Expression Of Excitatory Amino Acid Transporter Research Articles

Oxidative stress plays an important role in the central regulatory mechanism of orofacial hyperalgesia under low estrogen conditions

Hyperalgesia occurs in the orofacial region of rats when estrogen levels are low, although the specific mechanism needs to be investigated further. Furthermore, oxidative stress plays an important role in the transmission of pain signals. This study aimed to explore the role of oxidative stress in orofacial hyperalgesia under low estrogen conditions. We firstly found an imbalance between oxidative and antioxidant capacity within the spinal trigeminal subnucleus caudalis (SP5C) of rats after ovariectomy (OVX), resulting in oxidative stress and then a decrease in the orofacial pain threshold. To investigate the mechanism by which oxidative stress occurs, we used virus as a tool to inhibit or overexpress the excitatory amino acid transporter 3 (EAAT3) gene. Further investigation revealed that the regulation of glutathione (GSH) and reactive oxygen species (ROS) can be achieved by regulating EAAT3, which in turn impacts the occurrence of oxidative stress. In summary, our findings suggest that reduced expression of EAAT3 within the SP5C of rats in the low estrogen state may decrease GSH content and increase ROS levels, resulting in oxidative stress and ultimately lead to orofacial hyperalgesia. This suggests that antioxidants could be a potential therapeutic direction for orofacial hyperalgesia under low estrogen conditions, though more research is needed to understand its mechanism.

Increased excitatory amino acid transporter 2 levels in basolateral amygdala astrocytes mediate chronic stress-induced anxiety-like behavior

Abstract The conventional perception of astrocytes as mere supportive cells within the brain has recently been called into question by empirical evidence, which has revealed their active involvement in regulating brain function and encoding behaviors associated with emotions. Specifically, astrocytes in the basolateral amygdala (BLA) have been found to play a role in the modulation of anxiety-like behaviors triggered by chronic stress. Nevertheless, the precise molecular mechanisms by which BLA astrocytes regulate chronic stress–induced anxiety-like behaviors remain to be fully elucidated. In this study, we found that in a mouse model of anxiety triggered by unpredictable chronic mild stress (UCMS), the expression of excitatory amino acid transporter 2 (EAAT2) was upregulated in the BLA. Interestingly, our findings indicate that the targeted knockdown of EAAT2 specifically within the BLA astrocytes was able to rescue the anxiety-like behavior in mice subjected to stress. Furthermore, we found that the overexpression of EAAT2 in the BLA, whether achieved through intracranial administration of EAAT2 agonists or through injection of EAAT2-overexpressing viruses with GfaABC1D promoters, evoked anxiety-like behavior in mice. Our single-nucleus RNA sequencing analysis further confirmed that chronic stress induced an upregulation of EAAT2 specifically in astrocytes in the BLA. Moreover, through in vivo calcium signal recordings, we found that the frequency of calcium activity in the BLA of mice subjected to chronic stress was higher compared with normal mice. After knocking down the expression of EAAT2 in the BLA, the frequency of calcium activity was not significantly increased, and anxiety-like behavior was obviously mitigated. Additionally, administration of an EAAT2 inhibitor in the BLA yielded a notable reduction in anxiety level among mice subjected to stress. These results suggest that BLA astrocytic EAAT2 plays a role in in the regulation of UCMS-induced anxiety-like behavior by impacting the activity of local glutamatergic neurons, and targeting EAAT2 in the BLA holds therapeutic promise for addressing anxiety disorders.

Synthesis and Structure-Activity Relationships for Glutamate Transporter Allosteric Modulators.

Excitatory amino acid transporters (EAATs) are essential CNS proteins that regulate glutamate levels. Excess glutamate release and alteration in EAAT expression are associated with several CNS disorders. Previously, we identified positive allosteric modulators (PAM) of EAAT2, the main CNS transporter, and have demonstrated their neuroprotective properties in vitro. Herein, we report on the structure-activity relationships (SAR) for the analogs identified from virtual screening and from our medicinal chemistry campaign. This work identified several selective EAAT2 positive allosteric modulators (PAMs) such as compounds 4 (DA-023) and 40 (NA-014) from a library of analogs inspired by GT949, an early generation compound. This series also provides nonselective EAAT PAMs, EAAT inhibitors, and inactive compounds that may be useful for elucidating the mechanism of EAAT allosteric modulation.

Forebrain EAAT3 Overexpression Increases Susceptibility to Amphetamine-Induced Repetitive Behaviors.

Obsessive-compulsive disorder (OCD) is a debilitating psychiatric disorder characterized by intrusive obsessive thoughts and compulsive behaviors. Multiple studies have shown the association of polymorphisms in the SLC1A1 gene with OCD. The most common of these OCD-associated polymorphisms increases the expression of the encoded protein, excitatory amino acid transporter 3 (EAAT3), a neuronal glutamate transporter. Previous work has shown that increased EAAT3 expression results in OCD-relevant behavioral phenotypes in rodent models. In this study, we created a novel mouse model with targeted, reversible overexpression of Slc1a1 in forebrain neurons. The mice do not have a baseline difference in repetitive behavior but show increased hyperlocomotion following a low dose of amphetamine (3 mg/kg) and increased stereotypy following a high dose of amphetamine (8 mg/kg). We next characterized the effect of amphetamine on striatal cFos response and found that amphetamine increased cFos throughout the striatum in both control and Slc1a1-overexpressing (OE) mice, but Slc1a1-OE mice had increased cFos expression in the ventral striatum relative to controls. We used an unbiased machine classifier to robustly characterize the behavioral response to different doses of amphetamine and found a unique response to amphetamine in Slc1a1-OE mice, relative to controls. Lastly, we found that the differences in striatal cFos expression in Slc1a1-OE mice were driven by cFos expression specifically in D1 neurons, as Slc1a1-OE mice had increased cFos in D1 ventral medial striatal neurons, implicating this region in the exaggerated behavioral response to amphetamine in Slc1a1-OE mice.

Anesthetic Sevoflurane Induces Enlargement of Dendritic Spine Heads in Mouse Neurons via Tau-Dependent Mechanisms.

Sevoflurane induces neuronal dysfunction and cognitive impairment. However, the underlying mechanism remains largely to be determined. Tau, cyclophilin D, and dendritic spine contribute to cognitive function. But whether changes in dendritic spines are involved in the effects of sevoflurane and the potential association with tau and cyclophilin D is not clear. We harvested hippocampal neurons from wild-type mice, tau knockout mice, and cyclophilin D knockout mice. We treated these neurons with sevoflurane at day in vitro 7 and measured the diameter of dendritic spine head and the number of dendritic spines. Moreover, we determined the effects of sevoflurane on the expression of excitatory amino acid transporter 3 (EAAT3), extracellular glutamate levels, and miniature excitatory postsynaptic currents (mEPSCs). Finally, we used lithium, cyclosporine A, and overexpression of EAAT3 in the interaction studies. Sevoflurane-induced tau phosphgorylation increased the diameter of dendritic spine head and decreased the number of dendritic spines in neurons harvested from wild-type and cyclophilin D knockout mice, but not tau knockout mice. Sevoflurane decreased the expression of EAAT3, increased extracellular glutamate levels, and decreased the frequency of mEPSCs in the neurons. Overexpression of EAAT3 mitigated the effects of sevoflurane on dendritic spines. Lithium, but not cyclosporine A, attenuated the effects of sevoflurane on dendritic spines. Lithium also inhibited the effects of sevoflurane on EAAT3 expression and mEPSCs. These data suggest that sevoflurane induces a tau phosphorylation-dependent demtrimental effect on dendritic spine via decreasing EAAT3 expression and increasing extracellular glutamate levels, leading to neuronal dysfunction.

Orally Administrated Glutamate Restored EAAT1 and 3 Expression Levels Suppressed in 5-Fluorouracil-damaged Intestinal Epithelial Cells.

5-Fluorouracil (5-FU) treatment induces intestinal mucositis, with diarrhea as the primary symptom. Mucositis significantly reduces patients' quality of life (QOL). Amino acids such as glutamate are beneficial for treating gastrointestinal disorders; however, the underlying mechanism remains unclear. Therefore, this study aimed to clarify the role of excitatory amino acid transporters (EAATs) in 5-FU-induced intestinal injury. The rat intestinal epithelial cell line (IEC-6) was used to evaluate whether the EAAT inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC) affects 5-FU-induced cytotoxicity. Mice with 5-FU-induced mucositis were used to determine the effects of glutamate on EAATs expression levels. Treatment with L-trans-PDC suppressed IEC-6 cell growth. It also exacerbated the 5-FU-induced cell growth suppression and increased inflammatory cytokine expression. In addition, mice treated with 5-FU+Glutamate showed higher EAAT1,3 expression than 5-FU only-treated mice. Decreased EAAT levels worsen intestinal cell damage caused by 5-FU, suppress cell growth, and induce inflammation. This study contributes to the understanding EAAT and its relationship with intestinal mucositis, which can aid in the development of novel preventive strategies for cancer chemotherapy.

Citrullinated isomer of myelin basic protein can induce inflammatory responses in astrocytes

PurposeDuring the course of demyelinating inflammatory diseases, myelin-derived proteins, including myelin basic protein(MBP), are secreted into extracellular space. MBP shows extensive post-translational modifications, including deimination/citrullination. Deiminated MBP is structurally less ordered, susceptible to proteolytic attack, and more immunogenic than unmodified MBP. This study investigated the effect of the deiminated/citrullinated isomer of MBP(C8) and the unmodified isomer of MBP(C1) on cultured primary astrocytes. MethodsMBP charge isomers were isolated/purified from bovine brain. Primary astrocyte cultures were prepared from the 2-day-old Wistar rats. For evaluation of glutamate release/uptake a Fluorimetric glutamate assay was used. Expression of peroxisome proliferator-activated receptor-gamma(PPAR-γ), excitatory amino acid transporter 2(EAAT2), the inhibitor of the nuclear factor kappa-B(ikB) and high mobility group-B1(HMGB1) protein were assayed by Western blot analysis. IL-17A expression was determined in cell medium by ELISA. ResultsWe found that MBP(C8) and MBP(C1) acted differently on the uptake/release of glutamate in astrocytes: C1 increased glutamate uptake and did not change its release, whereas C8 decreased glutamate release but did not change its uptake. Both isomers increased the expression of PPAR-γ and EAAT2 to the same degree. Western blots of cell lysates revealed decreased expression of ikB and increased expression of HMGB1 proteins after treatment of astrocytes by C8. Moreover, C8-treated cells released more nitric oxide and proinflammatory IL-17A than C1-treated cells. ConclusionsThese data suggest that the most immunogenic deiminated isomer C8, in parallel to the decreases in glutamate release, elicits an inflammatory response and enhances the secretion of proinflammatory molecules via activation of nuclear factor kappa B(NF-kB). Summary statementThe most modified-citrullinated myelin basic protein charge isomer decreases glutamate release, elicits an inflammatory response and enhances the secretion of proinflammatory molecules via activation of nuclear factor kappa B in astrocytes.

A Review on Neuroinflammatory Pathway Mediating Through Ang-II/AT1 Receptors and a Novel Approach for the Treatment of Cerebral Ischemia in Combination with ARB's and Ceftriaxone.

Ischemic stroke is one of the prevalent neurodegenerative disorders; it is generally characterized by sudden abruption of blood flow due to thromboembolism and vascular abnormalities, eventually impairing the supply of oxygen and nutrients to the brain for its metabolic needs. Oxygen-glucose deprived conditions provoke the release of excessive glutamate, which causes excitotoxicity. Recent studies suggest that circulatory angiotensin-II (Ang-II) has an imperative role in initiating detrimental events through binding central angiotensin 1 (AT1) receptors. Insufficient energy metabolites and essential ions often lead to oxidative stress during ischemic reperfusion, which leads to the release of proinflammatory mediators such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and cytokines like interleukin-18 (IL-18) and interleukin- 1beta (IL-1β). The transmembrane glutamate transporters, excitatory amino acid transporter-2 (EAAT-2), which express in astroglial cells, have a crucial role in the clearance of glutamate from its releasing site and convert glutamate into glutamine in normal circumstances of brain physiology. During cerebral ischemia, an impairment or dysfunction of EAAT-2 attributes the risk of delayed neuronal cell death. Earlier studies evidencing that angiotensin receptor blockers (ARB) attenuate neuroinflammation by inhibiting the Ang-II/AT1 receptor-mediated inflammatory pathway and that ceftriaxone ameliorates the excitotoxicity-induced neuronal deterioration by enhancing the transcription and expression of EAAT-2 via the nuclear transcriptional factor kappa-B (NF-kB) signaling pathway. The present review will briefly discuss the mechanisms involved in Ang-II/AT1-mediated neuroinflammation, ceftriaxone-induced EAAT-2 expression, and the repurposing hypothesis of the novel combination of ARBs and ceftriaxone for the treatment of cerebral ischemia.

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

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. 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). 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. 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.

Postnatal exposure to trimethyltin chloride induces retinal developmental neurotoxicity in mice via glutamate and its transporter related changes

Exposure to toxic substances during postnatal period is one of the major factors causing retinal developmental defects. The developmental toxicity of trimethyltin chloride (TMT), a byproduct of an organotin compound widely used in agriculture and industrial fields, has been reported; however, the effect on the mammalian retina during postnatal development and the mechanism have not been elucidated to date. We exposed 0.75 and 1.5 mg/kg of TMT to neonatal ICR mice (1:1 ratio of male and female) up to postnatal day 14 and performed analysis of the retina: histopathology, apoptosis, electrophysiological function, glutamate concentration, gene expression, and fluorescence immunostaining. Exposure to TMT caused delayed eye opening, eye growth defect and thinning of retinal layer. In addition, apoptosis occurred in the retina along with b-wave and spiking activity changes in the micro-electroretinogram. These changes were accompanied by an increase in the concentration of glutamate, upregulation of astrocyte-related genes, and increased expression of glial excitatory amino acid transporter (EAAT) 1 and 2. Conversely, EAAT 3, 4, and 5, mainly located in the neurons, were decreased. Our results are the first to prove postnatal retinal developmental neurotoxicity of TMT at the mammalian model and analyze the molecular, functional as well as morphological aspects to elucidate possible mechanisms: glutamate toxicity with EAAT expression changes. These mechanisms may suggest not only a strategy to treat but also a clue to prevent postnatal retina developmental toxicity of toxic substances.

Chronic Vagotomy Alters Glutamate Transmission in the Nucleus Tractus Solitarii (nTS)

The nTS is the first site of integration and processing of sensory afferent information and is one of the crucial brainstem nuclei involved in cardiorespiratory reflexes. This nucleus receives afferent information from many visceral structures in the thorax and abdomen via the peripheral branches of the vagus nerve. Vagal afferents release glutamate (Glu) which initiates signaling processes that leads to chemo- and baroreflex-mediated changes in sympathetic and phrenic nerve activity. Previous studies by us have shown that decreasing afferent input via unilateral vagotomy augmented nTS astrocyte complexity. Astrocytes contribute to regulation of Glu concentration by removing Glu from the synaptic cleft via excitatory amino acid transporters (EAATs). We hypothesized that vagotomy attenuates Glu signaling in the nTS due to increased Glu uptake by greater EAAT expression or function. Surgical transection or sham surgery of the right cervical vagus nerve caudal to the nodose ganglion was performed in male Sprague-Dawley rats. One week after surgery, mean arterial pressure (MAP), heart rate (HR), and splanchnic sympathetic and phrenic nerve activity (SSNA and PhrNA) were recorded in anesthetized and ventilated rats. Intact vagus nerves and the right phrenic nerve were acutely transected. Nanoinjections of Glu (30 nl, 1-10 mM) into the nTS decreased MAP, HR, SSNA and PhNA in both sham and vagotomy groups. Vagotomy blunted the HR, SSNA and PhrNA responses, indicating that Glu signaling was attenuated. To determine if increased EAAT function contributed to blunted Glu responses, the EAAT blocker TFB-TBOA (10nM, 60nl) was nanoinjected into the nTS. EAAT blockade mimicked the effect of Glu, producing similar depressor, bradycardic, and apneic responses in both groups. TBOA also enhanced the cardiorespiratory responses to Glu injection in both groups. EAAT blockade did not restore Glu responses in the vagotomy group compared to sham. Taken together, these data suggest that increased EAAT function did not account for the attenuated Glu signaling in the nTS. Further studies are needed to uncover the mechanisms by which vagotomy affects Glu signaling in the nTS. NIH R01 HL098602 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Glucose supplementation improves intestinal amino acid transport and muscle amino acid pool in pigs during chronic cold exposure.

Mammals in northern regions chronically suffer from low temperatures during autumn-winter seasons. The aim of this study was to investigate the response of intestinal amino acid transport and the amino acid pool in muscle to chronic cold exposure via Min pig models (cold adaptation) and Yorkshire pig models (non-cold adaptation). Furthermore, this study explored the beneficial effects of glucose supplementation on small intestinal amino acid transport and amino acid pool in muscle of cold-exposed Yorkshire pigs. Min pigs (Exp. 1) and Yorkshire pigs (Exp. 2) were divided into a control group (17°C, n=6) and chronic cold exposure group (7°C, n=6), respectively. Twelve Yorkshire pigs (Exp. 3) were divided into a cold control group and cold glucose supplementation group (8°C). The results showed that chronic cold exposure inhibited peptide transporter protein 1 (PepT1) and excitatory amino acid transporter 3 (EAAT3) expression in ileal mucosa and cationic amino acid transporter-1 (CAT-1) in the jejunal mucosa of Yorkshire pigs (P<0.05). In contrast, CAT-1, PepT1 and EAAT3 expression was enhanced in the duodenal mucosa of Min pigs (P<0.05). Branched amino acids (BCAA) in the muscle of Yorkshire pigs were consumed by chronic cold exposure, accompanied by increased muscle RING-finger protein-1 (MuRF1) and muscle atrophy F-box (atrogin-1) expression (P<0.05). More importantly, reduced concentrations of dystrophin were detected in the muscle of Yorkshire pigs (P<0.05). However, glycine concentration in the muscle of Min pigs was raised (P<0.05). In the absence of interaction between chronic cold exposure and glucose supplementation, glucose supplementation improved CAT-1 expression in the jejunal mucosa and PepT1 expression in the ileal mucosa of cold-exposed Yorkshire pigs (P<0.05). It also improved BCAA and inhibited MuRF1 and atrogin-1 expression in muscle (P<0.05). Moreover, dystrophin concentration was improved by glucose supplementation (P<0.05). In summary, chronic cold exposure inhibits amino acid absorption in the small intestine, depletes BCAA and promotes protein degradation in muscle. Glucose supplementation ameliorates the negative effects of chronic cold exposure on amino acid transport and the amino acid pool in muscle.

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.

Structural insights into inhibitory mechanism of human excitatory amino acid transporter EAAT2

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states at 3.2 Å and 2.8 Å, respectively. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. We provide the characterization of the molecular features of EAAT2 and its selective inhibition mechanism that may facilitate structure-based drug design for EAAT2.

The Regulation of Glial Glutamate Transporters after in‐vitro Ischemic Stroke

Stroke is the 3rd leading cause of death in the United States but adequate treatment is still lacking. Moreover, the pathological mechanisms involved are still not fully understood, highlighting the need for further studies. In this work, we employed an in vitro model of ischemic stroke to study the regulation of excitatory amino acid transporters (EAATs), which are key proteins that control the extracellular concentration of glutamate in the brain and thereby help maintain brain homeostasis. Glutamate dysregulation is a fundamental feature of many neurological disorders, leading to neuronal death through a mechanism known as excitotoxicity. This makes EAATs key players in mitigating glutamate imbalance in the brain and thereby have a major role in excitotoxic outcomes following ischemia. Our goal was to understand how the activity and expression of EAATs are modulated following different severities of ischemic insults. Glia cultures were subjected to oxygen‐glucose deprivation (OGD) for 30‐minutes, 1‐hour and 2‐hours. Our results demonstrated that increasing lengths of insult result in decreased levels of glutamate transport, suggesting an inability of these transporters to effectively clear excess glutamate. The expression of glial transporters EAAT1 and EAAT2 are differentially regulated: EAAT1 is downregulated after ischemia, with a more profound effect after 1‐hour OGD, whereas EAAT2 expression is up regulated after 30‐minutes and 1‐hour OGD before decreasing below control levels after 2‐hour OGD. These results suggest that these transporters may work together, likely as a compensatory mechanism. We also investigated the effects of Clavulanic Acid (CA), a compound that enhances EAAT2, which is the main subtype of EAAT in the CNS. CA pretreatment was only able to rescue EAAT activity and significantly increase EAAT2 levels in control conditions and after 30‐minutes of OGD, suggesting that this strategy only offers benefit in mild ischemic insults. Furthermore, we investigated reactive gliosis, which can be defined by the activation of glia cells in response to a pathology. Previous work has shown that reactive gliosis is a phenotype associated with ischemia, however we wanted to further define and evaluate this type of astrocytic response. In specific, we wanted to determine how reactive gliosis is affected following different ischemic insults. Results show that reactive gliosis, which was determined by an increased expression and fluorescence of GFAP, increases with OGD insult, with a significant difference seen after 2‐hours of OGD. This suggests the reactive phenotype is more prominent as the severity of ischemia increases. These findings advanced our knowledge on EAAT regulation following different severities of ischemic stroke in‐vitro. Future studies will investigate potential mechanisms associated with their differential regulation, such as transport reversal, internalization, and degradation pathways.

Chandamarutha Chenduram, an Indian traditional Siddha preparation attenuated the neuronal degeneration in ischemic mice through ameliorating cytokines and oxy-radicals mediated EAAT-2 dysfunction

Ethnopharmacological relevanceChandamarutha Chenduram (CC), an Indian traditional Siddha preparation officially recorded in the Siddha formulary of India and its composition are widely used in the Siddha practice of neurological disorders like stroke/paralysis in India. However, the scientific validation and mechanistic evidence is lacking and yet to be elucidated. Aim of the studyTo establish the scientific evidences and to explore the possible neuroprotective mechanism of CC in cerebral ischemia. Materials and methodsChemical standardization of the CC was performed using atomic absorption spectroscopy and gravimetric analysis. Acute toxicity study for CC in mice was performed in accordance with OECD 423 guidelines. CC (5 mg/kg) and CC (10 mg/kg) were investigated in bilateral common carotid occlusion (BCCAo) model in mice. After, behavioral assessments, the brain samples were collected and the hippocampus region was micro-dissected for neurotransmitter, neurobiochemicals and inflammatory cytokines estimation. The excitatory amino acid transporter-2 (EAAT-2) expressions was analyzed by RT-PCR to understand the possible molecular mechanism. In addition, hematoxylin and eosin staining of CA1 hippocampal brain region was performed to support the neuroprotective effect of CC in ischemic condition. ResultsChemical standardization analysis showed that CC has acceptable range of mercury (0.82 ppm) and elemental sulphur (11% w/w). Also, other heavy metal limits were found to be less or not detectable. Toxicity study also evidenced the safety profile of CC. CC has significantly reversed the behavioral dysfunctions (p < 0.001) in global ischemic mice. Treatment with CC has attenuated the excitatory neurotransmitter glutamate, lipid peroxide, nitric oxide, cytokines (IL-1β, TNF-α) (p < 0.001) and increased the antioxidant enzymes (SOD, CAT, GSH) and EAAT-2 expression level (p < 0.001) in ischemic brain. The hematoxylin and eosin staining in CA1 region of hippocampus also evidence the neuroprotective effect exhibited by CC. ConclusionsTreatment with CC has exhibited dose dependent effect and CC10 has shown significant protective effect in comparison to CC5 in most of the parameters studied. CC prevented further degeneration of neurons in cerebral ischemic mice through ameliorating inflammatory cytokines and oxy-radicals mediated EAAT-2 dysfunction and subsequent excitotoxicity in neurons.

Astrocytic transcription factor REST upregulates glutamate transporter EAAT2, protecting dopaminergic neurons from manganese-induced excitotoxicity

Chronic exposure to high levels of manganese (Mn) leads to manganism, a neurological disorder with similar symptoms to those inherent to Parkinson's disease. However, the underlying mechanisms of this pathological condition have yet to be established. Since the human excitatory amino acid transporter 2 (EAAT2) (glutamate transporter 1 in rodents) is predominantly expressed in astrocytes and its dysregulation is involved in Mn-induced excitotoxic neuronal injury, characterization of the mechanisms that mediate the Mn-induced impairment in EAAT2 function is crucial for the development of novel therapeutics against Mn neurotoxicity. Repressor element 1-silencing transcription factor (REST) exerts protective effects in many neurodegenerative diseases. But the effects of REST on EAAT2 expression and ensuing neuroprotection are unknown. Given that the EAAT2 promoter contains REST binding sites, the present study investigated the role of REST in EAAT2 expression at the transcriptional level in astrocytes and Mn-induced neurotoxicity in an astrocyte–neuron coculture system. The results reveal that astrocytic REST positively regulates EAAT2 expression with the recruitment of an epigenetic modifier, cAMP response element-binding protein–binding protein/p300, to its consensus binding sites in the EAAT2 promoter. Moreover, astrocytic overexpression of REST attenuates Mn-induced reduction in EAAT2 expression, leading to attenuation of glutamate-induced neurotoxicity in the astrocyte–neuron coculture system. Our findings demonstrate that astrocytic REST plays a critical role in protection against Mn-induced neurotoxicity by attenuating Mn-induced EAAT2 repression and the ensuing excitotoxic dopaminergic neuronal injury. This indicates that astrocytic REST could be a potential molecular target for the treatment of Mn toxicity and other neurological disorders associated with EAAT2 dysregulation.

EAAT2 Expression in the Hippocampus, Subiculum, Entorhinal Cortex and Superior Temporal Gyrus in Alzheimer's Disease.

Alzheimer’s disease (AD) is a neuropathological disorder characterized by the presence and accumulation of amyloid-beta plaques and neurofibrillary tangles. Glutamate dysregulation and the concept of glutamatergic excitotoxicity have been frequently described in the pathogenesis of a variety of neurodegenerative disorders and are postulated to play a major role in the progression of AD. In particular, alterations in homeostatic mechanisms, such as glutamate uptake, have been implicated in AD. An association with excitatory amino acid transporter 2 (EAAT2), the main glutamate uptake transporter, dysfunction has also been described. Several animal and few human studies examined EAAT2 expression in multiple brain regions in AD but studies of the hippocampus, the most severely affected brain region, are scarce. Therefore, this study aims to assess alterations in the expression of EAAT2 qualitatively and quantitatively through DAB immunohistochemistry (IHC) and immunofluorescence within the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus (STG) regions, between human AD and control cases. Although no significant EAAT2 density changes were observed between control and AD cases, there appeared to be increased transporter expression most likely localized to fine astrocytic branches in the neuropil as seen on both DAB IHC and immunofluorescence. Therefore, individual astrocytes are not outlined by EAAT2 staining and are not easily recognizable in the CA1–3 and dentate gyrus regions of AD cases, but the altered expression patterns observed between AD and control hippocampal cases could indicate alterations in glutamate recycling and potentially disturbed glutamatergic homeostasis. In conclusion, no significant EAAT2 density changes were found between control and AD cases, but the observed spatial differences in transporter expression and their functional significance will have to be further explored.

Acetylcholine Receptor Stimulation Mediates Trafficking of the Dopamine and Glutamate Transporters through Activation of Protein Kinase C

The dopamine transporter (DAT) clears neurotransmitter from the extracellular space and serves as an important regulator of signal amplitude and duration at sites of dopamine release. Similarly, the neuronal excitatory amino acid transporter 3 (EAAT3) plays an important role in the control of glutamatergic tone. Several different intracellular signaling pathways have been observed to modulate activity of these neurotransmitter transporters through regulation of the trafficking of the carriers to and from the cell surface. In this study, we investigated how the activation of protein kinase C (PKC) facilitates internalization of DAT and insertion of EAAT3 by addressing the endogenous cell-surface receptors that activate PKC and regulate the DAT and EAAT3 in vivo. We report here that stimulation of M1/M5 muscarinic receptors in midbrain cultures decreases the ability of dopamine neurons to transport dopamine through DAT. The cholinergic agonist, carbachol decreases DAT activity in primary cultures while the M1/M5-specific antagonist, pirenzepine, blocks these effects. The M3 antagonist, DAU 5884, has no effect, but a positive modulator of M5, VU 0238429, enhances the loss of DAT function in response to carbachol and acetylcholine. These data implicate M1/M5 receptors on dopamine neurons in the modulation of DAT function. Bisindolylmaleimide, a PKC inhibitor, blocks the effects of carbachol stimulation on dopamine uptake, indicating a role for PKC in muscarinic receptor-mediated DAT internalization. We designed a specific Gq inhibitor peptide that also blocks the effects of carbachol on DAT in primary cultures, confirming Gq as the G-protein that couples M1/M5 receptors to PKC activation in these cells. It has been established that the activity of the neuronal glutamate transporter EAAT3 increases in response to PMA treatment in the C6 glioma cell line. By total internal reflection fluorescence (TIRF) microscopy of a GFP-tagged EAAT3 we were able to confirm that this was due to an increase in EAAT3 present at the cell surface following PMA treatment. Carbachol activation of endogenous muscarinic receptors in the C6 glioma line also resulted in increased cell-surface expression of EAAT3. This increase was inhibited by co-application of the Gq inhibitor peptide indicating that the increase in EAAT3 activity depends upon Gq-coupled PKC signaling through M1/M5 receptors. In acute midbrain slices, biotinylation of cell-surface proteins showed that the decreases in dopamine transport mediated by muscarinic receptor stimulation the result of decreases in DAT carriers a cell surface. In addition, increases in surface expression of EAAT3 in the same experiment also appear to be modulated by M1/M5 receptors. These data indicate that stimulation of cholinergic receptors can lead to downregulation and upregulation of dopamine and glutamate transporters, respectively through alterations in their membrane trafficking.

The role of LRRK2 in the modulation of the glutamate transporter EAAT2 in Parkinson's disease

Parkinson's disease (PD) is a common chronic and progressive neurodegenerative disorder characterized by a complex physiopathology. Although several known causes lead to the onset of the disease, a central role has been attributed to excitotoxicity and subsequent cellular damages. Glutamate-mediated excitatory synaptic signalling is mainly directed by excitatory amino acid transporter 2 (EAAT2), localized on astrocyte plasma membrane and involved in the glutamate clearance. Alterations in the function or in the expression of EAAT2 lead to extracellular glutamate accumulation, playing an important role in the PD's physiopathology. One of the most common mutation in PD patients is G2019S substitution in Leucin Rich Repeat Kinase 2 (LRRK2). Recently, an impaired cortico-striatal glutamatergic transmission in mice carrying G2019S mutation has been reported. The role of LRRK2 wild-type and mutated in regulating the EAAT2 function and cellular localization have been investigated by heterologous expression in Xenopus laevis oocytes. The Two Electrodes Voltage Clamp (TEVC) and immunochemistry were applied to collect data about the activity and the protein expression. The results demonstrate that LRRK2 acts on EAAT2 significantly increasing the inward glutamate transport current when the wild-type protein is present. Conversely, testing the transporter with the pathogenic LRRK2 G2019S kinase elicited transport currents with amplitude similar to that of EAAT2 expressed alone. The LRRK2 kinase inhibitor MLi-2 can revert the phenotype of the mutated protein G2012S, showing in this condition a transport current amplitude similar to that recorded in the presence of the LRRK2 wild-type. The apparent substrate affinity of EAAT2 for glutamate was higher when co-expressed with G2012S than with wild-type LRRK2, slowing down the glutamate transport cycle. The immunochemistry study on oocytes heterologously expressing EAAT2 with LRRK2 wild-type and G2012S suggested that the amount of transporter on the plasma membrane is reduced in the presence of the mutated protein, while MLi-2 treatment restored the transporter localization. The electrophysiological studies and immunochemistry results highlight that the reduced membrane localization of transporter and the higher affinity can lead to an impaired clearance of glutamate, resulting in the well-known pathological effects. Our data confirmed the functional interaction between EAAT2 and LRRK2 and support the idea that LRRK2 G2019S could be responsible of chronic glutamate accumulation and neurodegeneration in PD.