Glutamate Transporter Function Research Articles

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

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. 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. 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. Overall, these data support the growing body of evidence for the role of dysregulation of the glutamate system in the pathophysiology of SCZ.

Aging-Related Changes in Expression and Function of Glutamate Transporters in Rat Spinal Cord Astrocytes

Astrocytes make up the predominant cell population among glial cells in the mammalian brain, and they play a vital role in ensuring its optimal functioning. They promote neuronal health and survival and protect neurons from glutamate-induced excitotoxicity. In the spinal cord’s dorsal horn (DH) and ventral horn (VH) regions, astrocytes serve crucial roles. Notably, VH motor neurons exhibit a heightened sensitivity to glutamate-induced damage. It is posited that this selective sensitivity could be related to their localized presence within the VH, where astrocytes possess a distinct set of mechanisms for managing glutamate. As organisms age, the risk of damage from glutamate increases, indicating a potential decline in the efficiency of astrocytic glutamate regulation. Our research involved an analysis of astrocytic structure, glutamate transporter levels, and glutamate uptake capabilities within the DH and VH through immunohistochemical methods, protein analysis via Western blot, and patch-clamp studies in electrophysiology. The investigations revealed a decrease in both the number and coverage of astroglia in the spinal cord, more so within the VH as aging progressed. Notably, levels of the excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2) also decreased with age, particularly within the VH. Patch-clamp analyses of astrocytes from both spinal regions confirmed a significant reduction in glutamate uptake activity as age advanced, indicating an age-related impairment in glutamate processing. The findings indicate aging leads to distinct changes in DH and VH astrocytes, impairing their glutamate management abilities, which could contribute significantly to the development of late-onset neurodegenerative conditions.

Apo state pore opening as functional basis of increased EAAT anion channel activity in episodic ataxia 6.

SLC1A2 and SLC1A3 encode the glial glutamate transporters EAAT2 and EAAT1, which are not only the predominant glutamate uptake carriers in our brain, but also function as anion channels. Two homologous mutations, which predict substitutions of prolines in the center of the fifth transmembrane helix by arginine (P289R EAAT2, P290R EAAT1), have been identified in patients with epileptic encephalopathy (SLC1A2) or with episodic ataxia type 6 (SLC1A3). Both mutations have been shown to impair glutamate uptake and to increase anion conduction. The molecular processes that link the disease-causing mutations to two major alterations of glutamate transporter function remain insufficiently understood. The mutated proline is conserved in every EAAT. Since the pathogenic changes mainly affect the anion channel function, we here study the functional consequences of the homologous P312R mutation in the neuronal glutamate transporter EAAT4, a low capacity glutamate transporter with predominant anion channel function. To assess the impact of charge and structure of the inserted amino acid for the observed functional changes, we generated and functionally evaluated not only P312R, but also substitutions of P312 with all other amino acids. However, only exchange of proline by arginine, lysine, histidine and asparagine were functionally tolerated. We compared WT, P312R and P312N EAAT4 using a combination of cellular electrophysiology, fast substrate application and kinetic modelling. We found that WT and mutant EAAT4 anion currents can be described with a 11-state model of the transport cycle, in which several states are connected to branching anion channel states to account for the EAAT anion channel function. Substitutions of P312 modify various transitions describing substrate binding/unbinding, translocation or anion channel opening. Most importantly, P312R generates a new anion conducting state that is accessible in the outward facing apo state and that is the main determinant of the increased anion conduction of EAAT transporters carrying this mutation. Our work provides a quantitative description how a naturally occurring mutation changes glutamate uptake and anion currents in two genetic diseases.

Neuronal glutamate transporters control reciprocal inhibition and gain modulation in D1 medium spiny neurons.

Understanding the function of glutamate transporters has broad implications for explaining how neurons integrate information and relay it through complex neuronal circuits. Most of what is currently known about glutamate transporters, specifically their ability to maintain glutamate homeostasis and limit glutamate diffusion away from the synaptic cleft, is based on studies of glial glutamate transporters. By contrast, little is known about the functional implications of neuronal glutamate transporters. The neuronal glutamate transporter EAAC1 is widely expressed throughout the brain, particularly in the striatum, the primary input nucleus of the basal ganglia, a region implicated with movement execution and reward. Here, we show that EAAC1 limits synaptic excitation onto a population of striatal medium spiny neurons identified for their expression of D1 dopamine receptors (D1-MSNs). In these cells, EAAC1 also contributes to strengthen lateral inhibition from other D1-MSNs. Together, these effects contribute to reduce the gain of the input-output relationship and increase the offset at increasing levels of synaptic inhibition in D1-MSNs. By reducing the sensitivity and dynamic range of action potential firing in D1-MSNs, EAAC1 limits the propensity of mice to rapidly switch between behaviors associated with different reward probabilities. Together, these findings shed light on some important molecular and cellular mechanisms implicated with behavior flexibility in mice.

Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function.

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory Amino Acid Transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl−) channels, but the physiological role of Cl− conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl− channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. Here we investigated the effects of five additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behaviour. Our results indicate that mutations with decreased hEAAT1 Cl− channel activity and functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl− homeostasis in glial cells for proper CNS function. We also identified a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells, due to protein-lipid interactions between the EA6-related mutant and lipids in the membrane bilayer. Together, these results reveal how these mutant transporters contributes to the pathology of EA6 and strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.

Activation of Protease-Activated Receptor-1 Causes Chronic Pain in Lupus-Prone Mice Via Suppressing Spinal Glial Glutamate Transporter Function and Enhancing Glutamatergic Synaptic Activity

Systemic lupus erythematosus (SLE) is an unpredictable autoimmune disease where the body's immune system mistakenly attacks healthy tissues in many parts of the body. Chronic pain is one of the most frequently reported symptoms among SLE patients. We previously reported that MRL lupus prone (MRL/lpr) mice develop hypersensitivity to mechanical and heat stimulation. In the present study, we found that the spinal protease-activated receptor-1(PAR1) plays an important role in the genesis of chronic pain in MRL/lpr mice. Female MRL/lpr mice with chronic pain had activation of astrocytes, over-expression of thrombin and PAR1, enhanced glutamatergic synaptic activity, as well as suppressed activity of adenosine monophosphate-activated protein kinase (AMPK) and glial glutamate transport function in the spinal cord. Intrathecal injection of either the PAR1 antagonist, or AMPK activator attenuated heat hyperalgesia and mechanical allodynia in MRL/lpr mice. Furthermore, we also identified that the enhanced glutamatergic synaptic activity and suppressed activity of glial glutamate transporters in the spinal dorsal horn of MRL/lpr mice are caused by activation of the PAR1 and suppression of AMPK signaling pathways. These findings suggest that targeting the PAR1 and AMPK signaling pathways in the spinal cord may be a useful approach for treating chronic pain caused by SLE. PerspectiveOur study provides evidence suggesting activation of PAR1 and suppression of AMPK in the spinal cord induces thermal hyperalgesia and mechanical allodynia in a lupus mouse model. Targeting signaling pathways regulating the PAR1 and AMPK could potentially provide a novel approach to the management of chronic pain caused by SLE.

Nitrogen starvation induces genome-wide activation of transposable elements in Arabidopsis.

Nitrogen (N) availability is a major limiting factor for plant growth and agricultural productivity. Although the gene regulation network in response to N starvation has been extensively studied, it remains unknown whether N starvation has an impact on the activity of transposable elements (TEs). Here, we report that TEs can be transcriptionally activated in Arabidopsis under N starvation conditions. Through genetic screening of idm1-14 suppressors, we cloned GLU1, which encodes a glutamate synthase that catalyzes the synthesis of glutamate in the primary N assimilation pathway. We found that glutamate synthase 1 (GLU1) and its functional homologs GLU2 and glutamate transport 1 (GLT1) are redundantly required for TE silencing, suggesting that N metabolism can regulate TE activity. Transcriptome and methylome analyses revealed that N starvation results in genome-wide TE activation without inducing obvious alteration of DNA methylation. Genetic analysis indicated that N starvation-induced TE activation is also independent of other well-established epigenetic mechanisms, including histone methylation and heterochromatin decondensation. Our results provide new insights into the regulation of TE activity under stressful environments in planta.

Positive effects of running exercise on astrocytes in the medial prefrontal cortex in an animal model of depression.

Depression is one of the most common mental illnesses and seriously affects all aspects of life. Running exercise has been suggested to prevent or alleviate the occurrence and development of depression; however, the underlying mechanisms of these effects remain unclear. Independent studies have indicated that astrocytes play essential roles and that the medial prefrontal cortex (mPFC) is an important brain region involved in the pathology underlying depression. However, it is unknown whether running exercise achieves antidepressant effects by affecting the number of astrocytes and glutamate transport function in the mPFC. Here, animal models of depression were established using chronic unpredictable stress (CUS), and depression-like behavior was assessed by the sucrose preference test. After successfully establishing the depression model, experimental animals performed running exercise. Glial fibrillary acidic protein-positive (GFAP+ ) cell number in the mPFC was precisely quantified using immunohistochemical and stereological methods, and the densities of bromodeoxyuridine-positive (BrdU+ ) and BrdU+ /GFAP+ cells in the mPFC were measured using a semiquantitative immunofluorescence assay. Changes in glutamate transporter gene expression in mPFC astrocytes were detected by mRNA sequencing and qRT-PCR. We found that running exercise reversed CUS-induced decreases in sucrose preference, increased astrocyte number and the density of newborn astrocytes, and reversed decreases in gene expression levels of GFAP, S100b, and the glutamate transporters GLT-1 and GLAST in the mPFC of CUS animals. These results suggested that changes in astrocyte number and glutamate transporter function may be potential meditators of the effects of running exercise in the treatment of depression.

Erinacine A attenuates glutamate transporter 1 downregulation and protects against ischemic brain injury

Maintaining glutamate homeostasis through astrocyte-enriched glutamate transporter 1 (GLT-1) is critical for neuronal survival, but it is often disrupted after brain injury. Hericium erinaceus (HE), an edible mushroom, was reported to be anti-inflammatory and neuroprotective against brain ischemia, but its effect on glutamate homeostasis was unknown. Here we investigated the neuroprotective effect of erinacine A (EA), an active component of HE, with special focus on the GLT-1 function in the in vitro and in vivo cerebral ischemia mouse models. By using oxygen-glucose deprivation (OGD) to challenge mouse glia-neuron (GN) mixed culture as the in vitro model, we found that EA treatment significantly improved neuronal/astroglial survival and attenuated OGD-induced proinflammatory NFκB and AKT signaling activations. Notably, EA attenuated OGD-induced GLT-1 downregulation, and a selective GLT-1 inhibitor WAY-213613 reversed these EA-mediated neuroprotection. EA also ameliorated glutamate excitotoxicity effectively. In a transient hypoxia-ischemia (tHI) brain injury mouse model, we examined an EA treatment strategy by performing a pre-tHI daily oral gavage of EA (oEA) for 7 days followed by a post-tHI intranasal injection of EA (nEA) for 3 days, and found that this treatment significantly protected sensorimotor cortex and improved the post-tHI forepaw grip strength. Western blotting results further revealed that EA treatment also preserved astrocyte-enriched glutamate and aspartate transporter (GLAST) as well as a GLT-1 function-associated potassium channel Kir4.1 in the cerebral cortex and striatum after tHI. These results suggest that EA is effective for preserving GLT-1 and glutamate clearance machinery to protect against excitotoxicity after ischemic brain injury.

WNT/β-catenin Pathway: a Possible Link Between Hypertension and Alzheimer's Disease.

Recent research has shown that older people with high blood pressure (BP), or hypertension, are more likely to have biomarkers of Alzheimer's disease (AD). Essential hypertension represents the most common cardiovascular disease worldwide and is thought to be responsible for about 13% of all deaths. People with essential hypertension who regularly take prescribed BP medications are half as likely to develop AD as those who do not take them. What then is the connection? We know that high BP can damage small blood vessels in the brain, affecting those parts that are responsible for memory and thinking. However, the link between AD and hypertension remains unclear. Recent advances in the field of molecular and cellular biology have revealed a downregulation of the canonical WNT/β-catenin pathway in both hypertension and AD. In AD, the glutamate transport function is decreased, a decrease that is associated with a loss of synapse and neuronal death. β-catenin signaling appears to act as a major regulator of glutamate transporters (EAAT and GS) expression and can be harnessed to remove excess glutamate in AD. This review focuses on the possible link between hypertension and AD through the decreased WNT/β-catenin which interacts with the glutamatergic pathway.

Ceftriaxone as a Novel Therapeutic Agent for Hyperglutamatergic States: Bridging the Gap Between Preclinical Results and Clinical Translation.

Dysregulation of glutamate homeostasis is a well-established core feature of neuropsychiatric disorders. Extracellular glutamate concentration is regulated by glutamate transporter 1 (GLT-1). The discovery of a beta-lactam antibiotic, ceftriaxone (CEF), as a safe compound with unique ability to upregulate GLT-1 sparked the interest in testing its efficacy as a novel therapeutic agent in animal models of neuropsychiatric disorders with hyperglutamatergic states. Indeed, more than 100 preclinical studies have shown the efficacy of CEF in attenuating the behavioral manifestations of various hyperglutamatergic brain disorders such as ischemic stroke, amyotrophic lateral sclerosis (ALS), seizure, Huntington’s disease, and various aspects of drug use disorders. However, despite rich and promising preclinical data, only one large-scale clinical trial testing the efficacy of CEF in patients with ALS is reported. Unfortunately, in that study, there was no significant difference in survival between placebo- and CEF-treated patients. In this review, we discussed the translational potential of preclinical efficacy of CEF based on four different parameters: (1) initiation of CEF treatment in relation to induction of the hyperglutamatergic state, (2) onset of response in preclinical models in relation to onset of GLT-1 upregulation, (3) mechanisms of action of CEF on GLT-1 expression and function, and (4) non-GLT-1-mediated mechanisms for CEF. Our detailed review of the literature brings new insights into underlying molecular mechanisms correlating the preclinical efficacy of CEF. We concluded here that CEF may be clinically effective in selected cases in acute and transient hyperglutamatergic states such as early drug withdrawal conditions.

Cloning of a new form of EAAT2/GLT-1 from human and rodent brains

Glutamate transporter 1 is the principal transporter that mediates glutamate clearance in the mammalian brain. In rodents, it is referred to as GLT-1, whereas in humans it is referred to as EAAT2. We have cloned a novel and abundantly expressed carboxyl-terminal splice variant of this transporter in both rodents and humans, which we denote as GLT-1d/EAAT2d. The novel splice variant results from usage of internal splice sites and the splicing event leads to novel extra sequence spliced in after exon 10. The open reading frames of GLT-1d and EAAT2d encode proteins of 572 and 566 amino acids respectively; both contain a C-terminal PDZ motif. When expressed in COS7 cells, the proteins function as glutamate transporters that are inhibited by dihydrokainate (a GLT-1/EAAT2 transporter inhibitor). RT-PCR amplification using GLT-1d specific primers confirmed expression of message in all brain regions examined (forebrain, midbrain, hindbrain and cerebellum) as well as spinal cord, astrocyte cultures, retina and peripheral tissues (liver, testis, small intestine and lung). Quantitative RT-PCR analysis showed that expression of GLT-1d is developmentally regulated. In adult human brain, EAAT2d message is ∼ 30% of the level of EAAT2a message (the most abundant form), potentially making it the second most abundantly expressed form of EAAT2 in the brain. The amino terminal region of GLT-1d is also alternately spliced; the brain and testis forms contain a sequence corresponding to the amino acid sequence MASTEG whereas the corresponding liver sequence is MVS. In summary, we have cloned a novel EAAT2/GLT-1 splice variant from human and rodent brains. The splice variant is abundantly expressed in the brain, spinal cord, retina, liver and testis; it is a functional glutamate transporter; therefore, we conclude that it will likely have a functional role in glutamate homeostasis in the rodent and human nervous system, during development, adulthood, and plausibly in pathological states.

Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function.

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory amino acid transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl–) channels, but the physiological role of Cl– conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl– channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. Here we investigated the effects of 5 additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behavior. Our results indicate that mutations resulting in decreased hEAAT1 Cl– channel activity but with functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl– homeostasis in glial cells for proper CNS function. We also identified what we believe is a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.

Induction of Parkinsonian-Like Changes via Targeted Downregulation of Astrocytic Glutamate Transporter GLT-1 in the Striatum.

Previous investigations have suggested that decreased expression of glutamate transporter-1 (GLT-1) is involved in glutamate excitotoxicity and contribute to the development of Parkinson's disease (PD), GLT-1 is decreased in animal models of PD. GLT-1 is mainly expressed in astrocytes, and the striatum is a GLT-1-rich brain area. The aim was to explore the function and mechanism of astrocytic GLT-1 in PD-like changes. In the study, PD-like changes and their molecular mechanism in rodents were tested by a behavioral assessment, micro-positron emission tomography/computed tomography (PET/CT), western blotting, immunohistochemical and immunofluorescence staining, and high performance liquid chromatography pre-column derivatization with O-pthaldialdehida after downregulating astrocytic GLT-1 in vivo and in vitro. In vivo, after 6 weeks of brain stereotactic injection of adeno-associated virus into the striatum, rats in the astrocytic GLT-1 knockdown group showed poorer motor performance, abnormal gait, and depression-like feature; but no olfactory disorders. The results of micro-PET/CT and western blotting indicated that the dopaminergic system was impaired in astrocytic GLT-1 knockdown rats. Similarly, tyrosine hydroxylase (TH) positive immune-staining in neurons of astrocytic GLT-1 knockdown rats showed deficit in cell count. In vitro, knockdown of astrocytic GLT-1 via RNA interference led to morphological injury of TH-positive neurons, which may be related to the abnormal calcium signal induced by glutamate accumulation after GLT-1 knockdown. Furthermore, the GLT-1 agonist ceftriaxone showed a protective effect on TH-positive neuron impairment. The present findings may shed new light in the future prevention and treatment of PD based on blocking glutamate excitotoxicity.

DNA Methylation-Dependent Gene Expression Regulation of Glutamate Transporters in Cultured Radial Glial Cells.

Exposure to xenobiotics has a significant impact in brain physiology that could be liked to an excitotoxic process induced by a massive release of the main excitatory neurotransmitter, L-glutamate. Overstimulation of extra-synaptic glutamate receptors, mainly of the N-methyl-D-aspartate subtype leads to a disturbance of intracellular calcium homeostasis that is critically involved in neuronal death. Hence, glutamate extracellular levels are tightly regulated through its uptake by glial glutamate transporters. It has been observed that glutamate regulates its own removal, both in the short-time frame via a transporter-mediated decrease in the uptake, and in the long-term through the transcriptional control of its gene expression, a process mediated by glutamate receptors that involves the Ca2+/diacylglycerol-dependent protein kinase and the transcription factor Ying Yang 1. Taking into consideration that this transcription factor is a member of the Polycomb complex and thus, part of repressive and activating chromatin remodeling factors, it might direct the interaction of DNA methyltransferases or dioxygenases of methylated cytosines to their target sequences. Here we explored the role of dynamic DNA methylation in the expression and function of glial glutamate transporters. To this end, we used the well-characterized models of primary cultures of chick cerebellar Bergmann glia cells and a human retina-derived Müller glia cell line. A time and dose-dependent increase in global DNA methylation was evident upon glutamate exposure. Under hypomethylation conditions, the glial glutamate transporter protein levels and uptake activity were increased. These results favor the notion that a dynamic DNA methylation program triggered by glutamate in glial cells modulates one of its major functions: glutamate removal.

Validation of a System xc– Functional Assay in Cultured Astrocytes and Nervous Tissue Samples

Disruption of the glutamatergic homeostasis is commonly observed in neurological diseases and has been frequently correlated with the altered expression and/or function of astrocytic high-affinity glutamate transporters. There is, however, a growing interest for the role of the cystine-glutamate exchanger system xc– in controlling glutamate transmission. This exchanger is predominantly expressed in glial cells, especially in microglia and astrocytes, and its dysregulation has been documented in diverse neurological conditions. While most studies have focused on measuring the expression of its specific subunit xCT by RT-qPCR or by Western blotting, the activity of this exchanger in tissue samples remains poorly examined. Indeed, the reported use of sulfur- and carbon-radiolabeled cystine in uptake assays shows several drawbacks related to its short radioactive half-life and its relatively high cost. We here report on the elaborate validation of a method using tritiated glutamate as a substrate for the reversed transport mediated by system xc–. The uptake assay was validated in primary cultured astrocytes, in transfected cells as well as in crude synaptosomes obtained from fresh nervous tissue samples. Working in buffers containing defined concentrations of Na+, allowed us to differentiate the glutamate uptake supported by system xc– or by high-affinity glutamate transporters, as confirmed by using selective pharmacological inhibitors. The specificity was further demonstrated in primary astrocyte cultures from transgenic mice lacking xCT or in cell lines where xCT expression was genetically induced or reduced. As such, this assay appears to be a robust and cost-efficient solution to investigate the activity of this exchanger in physiological and pathological conditions. It also provides a reliable tool for the screening and characterization of new system xc– inhibitors which have been frequently cited as valuable drugs for nervous disorders and cancer.

Metabotropic Glutamate Receptors 1 Regulates Rat Carotid Body Response to Acute Hypoxia via Presynaptic Mechanism.

Background: The carotid body (CB) plays a critical role in oxygen sensing; however, the role of glutamatergic signaling in the CB response to hypoxia remains uncertain. We previously found that functional multiple glutamate transporters and inotropic glutamate receptors (iGluRs) are expressed in the CB. The aim of this present research is to investigate the expression of group I metabotropic glutamate receptors (mGluRs) (mGluR1 and 5) in the CB and its physiological function in rat CB response to acute hypoxia.Methods: RT-PCR and immunostaining were conducted to examine the mRNA and protein expression of group I mGluRs in the human and rat CB. Immunofluorescence staining was performed to examine the cellular localization of mGluR1 in the rat CB. In vitro carotid sinus nerve (CSN) discharge recording was performed to detect the physiological function of mGluR1 in CB response to acute hypoxia.Results: We found that (1) mRNAs of mGluR1 and 5 were both expressed in the human and rat CB. (2) mGluR1 protein rather than mGluR5 protein was present in rat CB. (3) mGluR1 was distributed in type I cells of rat CB. (4) Activation of mGluR1 inhibited the hypoxia-induced enhancement of CSN activity (CSNA), as well as prolonged the latency time of CB response to hypoxia. (5) The inhibitory effect of mGluR1 activation on rat CB response to hypoxia could be blocked by GABAB receptor antagonist.Conclusion: Our findings reveal that mGluR1 in CB plays a presynaptic feedback inhibition on rat CB response to hypoxia.

MC-100093, a Novel β-Lactam Glutamate Transporter-1 Enhancer Devoid of Antimicrobial Properties, Attenuates Cocaine Relapse in Rats.

Cocaine use disorder currently lacks Food and Drug Administration-approved treatments. In rodents, the glutamate transporter-1 (GLT-1) is downregulated in the nucleus accumbens after cocaine self-administration, and increasing the expression and function of GLT-1 reduces the reinstatement of cocaine seeking. The β-lactam antibiotic ceftriaxone upregulates GLT-1 and attenuates cue- and cocaine-induced cocaine seeking without affecting motivation for natural rewards. Although ceftriaxone shows promise for treating cocaine use disorder, it possesses characteristics that limit successful translation from bench to bedside, including poor brain penetration, a lack of oral bioavailability, and a risk of bacterial resistance when used chronically. Thus, we aimed to develop novel molecules that retained the GLT-1-enhancing effects of ceftriaxone but displayed superior drug-like properties. Here, we describe a new monocyclic β-lactam, MC-100093, as a potent upregulator of GLT-1 that is orally bioavailable and devoid of antimicrobial properties. MC-100093 was synthesized and tested in vitro and in vivo to determine physiochemical, pharmacokinetic, and pharmacodynamic properties. Next, adult male rats underwent cocaine self-administration and extinction training. During extinction training, rats received one of four doses of MC-100093 for 6-8 days prior to a single cue-primed reinstatement test. Separate cohorts of rats were used to assess nucleus accumbens GLT-1 expression and MC-100093 effects on sucrose self-administration. We found that 50 mg/kg MC-100093 attenuated cue-primed reinstatement of cocaine seeking while upregulating GLT-1 expression in the nucleus accumbens core. This dose did not produce sedation, nor did it decrease sucrose consumption or body weight. Thus, MC-100093 represents a potential treatment to reduce cocaine relapse. SIGNIFICANCE STATEMENT: Increasing GLT-1 activity reliably reduces drug-seeking across classes of drugs; however, existing GLT1-enhancers have side effects and lack oral bioavailability. To address this issue, novel GLT-1 enhancers were synthesized, and the compound with the most favorable pharmacokinetic and pharmacodynamic properties, MC-100093, was selected for further testing. MC-100093 attenuated cued cocaine seeking without reducing food seeking or locomotion and upregulated GLT-1 expression in the nucleus accumbens.

Senescence‐Associated Secretory Phenotype (SASP) factors down‐regulate glutamate transporter expression in human brainstem astrocytes through a paracrine fashion

Glia-neuron crosstalk in the brainstem is emerging as a critical player in the regulation of sympathetic nerve activity (SNA). Specifically, astrocytes modulate SNA by tightly regulating synaptic glutamate levels through transporter mediated re-uptake mechanisms. Pro-inflammatory mediators like TNFα have been shown to negatively affect glutamate transporter expression in astrocytes. Previous studies from our laboratory have provided evidence for oxidative stress-induced senescence and their secretory phenotype (SASP), a potential source of inflammation, in human brainstem astrocytes. Hence, in the present study we hypothesize that inflammatory mediators in SASP factors down-regulate glutamate re-uptake transporter expression in a paracrine manner. To test our hypothesis, we induced senescence in cultured human brainstem astrocytes with hydrogen peroxide (H2O2) treatment at a dose of 300 μM for 2 hours. Senescence induction was confirmed through PCR expression for senescence markers and SA-beta-gal staining 7 days after H2O2 treatment. Conditioned media collected from the senescent and control astrocytes were used to assess the paracrine effect of SASP factors on astrocytic glutamate transporter expression. Following 36 hours of treatment with conditioned media from senescent astrocytes, we observed a significant down-regulation of two of the major astrocytic glutamate re-uptake transporters, EAAT1 (GLAST-1) and EAAT2 (GLT-1) in human brainstem astrocytes. This suggests that soluble SASP factors in the secretome of senescent cells can alter the function of glutamate transporters in the astrocytes through a paracrine action. Increased activity of glutamatergic neurons in the rostral ventrolateral medulla due to reduced glutamate re-uptake at the synapse may be a possible glial mechanism underlying enhanced sympathoexcitation in obesity.

MiR-30a-5p Regulates GLT-1 Function via a PKCα-Mediated Ubiquitin Degradation Pathway in a Mouse Model of Parkinson's Disease.

Glutamate excitotoxicity is caused by dysfunctional glutamate transporters and plays an important role in the pathogenesis of Parkinson's disease (PD); however, the mechanisms that underlie the regulation of glutamate transporters in PD are still not fully elucidated. MicroRNAs(miRNA), which are abundant in astrocytes and neurons, have been reported to play key roles in regulating the translation of glutamate-transporter mRNA. In this study, we hypothesized that the miR-30a-5p contributes to the pathogenesis of PD by regulating the ubiquitin-mediated degradation of glutamate transporter 1 (GLT-1). We demonstrated that short-hairpin RNA-mediated knockdown of miR-30a-5p ameliorated motor deficits and pathological changes like astrogliosis and reactive microgliosis in a mouse model of PD (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice). Western blotting and immunofluorescent labeling revealed that miR-30a-5p suppressed the expression and function of GLT-1 in MPTP-treated mice and specifically in astrocytes treated with 1-methyl-4-phenylpyridinium (MPP+) (cell model of PD). Both in vitro and in vivo, we found that miR-30a-5p knockdown promoted glutamate uptake and increased GLT-1 expression by hindering GLT-1 ubiquitination and subsequent degradation in a PKCα-dependent manner. Therefore, we conclude that miR-30a-5p represents a potential therapeutic target for the treatment of PD.