduction to the distribution and function of this family of glutamate transporters. This will be followed by a discussion of the signals that rapidly control the activity and/or localization of these transporters, including protein kinase C, ubiquitination, glutamate transporter substrates, nitrosylation, and palmitoylation. We also include the results of our attempts to define the role of palmitoylation in the regulation of GLT-1 in crude synaptosomes. In some cases, the mechanisms have been fairly well-defined, but in others, the mechanisms are not understood. In several cases, contradictory phenomena have been observed by more than one group; we describe these studies with the goal of identifying the opportunities for advancing the field. Abnormal glutamatergic signaling has been implicated in a wide variety of psychiatric and neurologic disorders. Although recent studies have begun to link regulation of glutamate transporters to the pathogenesis of these disorders, it will be difficult to determine how regulation influences signaling or pathophysiology of glutamate without a better understanding of the mechanisms involved.

Regulation of NRG-1-ErbB4 signaling and neuroprotection by exogenous neuregulin-1 in a mouse model of epilepsy.

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy. Dysregulation of glutamate transporters has been a common finding across animal models of epilepsy and in patients with TLE. In this study, we investigate NRG-1/ErbB4 signaling in epileptogenesis and the neuroprotective effects of NRG-1 treatment in a mouse model of temporal lobe epilepsy. Using immunohistochemistry, we report the first evidence for NRG-1/ErbB4-dependent selective upregulation of glutamate transporter EAAC1 and bihemispheric neuroprotection by exogeneous NRG-1 in the intrahippocampal kainic acid (IHKA) model of TLE. Our findings provide evidence that dysregulation of glutamate transporter EAAC1 contributes to the development of epilepsy and can be therapeutically targeted to reduce neuronal death following IHKA-induced status epilepticus (SE).

OCD candidate gene SLC1A1/EAAT3 impacts basal ganglia-mediated activity and stereotypic behavior.

Obsessive-compulsive disorder (OCD) is a chronic, disabling condition with inadequate treatment options that leave most patients with substantial residual symptoms. Structural, neurochemical, and behavioral findings point to a significant role for basal ganglia circuits and for the glutamate system in OCD. Genetic linkage and association studies in OCD point to SLC1A1, which encodes the neuronal glutamate/aspartate/cysteine transporter excitatory amino acid transporter 3 (EAAT3)/excitatory amino acid transporter 1 (EAAC1). However, no previous studies have investigated EAAT3 in basal ganglia circuits or in relation to OCD-related behavior. Here, we report a model of Slc1a1 loss based on an excisable STOP cassette that yields successful ablation of EAAT3 expression and function. Using amphetamine as a probe, we found that EAAT3 loss prevents expected increases in (i) locomotor activity, (ii) stereotypy, and (iii) immediate early gene induction in the dorsal striatum following amphetamine administration. Further, Slc1a1-STOP mice showed diminished grooming in an SKF-38393 challenge experiment, a pharmacologic model of OCD-like grooming behavior. This reduced grooming is accompanied by reduced dopamine D1 receptor binding in the dorsal striatum of Slc1a1-STOP mice. Slc1a1-STOP mice also exhibit reduced extracellular dopamine concentrations in the dorsal striatum both at baseline and following amphetamine challenge. Viral-mediated restoration of Slc1a1/EAAT3 expression in the midbrain but not in the striatum results in partial rescue of amphetamine-induced locomotion and stereotypy in Slc1a1-STOP mice, consistent with an impact of EAAT3 loss on presynaptic dopaminergic function. Collectively, these findings indicate that the most consistently associated OCD candidate gene impacts basal ganglia-dependent repetitive behaviors.

Early Postnatal Exposure to a Low Dose of Nanoparticulate Silver Induces Alterations in Glutamate Transporters in Brain of Immature Rats.

Due to strong antimicrobial properties, silver nanoparticles (AgNPs) are used in a wide range of medical and consumer products, including those dedicated for infants and children. While AgNPs are known to exert neurotoxic effects, current knowledge concerning their impact on the developing brain is scarce. During investigations of mechanisms of neurotoxicity in immature rats, we studied the influence of AgNPs on glutamate transporter systems which are involved in regulation of extracellular concentration of glutamate, an excitotoxic amino acid, and compared it with positive control-Ag citrate. We identified significant deposition of AgNPs in brain tissue of exposed rats over the post-exposure time. Ultrastructural alterations in endoplasmic reticulum (ER) and Golgi complexes were observed in neurons of AgNP-exposed rats, which are characteristics of ER stress. These changes presumably underlie substantial long-lasting downregulation of neuronal glutamate transporter EAAC1, which was noted in AgNP-exposed rats. Conversely, the expression of astroglial glutamate transporters GLT-1 and GLAST was not affected by exposure to AgNPs, but the activity of the transporters was diminished. These results indicate that even low doses of AgNPs administered during an early stage of life create a substantial risk for health of immature organisms. Hence, the safety of AgNP-containing products for infants and children should be carefully considered.

Loss of CLTRN function produces a neuropsychiatric disorder and a biochemical phenotype that mimics Hartnup disease.

Hartnup disease is an autosomal recessive condition characterized by neutral aminoaciduria and behavioral problems. It is caused by a loss of B0 AT1, a neutral amino acid transporter in the kidney and intestine. CLTRN encodes the protein collectrin that functions in the transportation and activation of B0 AT1 in the renal apical brush bordered epithelium. Collectrin deficient mice have severe aminoaciduria. However, the phenotype associated with collectrin deficiency in humans has not been reported. Here we report two patients, an 11-year-old male who is hemizygous for a small, interstitial deletion on Xp22.2 that encompasses CLTRN and a 22-year-old male with a deletion spanning exons 1 to 3 of CLTRN. Both of them present with neuropsychiatric phenotypes including autistic features, anxiety, depression, compulsions, and motor tics, as well as neutral aminoaciduria leading to a clinical diagnosis of Hartnup disease and treatment with niacin supplementation. Plasma amino acids were normal in both patients. One patient had low 5-hydroxyindoleacetic acid levels, a serotoninergic metabolite. We explored the expression of collectrin in the murine brain and found it to be particularly abundant in the hippocampus, brainstem, and cerebellum. We propose that collectrin deficiency in humans can be associated with aminoaciduria and a clinical picture similar to that seen in Hartnup disease. Further studies are needed to explore the role of collectrin deficiency in the neurological phenotypes.

Excitatory Amino Acid Transporters in Physiology and Disorders of the Central Nervous System.

Excitatory amino acid transporters (EAATs) encompass a class of five transporters with distinct expression in neurons and glia of the central nervous system (CNS). EAATs are mainly recognized for their role in uptake of the amino acid glutamate, the major excitatory neurotransmitter. EAATs-mediated clearance of glutamate released by neurons is vital to maintain proper glutamatergic signalling and to prevent toxic accumulation of this amino acid in the extracellular space. In addition, some EAATs also act as chloride channels or mediate the uptake of cysteine, required to produce the reactive oxygen speciesscavenger glutathione. Given their central role in glutamate homeostasis in the brain, as well as their additional activities, it comes as no surprise that EAAT dysfunctions have been implicated in numerous acute or chronic diseases of the CNS, including ischemic stroke and epilepsy, cerebellar ataxias, amyotrophic lateral sclerosis, Alzheimer's disease and Huntington's disease. Here we review the studies in cellular and animal models, as well as in humans that highlight the roles of EAATs in the pathogenesis of these devastating disorders. We also discuss the mechanisms regulating EAATs expression and intracellular trafficking and new exciting possibilities to modulate EAATs and to provide neuroprotection in course of pathologies affecting the CNS.

Ethanol (E) Impairs Fetal Brain GSH Homeostasis by Inhibiting Excitatory Amino-Acid Carrier 1 (EAAC1)-Mediated Cysteine Transport.

Central among the fetotoxic responses to in utero ethanol (E) exposure is redox-shift related glutathione (GSH) loss and apoptosis. Previously, we reported that despite an E-generated Nrf2 upregulation, fetal neurons still succumb. In this study, we investigate if the compromised GSH results from an impaired inward transport of cysteine (Cys), a precursor of GSH in association with dysregulated excitatory amino acid carrier1 (EAAC1), a cysteine transporter. In utero binge model involves administration of isocaloric dextrose or 20% E (3.5 g/kg)/ by gavage at 12 h intervals to pregnant Sprague Dawley (SD) rats, starting gestation day (gd) 17 with a final dose on gd19, 2 h prior to sacrifice. Primary cerebral cortical neurons (PCNs) from embryonic day 16-17 fetal SD rats were the in vitro model. E reduced both PCN and cerebral cortical GSH and Cys up to 50% and the abridged GSH could be blocked by administration of N-acetylcysteine. E reduced EAAC1 protein expression in utero and in PCNs (p < 0.05). This was accompanied by a 60-70% decrease in neuron surface expression of EAAC1 along with significant reductions of EAAC1/Slc1a1 mRNA (p < 0.05). In PCNs, EAAC1 knockdown significantly decreased GSH but not oxidized glutathione (GSSG) illustrating that while not the sole provider of Cys, EAAC1 plays an important role in neuron GSH homeostasis. These studies strongly support the concept that in both E exposed intact fetal brain and cultured PCNs a mechanism underlying E impairment of GSH homeostasis is reduction of import of external Cys which is mediated by perturbations of EAAC1 expression/function.

Protein Interacting with C-Kinase 1 Deficiency Impairs Glutathione Synthesis and Increases Oxidative Stress via Reduction of Surface Excitatory Amino Acid Carrier 1.

Protein interacting with C-kinase 1 (PICK1) has received considerable attention, because it interacts with a broad range of neurotransmitter receptors, transporters, and enzymes and thereby influences their localization and function in the CNS. Although it is suggested that putative partners of PICK1 are involved in neurological diseases such as schizophrenia, Parkinson's disease, chronic pain, and amyotrophic lateral sclerosis, the functions of PICK1 in neurological disorders are not clear. Here, we show that oxidative stress, which is tightly associated with neurological diseases, occurs in PICK1(-/-) mice. The oxidation in PICK1(-/-) mice was found selectively in neurons and was age dependent, leading to microglial activation and the release of inflammatory factors. Neurons in the cortex and hippocampus from PICK1(-/-) mice showed increased vulnerability to oxidants and reduced capacity to metabolize reactive oxygen species (ROS); this was caused by reduced glutathione content and impaired cysteine transport. The dysregulated expression of glutathione was attributed to a decrease of the surface glutamate transporter excitatory amino acid carrier 1 (EAAC1). Overexpression of PICK1 could rescue the surface expression of EAAC1 and ameliorate the glutathione deficit in PICK1(-/-) neurons. Finally, reduced surface EAAC1 was associated with defective Rab11 activity. Transfection with dominant-negative Rab11 effectively suppressed surface EAAC1 and increased ROS production. Together, these results indicate that PICK1 is a crucial regulator in glutathione homeostasis and may play important roles in oxidative stress and its associated neurodegenerative diseases.

Neuregulin 1 Controls Glutamate Uptake by Up-regulating Excitatory Amino Acid Carrier 1 (EAAC1).

Neuregulin 1 (NRG1) is a trophic factor that is thought to have important roles in the regulating brain circuitry. Recent studies suggest that NRG1 regulates synaptic transmission, although the precise mechanisms remain unknown. Here we report that NRG1 influences glutamate uptake by increasing the protein level of excitatory amino acid carrier (EAAC1). Our data indicate that NRG1 induced the up-regulation of EAAC1 in primary cortical neurons with an increase in glutamate uptake. These in vitro results were corroborated in the prefrontal cortex (PFC) of mice given NRG1. The stimulatory effect of NRG1 was blocked by inhibition of the NRG1 receptor ErbB4. The suppressed expression of ErbB4 by siRNA led to a decrease in the expression of EAAC1. In addition, the ablation of ErbB4 in parvalbumin (PV)-positive neurons in PV-ErbB4(-/-) mice suppressed EAAC1 expression. Taken together, our results show that NRG1 signaling through ErbB4 modulates EAAC1. These findings link proposed effectors in schizophrenia: NRG1/ErbB4 signaling perturbation, EAAC1 deficit, and neurotransmission dysfunction.

MicroRNA 101b Is Downregulated in the Prefrontal Cortex of a Genetic Model of Depression and Targets the Glutamate Transporter SLC1A1 (EAAT3) in Vitro.

BACKGROUND: MicroRNAs (miRNAs) are small regulatory molecules that cause translational repression by base pairing with target mRNAs. Cumulative evidence suggests that changes in miRNA expression may in part underlie the pathophysiology and treatment of neuropsychiatric disorders, including major depressive disorder (MDD). METHODS: A miRNA expression assay that can simultaneously detect 423 rat miRNAs (miRBase v.17) was used to profile the prefrontal cortex (PFC) of a genetic rat model of MDD (the Flinders Sensitive Line [FSL]) and the controls, the Flinders Resistant Line (FRL). Gene expression data from the PFC of FSL/FRL animals (GEO accession no. GSE20388) were used to guide mRNA target selection. Luciferase reporter assays were used to verify miRNA targets in vitro. RESULTS: We identified 23 miRNAs that were downregulated in the PFC of the FSL model compared with controls. Interestingly, one of the identified miRNAs (miR-101b) is highly conserved between rat and human and was recently found to be downregulated in the PFC of depressed suicide subjects. Using a combination of in silico and in vitro analyses, we found that miR-101b targets the neuronal glutamate transporter SLC1A1 (also known as EAAC1 or EAAT3). Accordingly, both mRNA and protein levels of SLC1A1 were found to be upregulated in the PFC of the FSL model. CONCLUSIONS: Besides providing a list of novel miRNAs associated with depression-like states, this preclinical study replicated the human association of miR-101 with depression. In addition, since one of the targets of miR-101b appears to be a glutamate transporter, our preclinical data support the hypothesis of a glutamatergic dysregulation being implicated in the etiology of depression.

Decreased cysteine uptake by EAAC1 gene deletion exacerbates neuronal oxidative stress and neuronal death after traumatic brain injury.

Excitatory amino acid carrier type 1 (EAAC1), a high-affinity glutamate transporter, can expend energy to move glutamate into neurons. However, under normal physiological conditions, EAAC1 does not have a great effect on glutamate clearance but rather participates in the neuronal uptake of cysteine. This process is critical to maintaining neuronal antioxidant function by providing cysteine for glutathione synthesis. Previous study showed that mice lacking EAAC1 show increased neuronal oxidative stress following transient cerebral ischemia. In the present study, we sought to characterize the role of EAAC1 in neuronal resistance after traumatic brain injury (TBI). Young adult C57BL/6 wild-type or EAAC1 (-/-) mice were subjected to a controlled cortical impact model for TBI. Neuronal death after TBI showed more than double the number of degenerating neurons in the hippocampus in EAAC1 (-/-) mice compared with wild-type mice. Superoxide production, zinc translocation and microglia activation similarly showed a marked increase in the EAAC1 (-/-) mice. Pretreatment with N-acetyl cysteine (NAC) reduced TBI-induced neuronal death, superoxide production and zinc translocation. These findings indicate that cysteine uptake by EAAC1 is important for neuronal antioxidant function and survival following TBI. This study also suggests that administration of NAC has therapeutic potential in preventing TBI-induced neuronal death.

Spinal Glutamate Transporters Are Involved in the Development of Electroacupuncture Tolerance.

BACKGROUND: Electroacupuncture (EA) tolerance is a gradual decline in EA antinociception because of its repeated or prolonged use. This study aims to explore the role of spinal glutamate transporters (GTs) in EA tolerance (EAT). METHODS: Rats were treated with EA once per day for eight consecutive days, and their L4-5 spinal cords were collected at days 0, 2, 4, 6 and 8. The levels of three spinal GTs and their mRNAs were detected with Western blot and pPCR, respectively. Then, riluzole, a positive GT regulator, was administered intrathecally in order to observe its effect on EA analgesia after repeated EA. RESULTS: The expression levels of the spinal GTs increased at days 2 and 4, and gradually decreased as the times of EA increased. At day 8, no difference was observed in the spinal GTs between the sham treatment and the EA treatment. Intrathecal administration of riluzole dose-dependently attenuated the decreased EA analgesia. CONCLUSION: These results indicated the participation of the spinal GTs in EAT.

Glutathione in Cellular Redox Homeostasis: Association with the Excitatory Amino Acid Carrier 1 (EAAC1).

Reactive oxygen species (ROS) are by-products of the cellular metabolism of oxygen consumption, produced mainly in the mitochondria. ROS are known to be highly reactive ions or free radicals containing oxygen that impair redox homeostasis and cellular functions, leading to cell death. Under physiological conditions, a variety of antioxidant systems scavenge ROS to maintain the intracellular redox homeostasis and normal cellular functions. This review focuses on the antioxidant system's roles in maintaining redox homeostasis. Especially, glutathione (GSH) is the most important thiol-containing molecule, as it functions as a redox buffer, antioxidant, and enzyme cofactor against oxidative stress. In the brain, dysfunction of GSH synthesis leading to GSH depletion exacerbates oxidative stress, which is linked to a pathogenesis of aging-related neurodegenerative diseases. Excitatory amino acid carrier 1 (EAAC1) plays a pivotal role in neuronal GSH synthesis. The regulatory mechanism of EAAC1 is also discussed.

Erythroid Differentiation Regulator 1 as a Regulator of Neuronal GSH Synthesis.

Erythroid differentiation regulator 1 (Erdr1) is a cytokine known to play important roles in cell survival under stressful conditions, maintenance of cellular growth homeostasis, and activation of the immune system. However, the impact of Erdr1 on neurons remains undefined. In this study, we present novel evidence that Erdr1 plays a role in regulating glutathione (GSH) synthesis via glutamate transporter-associated protein 3-18 (GTRAP3-18), an anchor protein in the endoplasmic reticulum that holds excitatory amino acid carrier 1 (EAAC1) in neurons. Both DNA microarray and quantitative real-time PCR analyses revealed an approximately 2-fold increase in Erdr1 levels in the hippocampus of GTRAP3-18-deficient mice compared to those of wild-type mice. Knockdown of Erdr1 in vitro resulted in a decrease in GTRAP3-18 levels, leading to an increase in EAAC1 expression and intracellular GSH levels, and subsequently, cytoprotective effects against oxidative stress. Our findings shed light on the regulatory mechanisms involving Erdr1, GTRAP3-18, EAAC1, and GSH in the context of neuronal defense against oxidative stress. Understanding the intricate interplay among these molecules may pave the way for the development of promising therapeutic strategies for neurodegenerative disorders.

Inhibition of GTRAP3-18 may increase neuroprotective glutathione (GSH) synthesis.

Glutathione (GSH) is a tripeptide consisting of glutamate, cysteine, and glycine; it has a variety of functions in the central nervous system. Brain GSH depletion is considered a preclinical sign in age-related neurodegenerative diseases, and it promotes the subsequent processes toward neurotoxicity. A neuroprotective mechanism accomplished by increasing GSH synthesis could be a promising approach in the treatment of neurodegenerative diseases. In neurons, cysteine is the rate-limiting substrate for GSH synthesis. Excitatory amino acid carrier 1 (EAAC1) is a neuronal cysteine/glutamate transporter in the brain. EAAC1 translocation to the plasma membrane promotes cysteine uptake, leading to GSH synthesis, while being negatively regulated by glutamate transport associated protein 3-18 (GTRAP3-18). Our recent studies have suggested GTRAP3-18 as an inhibitory factor for neuronal GSH synthesis. Inhibiting GTRAP3-18 function is an endogenous mechanism to increase neuron-specific GSH synthesis in the brain. This review gives an overview of EAAC1-mediated GSH synthesis, and its regulatory mechanisms by GTRAP3-18 in the brain, and a potential approach against neurodegeneration.

Altered Grooming Syntax and Amphetamine-Induced Dopamine Release in EAAT3 Overexpressing Mice.

The excitatory amino acid transporter EAAT3 plays an important role in the neuronal uptake of glutamate regulating the activation of glutamate receptors. Polymorphisms in the gene-encoding EAAT3 have been associated with obsessive-compulsive disorder (OCD), although the mechanisms underlying this relationship are still unknown. We recently reported that mice with increased EAAT3 expression in forebrain neurons (EAAT3 g lo /CMKII) display behavioral and synaptic features relevant to OCD, including increased grooming, higher anxiety-like behavior and altered cortico-striatal synaptic function. The dopamine neurotransmitter system is implicated in ritualistic behaviors. Indeed, dopaminergic neurons express EAAT3, and mice lacking EAAT3 exhibit decreased dopamine release and decreased expression of the dopamine D1 receptor. Moreover, EAAT3 plays a role on the effect of the psychostimulant amphetamine. As such, we sought to determine if the OCD-like behavior in EAAT3 g lo /CMKII mice is accompanied by altered nigro-striatal dopaminergic transmission. The aim of this study was to analyze dopamine transmission both in basal conditions and after an acute challenge of amphetamine, using behavioral, neurochemical, molecular, and cellular approaches. We found that in basal conditions, EAAT3 g lo /CMKII mice performed more grooming events and that they remained in phase 1 of the grooming chain syntax compared with control littermates. Administration of amphetamine increased the number of grooming events in control mice, while EAAT3 g lo /CMKII mice remain unaffected. Interestingly, the grooming syntax of amphetamine-control mice resembled that of EAAT3 g lo /CMKII mice in basal conditions. Using in vivo microdialysis, we found decreased basal dopamine levels in EAAT3 g lo /CMKII compared with control mice. Unexpectedly, we found that after acute amphetamine, EAAT3 g lo /CMKII mice had a higher release of dopamine compared with that of control mice, suggesting that EAAT3 overexpression leads to increased dopamine releasability. To determine postsynaptic effect of EAAT3 overexpression over dopamine transmission, we performed Western blot analysis of dopaminergic proteins and found that EAAT3 g lo /CMKII mice have higher expression of D2 receptors, suggesting a higher inhibition of the indirect striatal pathway. Together, the data indicate that EAAT3 overexpression impacts on dopamine transmission, making dopamine neurons more sensitive to the effect of amphetamine and leading to a disbalance between the direct and indirect striatal pathways that favors the performance of repetitive behaviors.

Neuregulin-1 inhibits CoCl2-induced upregulation of excitatory amino acid carrier 1 expression and oxidative stress in SH-SY5Y cells and the hippocampus of mice.

Excitatory amino acid carrier 1 (EAAC1) is an important subtype of excitatory amino acid transporters (EAATs) and is the route for neuronal cysteine uptake. CoCl2 is not only a hypoxia-mimetic reagent but also an oxidative stress inducer. Here, we found that CoCl2 induced significant EAAC1 overexpression in SH-SY5Y cells and the hippocampus of mice. Transient transfection of EAAC1 reduced CoCl2-induced cytotoxicity in SH-SY5Y cells. Based on this result, upregulation of EAAC1 expression by CoCl2 is thought to represent a compensatory response against oxidative stress in an acute hypoxic state. We further demonstrated that pretreatment with Neuregulin-1 (NRG1) rescued CoCl2-induced upregulation of EAAC1 and tau expression. NRG1 plays a protective role in the CoCl2-induced accumulation of reactive oxygen species (ROS) and reduction in antioxidative enzyme (SOD and GPx) activity. Moreover, NRG1 attenuated CoCl2-induced apoptosis and cell death. NRG1 inhibited the CoCl2-induced release of cleaved caspase-3 and reduction in Bcl-XL levels. Our novel finding suggests that NRG1 may play a protective role in hypoxia through the inhibition of oxidative stress and thereby maintain normal EAAC1 expression levels.

NCX1 and EAAC1 transporters are involved in the protective action of glutamate in an in vitro Alzheimer's disease-like model.

Increasing evidence suggests that metabolic dysfunctions are at the roots of neurodegenerative disorders such as Alzheimer's disease (AD). In particular, defects in cerebral glucose metabolism, which have been often noted even before the occurrence of clinical symptoms and histopathological lesions, are now regarded as critical contributors to the pathogenesis of AD. Hence, the stimulation of energy metabolism, by enhancing the availability of specific metabolites, might be an alternative way to improve ATP synthesis and to positively affect AD progression. For instance, glutamate may serve as an intermediary metabolite for ATP synthesis through the tricarboxylic acid (TCA) cycle and the oxidative phosphorylation. We have recently shown that two transporters are critical for the anaplerotic use of glutamate: the Na+-dependent Excitatory Amino Acids Carrier 1 (EAAC1) and the Na+-Ca2+ exchanger 1 (NCX1). Therefore, in the present study, we established an AD-like phenotype by perturbing glucose metabolism in both primary rat cortical neurons and retinoic acid (RA)-differentiated SH-SY5Y cells, and we explored the potential of glutamate to halt cell damage by monitoring neurotoxicity, AD markers, ATP synthesis, cytosolic Ca2+ levels and EAAC1/NCX1 functional activities. We found that glutamate significantly increased ATP production and cell survival, reduced the increase of AD biomarkers (amyloid ß protein and the hyperphosphorylated form of tau protein), and recovered the increase of NCX reverse-mode activity. The RNA silencing of either EAAC1 or NCX1 caused the loss of the beneficial effects of glutamate, suggesting the requirement of a functional interplay between these transporters for glutamate-induced protection. Remarkably, our results indicate, as proof-of-principle, that facilitating the use of alternative fuels, like glutamate, may be an effective approach to overcome deficits in glucose utilization and significantly slow down neuronal degenerative process in AD.

Electroacupuncture Reduces Seizure Activity and Enhances GAD 67 and Glutamate Transporter Expression in Kainic Acid Induced Status Epilepticus in Infant Rats.

Status epilepticus (SE) is one of the most significant complications in pediatric neurology. Clinical studies have shown positive effects of electroacupuncture (EA) as a therapeutic alternative in the control of partial seizures and secondary generalized clonic seizures. EA promotes the release of neurotransmitters such as GABA and some opioids. The present study aimed to evaluate the anticonvulsive and neuromodulatory effects of Shui Gou DM26 (SG_DM26) acupuncture point electrostimulation on the expression of the glutamate decarboxylase 67 (GAD67) enzyme and the glutamate transporter EAAC1 in an early SE model. At ten postnatal days (10-PD), male rats weighing 22-26 g were divided into 16 groups, including control and treatment groups: Simple stimulation, electrostimulation, anticonvulsant drug treatment, and combined treatment-electrostimulation and pentobarbital (PB). SE was induced with kainic acid (KA), and the following parameters were measured: Motor behavior, and expression of GAD67 and EAAC1. The results suggest an antiepileptic effect derived from SG DM26 point EA. The possible mechanism is most likely the increased production of the inhibitory neurotransmitter GABA, which is observed as an increase in the expression of both GAD67 and EAAC1, as well as the potential synergy between the neuromodulator effects of EA and PB.