Elevated Glutamate Concentrations Research Articles

Rewired glutamate metabolism diminishes cytostatic action of L-asparaginase

Tumor cells often adapt to amino acid deprivation through metabolic rewiring, compensating for the loss with alternative amino acids/substrates. We have described such a scenario in leukemic cells treated with L-asparaginase (ASNase). Clinical effect of ASNase is based on nutrient stress achieved by its dual enzymatic action which leads to depletion of asparagine and glutamine and is accompanied with elevated aspartate and glutamate concentrations in serum of acute lymphoblastic leukemia patients. We showed that in these limited conditions glutamate uptake compensates for the loss of glutamine availability. Extracellular glutamate flux detection confirms its integration into the TCA cycle and its participation in nucleotide and glutathione synthesis. Importantly, it is glutamate-driven de novo synthesis of glutathione which is the essential metabolic pathway necessary for glutamate's pro-survival effect. In vivo findings support this effect by showing that inhibition of glutamate transporters enhances the therapeutic effect of ASNase. In summary, ASNase induces elevated extracellular glutamate levels under nutrient stress, which leads to a rewiring of intracellular glutamate metabolism and has a negative impact on ASNase treatment.

Cerebrovascular Dysfunction in a Mouse Model of Marfan Syndrome: Increased Vulnerability to Neuropathology & Mild Traumatic Brain Injury

Marfan syndrome (MFS) is a connective tissue disorder (CTD) associated with mutations in fibrillin-1 ( Fbn1), leading to chronic systemic increases in transforming growth factor-β (TGF-β) availability and signaling that could lead to aortic wall weakening and aneurysm/rupture. TGF-β has been implicated in cerebrovascular dysfunction, loss of blood-brain barrier (BBB) integrity, age-related neuroinflammation and neurological deficits. Improvements in MFS treatment and life expectancy have uncovered a risk for systemic vascular dysfunction including cerebrovascular complications such as stroke, and cerebral aneurysm. Therefore, we hypothesized that Fbn1 mutation ( Fbn1C1041G/+) is associated with systemic vascular dysfunction, BBB permeability, neuroinflammation, neurobehavioral alterations, and impaired glutamate homeostasis, leaving the brain more vulnerable to mild traumatic brain injury (mTBI). Our experimental groups included: 6M-old male and female MFS ( Fbn1C1041G/+) and C57BL/6 (WT) control, and middle-aged 12M WT mice (N=3-10/group, P < 0.05). In vivo ultrasound imaging analyzed by two blinded investigators demonstrated increased aortic root diameters and stiffness in MFS mice compared to 6M and 12M WT mice, where MFS male diameters were affected more than females. Vascular dysfunction measured by blood flow velocity (BFV) extended to the left pulmonary artery where 6M MFS and 12M WT mice were reduced compared to 6M WT mice. This reduction continued through decreased posterior cerebral artery BFV in MFS males, where MFS females were comparable to 6 and 12M WT females. In 6M MFS male mice, Evans blue extravasation and Immunoglobulin G staining demonstrated increased BBB permeability in the hippocampus to large and small molecules, respectively, as well as to small molecules in the lateral hypothalamus as compared to 6M WT and like 12M WT. In adjacent sections, iba-1-stained cells had morphologies indicative of reactive microglia. In vivo electrochemical recordings in the hippocampus showed a trend towards lowered peak glutamate amplitudes and elevated baseline glutamate concentrations in MFS and 12M WT mice demonstrating disrupted glutamate homeostasis. Furthermore, exogenous TGF-β applied in the lateral hippocampus of 6M WT males revealed an evoked glutamate response that may support this elevated baseline in MFS mice. Higher neurobehavioral severity scale (NSS) scores in 6M MFS mice suggested neurobehavioral alterations that were more like 12M WT. Midline fluid percussion was used to induce mTBI, where 6M MFS male mice and both female groups required a 15% lower pressure to induce mTBI righting reflex times (5-10 minutes) compared to 6M WT male mice. Mice were evaluated at 1-day post injury, where BBB permeability to large and small molecules and microglia activation were increased, glutamate clearance was slowed, and NSS scores were increased compared to 6M WT and were like 6M MFS and 12M WT mice. These novel findings signify MFS as a condition of systemic vascular dysfunction and pre-mature cerebrovascular aging phenotype with more vulnerability to brain injury. Funding: NIH-R36AG083385, Valley Research Partnership-P1A-5012, NIH-R15HL145646, NIH-R01NS100793, Midwestern Graduate Funds, Phoenix Children’s Hospital Leadership Circle, Graduate and Professional Council Research and Projects Grant. This is the full abstract presented at the American Physiology Summit 2024 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.

High-frequency repetitive transcranial magnetic stimulation improves depressive-like behaviors in CUMS-induced rats by modulating astrocyte GLT-1 to reduce glutamate toxicity

Impaired glutamate recycling plays an important role in the pathophysiology of depression, and it has been demonstrated that glutamate transporter-1 (GLT-1) on astrocytes is involved in glutamate uptake. Studies have shown that repetitive transcranial magnetic stimulation (rTMS) is effective in treating depression, however, the exact mechanism of rTMS treatment remains unclear. Here, we used a chronic unpredictable mild stress (CUMS) protocol to induce depression-like behaviors in rats followed by rTMS treatment. Behavioral assessment was primarily through SPT, FST, OFT and body weight. Histological analysis focused on GFAP and GLT-1 expression, synaptic plasticity, apoptosis and PI3K/Akt/CREB pathway-related proteins. The results showed that rTMS treatment increased sucrose preference, improved locomotor activity, shortened immobility time as well as increased body weight. And rTMS intervention reversed the elevated glutamate concentration in the hippocampus of CUMS rats using an ELISA kit. Moreover, rTMS ameliorated the reduction in GFAP and GLT-1 expression, alleviated the decrease in BDNF, PSD95 and synapsin-1 expression, also reversed the expression levels of BAX and Bcl2 in the hippocampus of CUMS-induced rats. Moreover, rTMS also increased the protein phosphorylation level of PI3K/Akt/CREB pathway. These results suggest that rTMS treatment ameliorates depression-like behaviors in the rat model by reversing the reduction of GLT-1 on astrocytes and reducing glutamate accumulation in the synaptic cleft, which in turn ameliorates synaptic plasticity damage and neuronal apoptosis. The regulation of GLT-1 by rTMS may be through the PI3K/Akt/CREB pathway.

Chronic neuropathic pain components in whiplash-associated disorders correlate with metabolite concentrations in the anterior cingulate and dorsolateral prefrontal cortex: a consensus-driven MRS re-examination.

Whiplash injury (WHI) is characterised by a forced neck flexion/extension, which frequently occurs after motor vehicle collisions. Previous studies characterising differences in brain metabolite concentrations and correlations with neuropathic pain (NP) components with chronic whiplash-associated disorders (WAD) have been demonstrated in affective pain-processing areas such as the anterior cingulate cortex (ACC). However, the detection of a difference in metabolite concentrations within these cortical areas with chronic WAD pain has been elusive. In this study, single-voxel magnetic resonance spectroscopy (MRS), following the latest MRSinMRS consensus group guidelines, was performed in the anterior cingulate cortex (ACC), left dorsolateral prefrontal cortex (DLPFC), and occipital cortex (OCC) to quantify differences in metabolite concentrations in individuals with chronic WAD with or without neuropathic pain (NP) components. Healthy individuals (n = 29) and participants with chronic WAD (n = 29) were screened with the Douleur Neuropathique 4 Questionnaire (DN4) and divided into groups without (WAD-noNP, n = 15) or with NP components (WAD-NP, n = 14). Metabolites were quantified with LCModel following a single session in a 3 T MRI scanner within the ACC, DLPFC, and OCC. Participants with WAD-NP presented moderate pain intensity and interference compared with the WAD-noNP group. Single-voxel MRS analysis demonstrated a higher glutamate concentration in the ACC and lower total choline (tCho) in the DLPFC in the WAD-NP versus WAD-noNP group, with no intergroup metabolite difference detected in the OCC. Best fit and stepwise multiple regression revealed that the normalised ACC glutamate/total creatine (tCr) (p = 0.01), DLPFC n-acetyl-aspartate (NAA)/tCr (p = 0.001), and DLPFC tCho/tCr levels (p = 0.02) predicted NP components in the WAD-NP group (ACC r 2 = 0.26, α = 0.81; DLPFC r 2 = 0.62, α = 0.98). The normalised Glu/tCr concentration was higher in the healthy than the WAD-noNP group within the ACC (p < 0.05), but not in the DLPFC or OCC. Neither sex nor age affected key normalised metabolite concentrations related to WAD-NP components when compared to the WAD-noNP group. This study demonstrates that elevated glutamate concentrations within the ACC are related to chronic WAD-NP components, while higher NAA and lower tCho metabolite levels suggest a role for increased neuronal-glial signalling and cell membrane dysfunction in individuals with chronic WAD-NP components.

Hyperglucagonaemia and amino acid alterations in individuals with type 2 diabetes and non-alcoholic fatty liver disease.

Hyperglucagonaemia contributes to the pathophysiology in type 2 diabetes (T2D), but the mechanisms behind the inappropriate glucagon secretion are not fully understood. Glucagon and amino acids are regulated in a feedback loop referred to as the liver-α cell axis. Individuals with non-alcoholic fatty liver disease (NAFLD) appear to be glucagon resistant, disrupting the liver-α cell axis resulting in hyperglucagonaemia and hyperaminoacidaemia. We investigated the associations between circulating glucagon, amino acids, and liver fat content in a cohort of individuals with T2D. We included 110 individuals with T2D in this cross-sectional study. Liver fat content was quantified using 1H magnetic resonance spectroscopy (MRS). Associations between liver fat content and plasma glucagon and amino acids, respectively, were estimated in multivariate linear regression analyses. Individuals with NAFLD (n = 52) had higher plasma glucagon concentrations than individuals without NAFLD (n = 58). The positive association between plasma glucagon concentrations and liver fat content was confirmed in the multivariable regression analyses. Plasma concentrations of isoleucine and glutamate were increased, and glycine and serine concentrations were decreased in individuals with NAFLD. Concentrations of other amino acids were similar between individuals with and without NAFLD, and no clear association was seen between liver fat content and amino acids in the regression analyses. MRS-diagnosed NAFLD in T2D is associated with hyperglucagonaemia and elevated plasma concentrations of isoleucine and glutamate and low plasma concentrations of glycine and serine. Whether NAFLD and glucagon resistance per se induce these changes remains to be elucidated.

Sex-dependent Cerebrovascular Aging and Vulnerability to Traumatic Brain Injury in a Transgenic Mouse Model of a Fibrillin-1 Associated Connective Tissue Disorder

Marfan syndrome (MFS) is a connective tissue disorder (CTD) associated with mutations in fibrillin-1 (Fbn1), leading to systemic increases in transforming growth factor-β (TGF-β) availability and signaling that induces an accelerated vascular aging phenotype with known peripheral vascular dysfunction by 6-months (6M) of age in Fbn1 C1041G/+ (MFS) mice. TGF-β has been implicated in cerebrovascular dysfunction, loss of blood-brain barrier (BBB) integrity, age-related neuroinflammation and neurological deficits. Improvements in MFS treatment and life expectancy have uncovered a risk for cerebrovascular complications such as stroke, and cerebral aneurysm, predominantly in the posterior cerebral artery (PCA). CTDs have shown unmasking events via mild traumatic brain injury (mTBI), where patients display increased severity and recovery times. Cerebrovascular dysfunction and neuropathology have been minimally investigated in MFS. We hypothesized that abnormal Fbn1 protein expression is associated with a sex-dependent accelerated aging phenotype in the aorta, cerebrovasculature, BBB permeability, neuroinflammation, behavior, and glutamate homeostasis, leaving the brain more vulnerable to mTBI. Male and female 6M MFS, 6M C57BL/6 (WT) control, and middle-aged 12M WT mice were utilized. In vivo ultrasound imaging demonstrated increased aortic root diameters and stiffness in MFS mice compared to 6M WT mice, like 12M WT, where MFS males were affected more than females. Aortic results were associated with decreased PCA blood flow velocity in MFS males, where MFS females were similar to 6 and 12M WT female mice. In 6M MFS mice, Evans blue extravasation and Immunoglobulin G staining demonstrated increased BBB permeability in the hippocampus to large and small molecules, respectively, as compared to WT and like 12M WT. In adjacent sections, iba-1 stained cells had morphologies indicative of reactive microglia. Acute glial activation and BBB permeability can disrupt glutamate homeostasis, where in vivo electrochemical recordings in the hippocampus showed slower glutamate clearance and elevated baseline glutamate concentrations in MFS and 12M WT mice. Higher neurobehavioral severity scale (NSS) scores in 6M MFS mice suggested neurobehavioral alterations that were more like 12M WT. Midline fluid percussion was used to induce mTBI, where 6M MFS male mice and both female groups required a 15% lower pressure to induce mTBI righting reflex times (5-10 minutes) compared to 6M WT male mice. Mice were evaluated at 1-day post injury, where BBB permeability to large and small molecules and microglia activation were increased, glutamate clearance was slowed, and NSS scores were increased compared to 6M WT and were similar to 6M MFS and 12M WT mice. These novel findings signify MFS as a disease of accelerated cerebrovascular aging phenotype and demonstrate potential vulnerability to injury, where chronic TGF-β dysregulation may be a critical regulator of age-related pathology. Valley_Research_Partnership-P1A-5012, NIH-R15HL145646, NIH-R01NS100793, Midwestern Graduate Funds 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.

1H NMR-Based metabolomics and 13C isotopic ratio evaluation to differentiate conventional and organic soy sauce

Organic foods gaining popularity due its connection with the health, high nutritional value, and high quality. Therefore, it is of prime importance to develop the analytical methods to combat with food authentication and protect consumer from the food fraud. Conventional soy sauce (CS) and organic soy sauce (OS) were characterized and differentiate for the first time using 1 H NMR-based metabolomics approach. Classification methods like orthogonal partial least squares of discriminant analysis (OPLS-DA) showed a significant ( p < 0.01) separation among CS and OS yielding important metabolites contributing towards this separation. OS was characterized by increased levels of leucine, threonine, isoleucine, valine, choline, phenylalanine, tyrosine, lactate, and acetate whereas, CS showed elevated concentrations of glutamate, glucose, and sucrose. The abundance ratio of 13 C to 12 C was determined by 1 H NMR spectroscopy which showed an increased ratio of 13 C isotope in OS samples indicating the organically grown wheat and soybean used for the preparation of OS. The results can be helpful to convey the information to the end-user to prevent them from economically motivated adulteration. This information could also pave the way to further trace and authenticate the raw materials used in the production of soy.

Dysembryoplastic neuroepithelial tumors: A single-institutional series with special reference to glutamine synthetase expression

Dysembryoplastic neuroepithelial tumors (DNT) is a benign (World Health Organisation, WHO, grade I) glioneuronal tumor and it represent one of the most frequent neoplasm in patient affected by seizures. The epileptic neuronal activity can be determined by abnormal synchronization, excessive glutamate excitation and\\or inadequate GABA inhibition. Increasing evidence suggests that the astrocytes might be involved in this process even if neurons play a relevant role. In particular astrocytes promote the clearance of glutamate, a potent excitatory neurotransmitter of the central nervous system. Indeed, elevated concentrations of extracellular glutamate may determine iper-excitability and seizures as well as other neurological disorders. So, astrocytes, converting glutamate into glutamine via the enzyme glutamine synthetase (GS), could play a protective anti-seizures role. In the present study, we analyzed the immunohistochemical expression of GS in 20 DNTs specimens documenting a constant immunoistochemical expression of GS in astrocytes of the lesional tissue and of the cerebral cortex.

Repeated endosulfan exposure induces changes in neurochemicals, decreases ATPase transmembrane ionic-pumps, and increased oxidative/nitrosative stress in the brains of rats: Reversal by quercetin

Neurochemical and ATPase deregulations play important role in toxicant-induced neurodegeneration. Previous studies have shown that loss of ATPase ionic-pumps alters neurochemical balance via increased ammonia, oxidative and nitrosative stress. Thus, this study investigated the ameliorative potentials of quercetin on neurochemical, ATPase changes, hyperammonemia and oxidative/nitrosative status in the brains of Wistar rats exposed to endosulfan, a known toxic environmental pesticide that is casually used in many developing countries. Adult rats were divided into five treatment groups (n = 5). Groups 1–2 received normal saline and corn oil (vehicle) (10 mL/kg/day), group 3 received quercetin (20 mg/kg/day) orally for 28 days consecutively. However, animals in groups 4–5 were given endosulfan (5 mg/kg/day, p.o) for 28 days. But, from the 14th to 28th day, group 4 additionally received vehicle (10 mL/kg/day, p.o.), while group 5 was treated with quercetin (20 mg/kg/day, p.o.). Thereafter, brain levels of neurochemicals, ATPase activities, ammonia and oxidative/nitrosative stress were investigated by employing standardized biochemical assay protocols. Quercetin increased endosulfan-induced decreased levels of norepinephrine, dopamine, GABA, and decreased elevated concentrations of glutamate and serotonin. Quercetin normalized the increased levels of acetylcholinesterase and ammonia. Furthermore, quercetin significantly reversed the decrease in Na+/K+, Ca2+, Mg2+-ATPase activities induced by endosulfan. Also, quercetin increased superoxide dismutase, catalase and glutathione peroxidase activities, and reduced nitrite and peroxynitrite levels in brains of rats. These findings further provide evidence of the ameliorative potential of quercetin against endosulfan-induced neurotoxicity via attenuation of neurochemical, ATPase changes, and inhibition of acetylcholinesterase activity, ammonia release and oxidative/nitrosative stress in rat brains.

Plasma Amino Acid Concentrations at Birth and Patent Ductus Arteriosus in Very and Extremely Preterm Infants

Background: Amino acids are increasingly recognized as bioactive molecules in numerous physiological and pathophysiological pathways. The non-essential amino acid glutamate is vasoactive in the rat ductus arteriosus (DA) and a decrease in its levels within the 1st days of life has been associated with the presence of patent DA (PDA) in extremely preterm infants. However, these findings have not been confirmed in other studies.Objective: To investigate the possible association between amino acid concentrations in the 1st day of life and the presence of PDA in a cohort of 121 newborns with gestational age (GA) below 30 weeks and birth weight (BW) below 1,500 g.Methods: Plasma samples were collected 6–12 h after birth and amino acid concentrations were determined by tandem mass spectrometry. Besides PDA, we analyzed the potential association of amino acid concentrations with infant sex, small for GA (SGA, defined as BW < third percentile), antenatal corticosteroids, chorioamnionitis, and preeclampsia. Group differences were analyzed by ANOVA adjusted for GA and BW. A Bonferroni significance threshold of P < 0.0024 was used to correct for multiple testing.Results: PDA was found in 48 of the 121 infants examined. We observed higher mean levels of glutamate in infants with PDA (147.0 μmol/L, SD 84.0) as compared with those without (106.7 μmol/L, SD 49.1, P = 0.0006). None of the other amino acid concentrations in the PDA group reached the level of statistical significance that was pre-set to correct for multiple comparisons. Glutamate levels were not significantly affected by infant sex, being SGA, or by exposure to antenatal corticosteroids, clinical chorioamnionitis, or preeclampsia.Conclusion: Our study not only does not confirm the previous findings of low glutamate levels in preterm infants with PDA, but we have even found elevated glutamate concentrations associated with PDA. Nevertheless, despite the high statistical significance, the difference in glutamate levels may lack clinical significance or may be an epiphenomenon associated with the particular clinical condition of infants with PDA.

Up-regulation of astrocyte excitatory amino acid transporter 2 alleviates central sensitization in a rat model of chronic migraine.

Central sensitization is the potential pathogenesis of chronic migraine (CM) and is related to persistent neuronal hyperexcitability. Dysfunction of excitatory amino acid transporter 2 (EAAT2) leads to the accumulation of glutamate in the synaptic cleft, which may contribute to central sensitization by overactivating glutamate N-methyl-D-aspartate receptors and enhancing synaptic plasticity. However, the therapeutic potential of CM by targeting glutamate clearance remains largely unexplored. The purpose of this study was to investigate the role of EAAT2 in CM central sensitization and explore the effect of EAAT2 expression enhancer LDN-212320 in CM rats. The glutamate concentration was measured by high-performance liquid chromatography in a rat model of CM. Then, q-PCR and western blots were performed to detect EAAT2 expression, and the immunoreactivity of astrocytes was detected by immunofluorescence staining. To understand the effect of EAAT2 on central sensitization of CM, mechanical and thermal hyperalgesia and central sensitization-associated proteins were examined after administration of LDN-212320. In addition, the expression of synaptic-associated proteins was examined and Golgi-Cox staining was used to observe the dendritic spine density of trigeminal nucleus caudalis neurons. Also, the synaptic ultrastructure was observed by transmission electron microscope (TEM) to explore the changes of synaptic plasticity. In our study, elevated glutamate concentration and decreased EAAT2 expression were found in the trigeminal nucleus caudalis of CM rats, administration of LDN-212320 greatly up-regulated the protein expression of EAAT2, alleviated hyperalgesia, decreased the concentration of glutamate and the activation of astrocytes. Furthermore, reductions in calcitonin gene-related peptide, substance P(SP), and phosphorylated NR2B were examined after administration of LDN-212320. Moreover evaluation of the synaptic structure, synaptic plasticity-, and central sensitization-related proteins indicated that EAAT2 might participate in the CM central sensitization process by regulating synaptic plasticity. Taken together, up-regulation of EAAT2 expression has a protective effect in CM rats, and LDN-212320 may have clinical therapeutic potential. Cover Image for this issue: https://doi.org/10.1111/jnc.14769.

Hippocampal Pathology in Clinical High-Risk Patients and the Onset of Schizophrenia

BackgroundWe examined neuroimaging-derived hippocampal biomarkers in subjects at clinical high risk (CHR) for psychosis to further characterize the pathophysiology of early psychosis. We hypothesized that glutamate hyperactivity, reflected by increased metabolic activity derived from functional magnetic resonance imaging in the CA1 hippocampal subregion and from proton magnetic resonance spectroscopy–derived hippocampal levels of glutamate/glutamine, represents early hippocampal dysfunction in CHR subjects and is predictive of conversion to syndromal psychosis. MethodsWe enrolled 75 CHR individuals with attenuated positive symptom psychosis-risk syndrome as defined by the Structured Interview for Psychosis-risk Syndromes. We used optimized magnetic resonance imaging techniques to measure 3 validated in vivo pathologies of hippocampal dysfunction—focal cerebral blood volume, focal atrophy, and evidence of elevated glutamate concentrations. All patients were imaged at baseline and were followed for up to 2 years to assess for conversion to psychosis. ResultsAt baseline, compared with control subjects, CHR individuals had high glutamate/glutamine and elevated focal cerebral blood volume on functional magnetic resonance imaging, but only baseline focal hippocampal atrophy predicted progression to syndromal psychosis. ConclusionsThese findings provide evidence that CHR patients with attenuated psychotic symptoms have glutamatergic abnormalities, although only CHR patients who develop syndromal psychosis exhibit focal hippocampal atrophy. Furthermore, these results support the growing evidence that hippocampal dysfunction is an early feature of schizophrenia and related psychotic disorders.

Ceftriaxone- and N-acetylcysteine-induced brain tolerance to ischemia: Influence on glutamate levels in focal cerebral ischemia.

One of the major players in the pathophysiology of cerebral ischemia is disrupted homeostasis of glutamatergic neurotransmission, resulting in elevated extracellular glutamate (Glu) concentrations and excitotoxicity-related cell death. In the brain, Glu concentrations are regulated by Glu transporters, including Glu transporter-1 (GLT-1) and cystine/Glu antiporter (system xc-). Modulation of these transporters by administration of ceftriaxone (CEF, 200 mg/kg, i.p.) or N-acetylcysteine (NAC, 150 mg/kg, i.p.) for 5 days before focal cerebral ischemia may induce brain tolerance to ischemia by significantly limiting stroke-related damage and normalizing Glu concentrations. In the present study, focal cerebral ischemia was induced by 90-minute middle cerebral artery occlusion (MCAO). We compared the effects of CEF and NAC pretreatment on Glu concentrations in extracellular fluid and cellular-specific expression of GLT-1 and xCT with the effects of two reference preconditioning methods, namely, ischemic preconditioning and chemical preconditioning in rats. Both CEF and NAC significantly reduced Glu levels in the frontal cortex and hippocampus during focal cerebral ischemia, and this decrease was comparable with the Glu level achieved with the reference preconditioning strategies. The results of immunofluorescence staining of GLT-1 and xCT on astrocytes, neurons and microglia accounted for the observed changes in extracellular Glu levels to a certain extent. Briefly, after MCAO, the expression of GLT-1 on astrocytes decreased, but pretreatment with CEF seemed to prevent this downregulation. In addition, every intervention used in this study seemed to reduce xCT expression on astrocytes and neurons. The results of this study indicate that modulation of Glu transporter expression may restore Glu homeostasis. Moreover, our results suggest that CEF and NAC may induce brain tolerance to ischemia by influencing GLT-1 and system xc- expression levels. These transporters are presumably good targets for the development of novel therapies for brain ischemia.

Dramatic increases in blood glutamate concentrations are closely related to traumatic brain injury-induced acute lung injury

Traumatic brain injury-induced acute lung injury (TBI-ALI) is a serious complication after brain injury for which predictive factors are lacking. In this study, we found significantly elevated blood glutamate concentrations in patients with TBI or multiple peripheral trauma (MPT), and patients with more severe injuries showed higher blood glutamate concentrations and longer durations of elevated levels. Although the increase in amplitude was similar between the two groups, the duration was longer in the patients with TBI. There were no significant differences in blood glutamate concentrations in the patients with MPT with regard to ALI status, but the blood glutamate levels were significantly higher in the patients with TBI-ALI than in those without ALI. Moreover, compared to patients without ALI, patients with TBI showed a clearly enhanced inflammatory response that was closely correlated with the blood glutamate levels. The blood glutamate concentration was also found to be a risk factor (adjusted odds ratio, 2.229; 95% CI, 1.082–2.634) and was a better predictor of TBI-ALI than the Glasgow Coma Scale (GCS) score. These results indicated that dramatically increased blood glutamate concentrations were closely related to the occurrence of TBI-ALI and could be used as a predictive marker for “at-risk” patients.

Gut microbiome and serum metabolome alterations in obesity and after weight-loss intervention.

Emerging evidence has linked the gut microbiome to human obesity. We performed a metagenome-wide association study and serum metabolomics profiling in a cohort of lean and obese, young, Chinese individuals. We identified obesity-associated gut microbial species linked to changes in circulating metabolites. The abundance of Bacteroides thetaiotaomicron, a glutamate-fermenting commensal, was markedly decreased in obese individuals and was inversely correlated with serum glutamate concentration. Consistently, gavage with B. thetaiotaomicron reduced plasma glutamate concentration and alleviated diet-induced body-weight gain and adiposity in mice. Furthermore, weight-loss intervention by bariatric surgery partially reversed obesity-associated microbial and metabolic alterations in obese individuals, including the decreased abundance of B. thetaiotaomicron and the elevated serum glutamate concentration. Our findings identify previously unknown links between intestinal microbiota alterations, circulating amino acids and obesity, suggesting that it may be possible to intervene in obesity by targeting the gut microbiota.

Phosphorylation of tau at Y18, but not tau-fyn binding, is required for tau to modulate NMDA receptor-dependent excitotoxicity in primary neuronal culture

BackgroundHyperexcitability of neuronal networks can lead to excessive release of the excitatory neurotransmitter glutamate, which in turn can cause neuronal damage by overactivating NMDA-type glutamate receptors and related signaling pathways. This process (excitotoxicity) has been implicated in the pathogenesis of many neurological conditions, ranging from childhood epilepsies to stroke and neurodegenerative disorders such as Alzheimer’s disease (AD). Reducing neuronal levels of the microtubule-associated protein tau counteracts network hyperexcitability of diverse causes, but whether this strategy can also diminish downstream excitotoxicity is less clear.MethodsWe established a cell-based assay to quantify excitotoxicity in primary cultures of mouse hippocampal neurons and investigated the role of tau in exicitotoxicity by modulating neuronal tau expression through genetic ablation or transduction with lentiviral vectors expressing anti-tau shRNA or constructs encoding wildtype versus mutant mouse tau.ResultsWe demonstrate that shRNA-mediated knockdown of tau reduces glutamate-induced, NMDA receptor-dependent Ca2+ influx and neurotoxicity in neurons from wildtype mice. Conversely, expression of wildtype mouse tau enhances Ca2+ influx and excitotoxicity in tau-deficient (Mapt−/−) neurons. Reconstituting tau expression in Mapt−/− neurons with mutant forms of tau reveals that the tau-related enhancement of Ca2+ influx and excitotoxicity depend on the phosphorylation of tau at tyrosine 18 (pY18), which is mediated by the tyrosine kinase Fyn. These effects are most evident at pathologically elevated concentrations of glutamate, do not involve GluN2B–containing NMDA receptors, and do not require binding of Fyn to tau’s major interacting PxxP motif or of tau to microtubules.ConclusionsAlthough tau has been implicated in diverse neurological diseases, its most pathogenic forms remain to be defined. Our study suggests that reducing the formation or level of pY18-tau can counteract excitotoxicity by diminishing NMDA receptor-dependent Ca2+ influx.

Therapeutic targeting of the pathological triad of extrasynaptic NMDA receptor signaling in neurodegenerations.

Activation of extrasynaptic N-methyl-d-aspartate (NMDA) receptors causes neurodegeneration and cell death. The disease mechanism involves a pathological triad consisting of mitochondrial dysfunction, loss of integrity of neuronal structures and connectivity, and disruption of excitation-transcription coupling caused by CREB (cyclic adenosine monophosphate-responsive element-binding protein) shut-off and nuclear accumulation of class IIa histone deacetylases. Interdependency within the triad fuels an accelerating disease progression that culminates in failure of mitochondrial energy production and cell loss. Both acute and slowly progressive neurodegenerative conditions, including stroke, Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease, share increased death signaling by extrasynaptic NMDA receptors caused by elevated extracellular glutamate concentrations or relocalization of NMDA receptors to extrasynaptic sites. Six areas of therapeutic objectives are defined, based on which a broadly applicable combination therapy is proposed to combat the pathological triad of extrasynaptic NMDA receptor signaling that is common to many neurodegenerative diseases.

Brain neurochemical and hemodynamic findings in the NY1DD mouse model of mild sickle cell disease.

To characterize the cerebral profile associated with sickle cell disease (SCD), we used in vivo proton MRI and MRS to quantify hemodynamics and neurochemicals in the thalamus of NY1DD mice, a mild model of SCD, and compared them with wild-type (WT) control mice. Compared with WT mice, NY1DD mice at steady state had elevated cerebral blood flow (CBF) and concentrations of N-acetylaspartate (NAA), glutamate (Glu), alanine, total creatine and N-acetylaspartylglutamate. Concentrations of glutathione (GSH) at steady state showed a negative correlation with BOLD signal change in response to 100% oxygen, a marker for oxidative stress, and mean diffusivity assessed using diffusion-tensor imaging, a marker for edematous inflammation. In NY1DD mice, elevated basal CBF was correlated negatively with [NAA], but positively with concentration of glutamine ([Gln]). Immediately after experimental hypoxia (at reoxygenation after 18hours of 8% O2 ), concentrations of NAA, Glu, GSH, Gln and taurine (Tau) increased only in NY1DD mice. [NAA], [Glu], [GSH] and [Tau] all returned to baseline levels two weeks after the hypoxic episode. The altered neurochemical profile in the NY1DD mouse model of SCD at steady state and following experimental hypoxia/reoxygenation suggests a state of chronic oxidative stress leading to compensatory cerebral metabolic adjustments.

Biomarkers for glaucoma: from the lab to the clinic

Glaucoma, a leading cause of irreversible blindness worldwide, is often not diagnosed until many years after disease onset. Early and objective diagnostic measures are yet missing. Besides the main risk factor, an elevated intraocular pressure (IOP), age, sex, and ethnicity are known to affect disease progression and severity. Furthermore, oxidative stress, elevated glutamate concentrations, and an autoimmune component are considered possible risk factors. We could identify several potential proteomic biomarkers in glaucoma and examine distinct changes in the glaucomatous human retina proteome. Using an experimental autoimmune glaucoma animal (EAG) model we could demonstrate an IOP-independent loss of retinal ganglion cells (RGC), which is accompanied by antibody depositions and increased levels of microglia. In a different animal model we showed that intermittent IOP elevations provoke neurodegeneration in the optic nerve and the retina and elicit changes of IgG autoantibody reactivities. The correlation between neuronal damage and changes in autoantibody reactivity suggests that autoantibody profiling could be a useful biomarker for glaucoma. In vivo studies on neuroretinal cells and porcine retinal explants demonstrated a protective effect of antibodies (eg, anti-GFAP) on RGC, which seems to be the result of reduced stress levels in the retina. We conclude that the absence of some autoantibodies in glaucoma patients reflects a loss of the protective potential of natural autoimmunity and may thus encourage neurodegenerative processes. Concluding, autoantibody profiles resemble useful biomarkers for diagnosis, progression and severity of glaucoma. Future longitudinal studies will help to improve early detection and enable better monitoring of disease progression.

The conversion of glutamate by glutamine synthase in neocortical astrocytes from juvenile rat is important to limit glutamate spillover and peri/extrasynaptic activation of NMDA receptors.

Glutamate transporters (EAATs) are important to maintain spatial and temporal specificity of synaptic transmission. Their efficiency to uptake and transport glutamate into the intracellular space depends on several parameters including the intracellular concentrations of Na+ and glutamate, the elevations of which may slow down the cycling rate of EAATs. In astrocytes, glutamate is maintained at low concentration due to the presence of specific enzymes such as glutamine synthase (GS). GS inhibition results in cytosolic accumulation of glutamate suggesting that the conversion of glutamate by GS is important for EAATs operation. Here we recorded astrocytes from juvenile rat neocortical slices and analyzed the consequences of elevated intracellular glutamate concentrations and of GS inhibition on the time course of synaptically evoked transporter current (STC). In slices from rats treated with methionine sulfoximine (MSO), a GS inhibitor, STC evoked by short burst of high frequency stimulation (HFS; 100 Hz for 100 ms) but not by low frequency stimulation (LFS; 0.1 Hz) was twice slower than STC evoked from saline injected rats. Same results were obtained for astrocytes recorded with pipette containing 3-10 mM glutamate and compared with cells recorded with 0 or1 mM glutamate in the patch pipette. We also showed that HFS elicited significantly larger NMDAR-excitatory postsynaptic currents (EPSCs) with a stronger peri/extrasynaptic component in pyramidal cells from MSO-treated compared with saline treated rats. Taken together our data demonstrate that the conversion of glutamate by GS is fundamental to ensure an efficient clearance of glutamate by EAATs and to prevent glutamate spillover. GLIA 2017;65:401-415.