Computational Investigation of BMAA and Its Carbamate Adducts as Potential GluR2 Modulators.

Beta-N-methylamino-l-alanine (BMAA) is a potential neurotoxic nonprotein amino acid, which can reach the human body through the food chain. When BMAA interacts with bicarbonate in the human body, carbamate adducts are produced, which share a high structural similarity with the neurotransmitter glutamate. It is believed that BMAA and its l-carbamate adducts bind in the glutamate binding site of ionotropic glutamate receptor 2 (GluR2). Chronic exposure to BMAA and its adducts could cause neurological illness such as neurodegenerative diseases. However, the mechanism of BMAA action and its carbamate adducts bound to GluR2 has not yet been elucidated. Here, we investigate the binding modes and the affinity of BMAA and its carbamate adducts to GluR2 in comparison to the natural agonist, glutamate, to understand whether these can act as GluR2 modulators. Initially, we perform molecular dynamics simulations of BMAA and its carbamate adducts bound to GluR2 to examine the stability of the ligands in the S1/S2 ligand-binding core of the receptor. In addition, we utilize alchemical free energy calculations to compute the difference in the free energy of binding of the beta-carbamate adduct of BMAA to GluR2 compared to that of glutamate. Our findings indicate that carbamate adducts of BMAA and glutamate remain stable in the binding site of the GluR2 compared to BMAA. Additionally, alchemical free energy results reveal that glutamate and the beta-carbamate adduct of BMAA have comparable binding affinity to the GluR2. These results provide a rationale that BMAA carbamate adducts may be, in fact, the modulators of GluR2 and not BMAA itself.

Glutamate neurotransmission from leptin receptor cells is required for typical puberty and reproductive function in female mice.

The hypothalamic ventral premammillary nucleus (PMv) is a glutamatergic nucleus essential for the metabolic control of reproduction. However, conditional deletion of leptin receptor long form (LepRb) in vesicular glutamate transporter 2 (Vglut2) expressing neurons results in virtually no reproductive deficits. In this study, we determined the role of glutamatergic neurotransmission from leptin responsive PMv neurons on puberty and fertility. We first assessed if stimulation of PMv neurons induces luteinizing hormone (LH) release in fed adult females. We used the stimulatory form of designer receptor exclusively activated by designer drugs (DREADDs) in LeprCre (LepRb-Cre) mice. We collected blood sequentially before and for 1 hr after intravenous clozapine-N-oxide injection. LH level increased in animals correctly targeted to the PMv, and LH level was correlated to the number of Fos immunoreactive neurons in the PMv. Next, females with deletion of Slc17a6 (Vglut2) in LepRb neurons (LeprΔVGlut2) showed delayed age of puberty, disrupted estrous cycles, increased gonadotropin-releasing hormone (GnRH) concentration in the axon terminals, and disrupted LH secretion, suggesting impaired GnRH release. To assess if glutamate is required for PMv actions in pubertal development, we generated a Cre-induced reexpression of endogenous LepRb (LeprloxTB) with concomitant deletion of Slc17a6 (Vglut2flox) mice. Rescue of Lepr and deletion of Slc17a6 in the PMv was obtained by stereotaxic injection of an adeno-associated virus vector expressing Cre recombinase. Control LeprloxTB mice with PMv LepRb rescue showed vaginal opening, follicle maturation, and became pregnant, while LeprloxTB;Vglut2flox mice showed no pubertal development. Our results indicate that glutamatergic neurotransmission from leptin sensitive neurons regulates the reproductive axis, and that leptin action on pubertal development via PMv neurons requires Vglut2.

Genome-scale metabolic network of human carotid plaque reveals the pivotal role of glutamine/glutamate metabolism in macrophage modulating plaque inflammation and vulnerability.

BACKGROUND: Metabolism is increasingly recognized as a key regulator of the function and phenotype of the primary cellular constituents of the atherosclerotic vascular wall, including endothelial cells, smooth muscle cells, and inflammatory cells. However, a comprehensive analysis of metabolic changes associated with the transition of plaque from a stable to a hemorrhaged phenotype is lacking. METHODS: In this study, we integrated two large mRNA expression and protein abundance datasets (BIKE, n = 126; MaasHPS, n = 43) from human atherosclerotic carotid artery plaque to reconstruct a genome-scale metabolic network (GEM). Next, the GEM findings were linked to metabolomics data from MaasHPS, providing a comprehensive overview of metabolic changes in human plaque. RESULTS: Our study identified significant changes in lipid, cholesterol, and inositol metabolism, along with altered lysosomal lytic activity and increased inflammatory activity, in unstable plaques with intraplaque hemorrhage (IPH+) compared to non-hemorrhaged (IPH-) plaques. Moreover, topological analysis of this network model revealed that the conversion of glutamine to glutamate and their flux between the cytoplasm and mitochondria were notably compromised in hemorrhaged plaques, with a significant reduction in overall glutamate levels in IPH+ plaques. Additionally, reduced glutamate availability was associated with an increased presence of macrophages and a pro-inflammatory phenotype in IPH+ plaques, suggesting an inflammation-prone microenvironment. CONCLUSIONS: This study is the first to establish a robust and comprehensive GEM for atherosclerotic plaque, providing a valuable resource for understanding plaque metabolism. The utility of this GEM was illustrated by its ability to reliably predict dysregulation in the cholesterol hydroxylation, inositol metabolism, and the glutamine/glutamate pathway in rupture-prone hemorrhaged plaques, a finding that may pave the way to new diagnostic or therapeutic measures.

Polycomb repressive complex 2 is critical for mouse cortical glutamatergic neuron development.

The Polycomb Repressive Complex 2 (PRC2) regulates corticogenesis, yet the consequences of mutations to this epigenetic modifier in the mature brain are poorly defined. Importantly, PRC2 core genes are haploinsufficient and causative of several human neurodevelopmental disorders. To address the role of PRC2 in mature cortical structure and function, we conditionally deleted the PRC2 gene Eed from the developing mouse dorsal telencephalon. Adult homozygotes displayed smaller forebrain structures. Single-nucleus transcriptomics revealed that glutamatergic neurons were particularly affected, exhibiting dysregulated gene expression profiles, accompanied by aberrations in neuronal morphology and connectivity. Remarkably, homozygous mice performed well on challenging cognitive tasks. In contrast, while heterozygous mice did not exhibit clear anatomical or behavioral differences, they displayed dysregulation of neuronal genes and altered neuronal morphology that was strikingly different from homozygous phenotypes. Collectively, these data reveal how alterations to PRC2 function shape the mature brain and reveal a dose-specific role for PRC2 in determining glutamatergic neuron identity.

Effects of Cannabis on Glutamatergic Neurotransmission: The Interplay between Cannabinoids and Glutamate.

There has been a significant increase in the consumption of cannabis for both recreational and medicinal purposes in recent years, and its use can have long-term consequences on cognitive functions, including memory. Here, we review the immediate and long-term effects of cannabis and its derivatives on glutamatergic neurotransmission, with a focus on both the presynaptic and postsynaptic alterations. Several factors can influence cannabinoid-mediated changes in glutamatergic neurotransmission, including dosage, sex, age, and frequency of use. Acute exposure to cannabis typically inhibits glutamate release, whereas chronic use tends to increase glutamate release. Conversely, the postsynaptic alterations are more complicated than the presynaptic effects, as cannabis can affect the glutamate receptor expression and the downstream signaling of glutamate. All these effects ultimately influence cognitive functions, particularly memory. This review will cover the current research on glutamate-cannabis interactions, as well as the future directions of research needed to understand cannabis-related health effects and neurological and psychological aspects of cannabis use.

RNA editing regulates glutamatergic synapses in the frontal cortex of a molecular subtype of Amyotrophic Lateral Sclerosis.

BACKGROUND: Amyotrophic Lateral Sclerosis (ALS) is a highly heterogenous neurodegenerative disorder that primarily affects upper and lower motor neurons, affecting additional cell types and brain regions. Underlying molecular mechanisms are still elusive, in part due to disease heterogeneity. Molecular disease subtyping through integrative analyses including RNA editing profiling is a novel approach for identification of molecular networks involved in pathogenesis. METHODS: We aimed to highlight the role of RNA editing in ALS, focusing on the frontal cortex and the prevalent molecular disease subtype (ALS-Ox), previously determined by transcriptomic profile stratification. We established global RNA editing (editome) and gene expression (transcriptome) profiles in control and ALS-Ox cases, utilizing publicly available RNA-seq data (GSE153960) and an in-house analysis pipeline. Functional annotation and pathway analyses identified molecular processes affected by RNA editing alterations. Pearson correlation analyses assessed RNA editing effects on expression. Similar analyses on additional ALS-Ox and control samples (GSE124439) were performed for verification. Targeted re-sequencing and qRT-PCR analysis targeting CACNA1C, were performed using frontal cortex tissue from ALS and control samples (n = 3 samples/group). RESULTS: We identified reduced global RNA editing in the frontal cortex of ALS-Ox cases. Differentially edited transcripts are enriched in synapses, particularly in the glutamatergic synapse pathway. Bioinformatic analyses on additional ALS-Ox and control RNA-seq data verified these findings. We identified increased recoding at the Q621R site in the GRIK2 transcript and determined positive correlations between RNA editing and gene expression alterations in ionotropic receptor subunits GRIA2, GRIA3 and the CACNA1C transcript, which encodes the pore forming subunit of a post-synaptic L-type calcium channel. Experimental data verified RNA editing alterations and editing-expression correlation in CACNA1C, highlighting CACNA1C as a target for further study. CONCLUSIONS: We provide evidence on the involvement of RNA editing in the frontal cortex of an ALS molecular subtype, highlighting a modulatory role mediated though recoding and gene expression regulation on glutamatergic synapse related transcripts. We report RNA editing effects in disease-related transcripts and validated editing alterations in CACNA1C. Our study provides targets for further functional studies that could shed light in underlying disease mechanisms enabling novel therapeutic approaches.

Label-Free Assessment of Neuronal Activity Using Raman Micro-Spectroscopy.

Given the pivotal role of neuronal populations in various biological processes, assessing their collective output is crucial for understanding the nervous system's complex functions. Building on our prior development of a spiral scanning mechanism for the rapid acquisition of Raman spectra from single cells and incorporating machine learning for label-free evaluation of cell states, we investigated whether the Paint Raman Express Spectroscopy System (PRESS) can assess neuronal activities. We tested this hypothesis by examining the chemical responses of glutamatergic neurons as individual neurons and autonomic neuron ganglia as neuronal populations derived from human-induced pluripotent stem cells. The PRESS successfully acquired Raman spectra from both individual neurons and ganglia within a few seconds, achieving a signal-to-noise ratio sufficient for detailed analysis. To evaluate the ligand responsiveness of the induced neurons and ganglia, the Raman spectra were subjected to principal component and partial least squares discriminant analyses. The PRESS detected neuronal activity in response to glutamate and nicotine, which were absent in the absence of calcium. Additionally, the PRESS induced dose-dependent neuronal activity changes. These findings underscore the capability of the PRESS to assess individual neuronal activity and elucidate neuronal population dynamics and pharmacological responses, heralding new opportunities for drug discovery and regenerative medicine advancement.

Metabolomics-based search for lung cancer markers among patients with different smoking status.

Tobacco smoking is the main etiological factor of lung cancer (LC), which can also cause metabolome disruption. This study aimed to investigate whether the observed metabolic shift in LC patients was also associated with their smoking status. Untargeted metabolomics profiling was applied for the initial screening of changes in serum metabolic profile between LC and chronic obstructive pulmonary disease (COPD) patients, selected as a non-cancer group. Differences in metabolite profiles between current and former smokers were also tested. Then, targeted metabolomics methods were applied to verify and validate the proposed LC biomarkers. For untargeted metabolomics, a single extraction-dual separation workflow was applied. The samples were analyzed using a liquid chromatograph-high resolution quadrupole time-of-flight mass spectrometer. Next, the selected metabolites were quantified using liquid chromatography-triple-quadrupole mass spectrometry. The acquired data confirmed that patients' stratification based on smoking status impacted the discriminating ability of the identified LC marker candidates. Analyzing a validation set of samples enabled us to determine if the putative LC markers were truly robust. It demonstrated significant differences in the case of four metabolites: allantoin, glutamic acid, succinic acid, and sphingosine-1-phosphate. Our research showed that studying the influence of strong environmental factors, such as tobacco smoking, should be considered in cancer marker research since it reduces the risk of false positives and improves understanding of the metabolite shifts in cancer patients.

ß-amyloid monomer scavenging by an anticalin protein prevents neuronal hyperactivity in mouse models of Alzheimer's Disease.

Hyperactivity mediated by synaptotoxic ß-amyloid (Aß) oligomers is one of the earliest forms of neuronal dysfunction in Alzheimer's disease. In the search for a preventive treatment strategy, we tested the effect of scavenging Aß peptides before Aß plaque formation. Using in vivo two-photon calcium imaging and SF-iGluSnFR-based glutamate imaging in hippocampal slices, we demonstrate that an Aß binding anticalin protein (Aß-anticalin) can suppress early neuronal hyperactivity and synaptic glutamate accumulation in the APP23xPS45 mouse model of ß-amyloidosis. Our results suggest that the sole targeting of Aß monomers is sufficient for the hyperactivity-suppressing effect of the Aß-anticalin at early disease stages. Biochemical and neurophysiological analyses indicate that the Aß-anticalin-dependent depletion of naturally secreted Aß monomers interrupts their aggregation to neurotoxic oligomers and, thereby, reverses early neuronal and synaptic dysfunctions. Thus, our results suggest that Aß monomer scavenging plays a key role in the repair of neuronal function at early stages of AD.

[Exogenous leptin improves cerebral ischemia-reperfusion-induced glutamate excitotoxic injury in mice by up-regulating GLT-1 and GLAST expression in astrocytes].

OBJECTIVE: To investigate the protective effect of exogenous leptin against focal cerebral ischemia-reperfusion (I/R) injury in mice and explore the underlying mechanism. METHODS: A total of 100 C57BL/6 mice were randomly divided into 5 groups, including a sham-operated group, cerebral I/R model group, and 3 leptin treatment groups with intraperitoneal injections of 0.5, 1.0 or 2.0 leptin immediately after occlusion of the internal carotid artery. At 24 h after reperfusion, neurological function scores of the mice were assessed, and TTC staining was used to determine the area of cerebral infarction. The pathological changes in the cortical brain tissue of the mice were observed using HE staining, and degenerative damage of the cortical neurons were assessed with Fluoro-Jade C staining. The expression of glial fibrillary acidic protein in cortical brain tissues was detected using immunohistochemistry and Western blotting. In another 45 C57BL/6 mice with sham operation, I/R modeling, or leptin (1 mg/kg) treatment, glutamic acid in the cortical brain tissue was detected using glutamate assay, and cortical glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) protein expressions were detected using immunohistochemistry. RESULTS: Compared with the I/R model mice, the leptin-treated mice had significantly lower neurological deficit scores, smaller cerebral infarct area, milder pathologies in the cortical brain tissue, and lessened cortical neuronal damage with normal morphology and less excessive proliferation of the astrocytes. Leptin treatment significantly up-regulated the expressions of GLT-1 and GLAST and lowered the content of glutamic acid in the brain tissue of the I/R mice. CONCLUSION: Exogenous leptin has obvious neuroprotective effect against cerebral I/R injury in mice, mediated probably by controlling excessive astrocyte proliferation and up-regulating cortical GLT-1 and GLAST expressions to reduce glutamate-mediated excitotoxic injury of the astrocytes.

The human VGLUT3-pT8I mutation elicits uneven striatal DA signaling, food or drug maladaptive consumption in male mice.

Cholinergic striatal interneurons (ChIs) express the vesicular glutamate transporter 3 (VGLUT3) which allows them to regulate the striatal network with glutamate and acetylcholine (ACh). In addition, VGLUT3-dependent glutamate increases ACh vesicular stores through vesicular synergy. A missense polymorphism, VGLUT3-p.T8I, was identified in patients with substance use disorders (SUDs) and eating disorders (EDs). A mouse line was generated to understand the neurochemical and behavioral impact of the p.T8I variant. In VGLUT3T8I/T8I male mice, glutamate signaling was unchanged but vesicular synergy and ACh release were blunted. Mutant male mice exhibited a reduced DA release in the dorsomedial striatum but not in the dorsolateral striatum, facilitating habit formation and exacerbating maladaptive use of drug or food. Increasing ACh tone with donepezil reversed the self-starvation phenotype observed in VGLUT3T8I/T8I male mice. Our study suggests that unbalanced dopaminergic transmission in the dorsal striatum could be a common mechanism between SUDs and EDs.

Mass spectrometry-based metabolomics study of nicotine exposure in THP-1 monocytes.

The tobacco alkaloid nicotine is known for its activation of neuronal nicotinic acetylcholine receptors. Nicotine is consumed in different ways such as through conventional smoking, e-cigarettes, snuff or nicotine pouches. The use of snuff has been associated with several adverse health effects, such as inflammatory reactions of the oral mucosa and oral cavity cancer. We performed a metabolomic analysis of nicotine-exposed THP-1 human monocytes. Cells were exposed to 5 mM of the alkaloid for up to 4 h, and cell extracts and medium subjected to untargeted liquid chromatography high-resolution mass spectrometry. Raw data processing revealed 17 nicotine biotransformation products. Among these, cotinine and nornicotine were identified as the two major cellular biotransformation products. The application of multi- and univariate statistical analyses resulted in the annotation, up to a certain level of identification, of 12 compounds in the cell extracts and 13 compounds in the medium that were altered by nicotine exposure. Of these, four were verified as methylthioadenosine, cytosine, uric acid, and L-glutamate. Methylthioadenosine levels were affected in both cells and the medium, while cytosine, uric acid, and L-glutamate levels were affected in the medium only. The effects of smoking on the pathways involving these metabolites have been previously demonstrated in humans. Most of the other discriminating compounds, which were merely tentatively or not fully identified, were amino acids or amino acid derivatives. In conclusion, our preliminary data suggest that some of the potentially adverse effects related to smoking may also be expected when nicotine is consumed via snuff or nicotine pouches.

Effect of Dietary L-Theanine on Protein Expression in the Hippocampus of Senescence-Accelerated Mice (SAMP8).

L-Theanine is contained in green tea at 1-3% per dry matter as an amino acid with an umami taste, and the antidepressant effect and protective effect against stress-induced brain atrophy in mice, as well as the related mechanism have been reported. However, effects of theanine on the hippocampus from the proteome analysis and the action mechanism have not been examined. In this study, we mainly investigated the possibility of theanine's cognitive impairment-preventing function and the action mechanism by proteomics in the hippocampus of SAMP8 administered with theanine. In addition to improvement in the aging score with theanine administration, in proteomics, significant suppressions in the expressions of synapsin 2, α-synuclein, ß-synuclein, and protein tau were observed by theanine administration, and the expression of CAM kinase II beta and alpha exhibited a significant increase and increasing tendency with theanine administration, respectively. The expression of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein tended to increase by theanine administration. On the other hand, serotonin/tryptophan, GABA/glutamic acid and glutamine/glutamic acid ratios in the hippocampus showed an increasing tendency, a significant increase, and an increasing tendency with theanine administration, respectively. These results suggested that theanine might have been involved in the improvement of neurodegeneration or cognitive impairment by suppressing the productions of synapsin, synuclein and protein tau which are considered to be produced along with aging and oxidation, and by enhancing the production of serotonin by increasing the expression of CAM kinase II, and further by affecting the metabolism of glutamate.

Influence of the glutamate-glutamine cycle on valproic acid-associated hepatotoxicity in pediatric patients with epilepsy.

INTRODUCTION: Although valproic acid is generally well tolerated, hepatotoxicity is a common side effect in patients receiving long-term treatment. However, the mechanisms underlying valproic acid-associated hepatotoxicity remain elusive. METHODS: To investigate the mechanisms and explore the potential risk factors for valproic acid-associated hepatotoxicity, 165 age-matched pediatric patients were recruited for laboratory tests and glutamate-glutamine cycle analysis. RESULTS: The concentration of glutamate in patients with hepatotoxicity was significantly greater than that in control patients, while the concentration of glutamine in patients with hepatotoxicity was significantly lower than that in control patients (P <0.05). In addition, the frequencies of the heterozygous with one mutant allele and homozygous with two mutant alleles genotypes in glutamate-ammonia ligase rs10911021 were significantly higher in the hepatotoxicity group than those in the control group (47.1 percent versus 32.5 percent, P = 0.010; 17.6 percent versus 5.2 percent, P = 0.001, respectively). Moreover, heterozygous carriers with one mutant allele and homozygous carriers with two mutant alleles genotypes of glutamate-ammonia ligase rs10911021 exhibited significant differences in the concentrations of glutamine and glutamate concentrations (P ˂ 0.001 and P = 0.001, respectively) and liver function indicators (activities of aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transferase, P <0.001, respectively). Furthermore, logistic regression analysis indicated that glutamate-ammonia ligase rs10911021 (P = 0.002, odds ratio: 3.027, 95 percent confidence interval, 1.521 - 6.023) and glutamate (P = 0.001, odds ratio: 2.235, 95 percent confidence interval, 1.369 - 3.146) were associated with a greater risk for hepatotoxicity, while glutamine concentrations were negatively associated with hepatotoxicity (P = 0.001, odds ratio: 0.711, 95 percent confidence interval, 0.629 - 0.804). DISCUSSION: Understanding pharmacogenomic risks for valproic acid induced hepatotoxicity might help direct patient specific care. Limitations of our study include the exclusive use of children from one location and concomitant medication use in many patients. CONCLUSION: Perturbation of the glutamate-glutamine cycle is associated with valproic acid-associated hepatotoxicity. Moreover, glutamate-ammonia ligase rs10911021, glutamate and glutamine concentrations are potential risk factors for valproic acid-associated hepatotoxicity.

Association of Cortical Lesions With Regional Glutamate, GABA, N-Acetylaspartate, and Myoinositol Levels in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Cortical lesions contribute to disability in multiple sclerosis (MS), but their impact on regional neurotransmitter levels remains to be clarified. We tested the hypothesis that cortical lesions are associated with regional glutamate and gamma-aminobutyric acid (GABA) concentrations within the affected cortical region. METHODS: In this cross-sectional study, we used structural 7T MRI to segment cortical lesions and 7T proton MR-spectroscopy of the bilateral sensorimotor hand areas to quantify regional GABA, glutamate, N-acetylaspartate, and myoinositol concentrations in patients with MS (inclusion criteria: diagnosis of relapsing-remitting [RR] or secondary progressive MS [SPMS]; age 18-80 years) and age and sex-matched healthy controls. Data were collected at a single center between August 2018 and September 2020. Linear mixed-effects models were used to test for associations between metabolite concentrations and cortical lesion volumes within the same MR-spectroscopy voxel. RESULTS: Forty-seven patients with MS (34 RRMS, 13 SPMS; 45.1 ± 12.5 years; 31 women) and 23 healthy controls (44.4 ± 13 years, 15 women) were studied. In patients, higher regional glutamate and lower regional GABA concentrations were associated with larger cortical lesion volume within the MR-spectroscopy voxel [glutamate: 0.61 (95% CI 0.19-1.03) log(mm3), p = 0.005, GABA: -0.71 (-1.24 to -0.18) log(mm3), p = 0.01]. In addition, lower N-acetylaspartate levels [-0.37 (-0.67 to -0.07) log(mm3), p = 0.016] and higher myoinositol levels [0.48 (0.03-0.93) log(mm3), p = 0.037] were associated with a larger regional cortical lesion volume. Furthermore, glutamate concentrations were reduced in patients with SPMS compared with healthy participants [-0.75 (-1.3 to -0.19) mM, p = 0.005] and patients with RRMS [-0.55 (-1.07 to -0.02) mM, p = 0.04]. N-acetylaspartate levels were lower in both patients with RRMS [-0.81 (-1.39 to -0.24) mM, p = 0.003] and SPMS [-1.31 (-2.07 to -0.54) mM, p < 0.001] when compared with healthy controls. Creatine-normalized N-acetylaspartate levels were associated with performance in the 9-hole peg test of the contralateral hand [-0.004 (-0.007 to -0.002) log(s), p = 0.002], and reduced mean creatine-normalized glutamate was associated with increased Expanded Disability Status Scale (R = -0.39, p = 0.02). DISCUSSION: Cortical lesions are associated with local increases in glutamate and a reduction in GABA concentration within the lesional or perilesional tissue. Further studies are needed to investigate the causal relationship between cortical lesions and changes in neurotransmitter concentrations.

Spectrophotometric Assays for Measuring Photorespiratory Glutamate:Glyoxylate and Serine:Glyoxylate Aminotransferase Reactions.

Glutamate:glyoxylate aminotransferase (GGAT; EC 2.6.1.4) and serine:glyoxylate aminotransferase activities (SGAT; EC 2.6.1.45) are central photorespiratory reactions within plant peroxisomes. Both enzymatic reactions convert glyoxylate, a product of glycolate oxidase, to glycine, a substrate of the mitochondrial glycine decarboxylase complex. The GGAT reaction uses glutamate as an amino group donor and also produces α-ketoglutarate, which is recycled to glutamate in plastids by ferredoxin-dependent glutamate synthase. Using serine, a product of mitochondrial serine hydroxymethyltransferase, as an amino group donor, the SGAT reaction also produces hydroxypyruvate, a substrate of hydroxypyruvate reductase. The activities of these photorespiratory aminotransferases can be measured using indirect, coupled, spectrophotometric assays, detailed herein.

Enhanced long-term potentiation in the anterior cingulate cortex of tree shrew.

Synaptic plasticity is a key cellular model for learning, memory and chronic pain. Most previous studies were carried out in rats and mice, and less is known about synaptic plasticity in non-human primates. In the present study, we used integrative experimental approaches to study long-term potentiation (LTP) in the anterior cingulate cortex (ACC) of adult tree shrews. We found that glutamate is the major excitatory transmitter and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid (AMPA) receptors mediate postsynaptic responses. LTP in tree shrews was greater than that in adult mice and lasted for at least 5 h. N-methyl-d-aspartic acid (NMDA) receptors, Ca2+ influx and adenylyl cyclase 1 (AC1) contributed to tree shrew LTP. Our results suggest that LTP is a major form of synaptic plasticity in the ACC of primate-like animals. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Glutamatergic supramammillary nucleus neurons respond to threatening stressors and promote active coping.

Threat-response neural circuits are conserved across species and play roles in normal behavior and psychiatric diseases. Maladaptive changes in these neural circuits contribute to stress, mood, and anxiety disorders. Active coping in response to stressors is a psychosocial factor associated with resilience against stress-induced mood and anxiety disorders. The neural circuitry underlying active coping is poorly understood, but the functioning of these circuits could be key for overcoming anxiety and related disorders. The supramammillary nucleus (SuM) has been suggested to be engaged by threat. SuM has many projections and a poorly understood diversity of neural populations. In studies using mice, we identified a unique population of glutamatergic SuM neurons (SuMVGLUT2+::POA) based on projection to the preoptic area of the hypothalamus (POA) and found SuMVGLUT2+::POA neurons have extensive arborizations. SuMVGLUT2+::POA neurons project to brain areas that mediate features of the stress and threat responses including the paraventricular nucleus thalamus (PVT), periaqueductal gray (PAG), and habenula (Hb). Thus, SuMVGLUT2+::POA neurons are positioned as a hub, connecting to areas implicated in regulating stress responses. Here we report SuMVGLUT2+::POA neurons are recruited by diverse threatening stressors, and recruitment correlated with active coping behaviors. We found that selective photoactivation of the SuMVGLUT2+::POA population drove aversion but not anxiety like behaviors. Activation of SuMVGLUT2+::POA neurons in the absence of acute stressors evoked active coping like behaviors and drove instrumental behavior. Also, activation of SuMVGLUT2+::POA neurons was sufficient to convert passive coping strategies to active behaviors during acute stress. In contrast, we found activation of GABAergic (VGAT+) SuM neurons (SuMVGAT+) neurons did not alter drive aversion or active coping, but termination of photostimulation was followed by increased mobility in the forced swim test. These findings establish a new node in stress response circuitry that has projections to many brain areas and evokes flexible active coping behaviors.

Hyperactivation of MEK1 in cortical glutamatergic neurons results in projection axon deficits and aberrant motor learning.

Abnormal extracellular signal-regulated kinase 1/2 (ERK1/2, encoded by Mapk3 and Mapk1, respectively) signaling is linked to multiple neurodevelopmental diseases, especially the RASopathies, which typically exhibit ERK1/2 hyperactivation in neurons and non-neuronal cells. To better understand how excitatory neuron-autonomous ERK1/2 activity regulates forebrain development, we conditionally expressed a hyperactive MEK1 (MAP2K1) mutant, MEK1S217/221E, in cortical excitatory neurons of mice. MEK1S217/221E expression led to persistent hyperactivation of ERK1/2 in cortical axons, but not in soma/nuclei. We noted reduced axonal arborization in multiple target domains in mutant mice and reduced the levels of the activity-dependent protein ARC. These changes did not lead to deficits in voluntary locomotion or accelerating rotarod performance. However, skilled motor learning in a single-pellet retrieval task was significantly diminished in these MEK1S217/221E mutants. Restriction of MEK1S217/221E expression to layer V cortical neurons recapitulated axonal outgrowth deficits but did not affect motor learning. These results suggest that cortical excitatory neuron-autonomous hyperactivation of MEK1 is sufficient to drive deficits in axon outgrowth, which coincide with reduced ARC expression, and deficits in skilled motor learning. Our data indicate that neuron-autonomous decreases in long-range axonal outgrowth may be a key aspect of neuropathogenesis in RASopathies.

CD3ζ-Mediated Signaling Protects Retinal Ganglion Cells in Glutamate Excitotoxicity of the Retina.

Excessive levels of glutamate activity could potentially damage and kill neurons. Glutamate excitotoxicity is thought to play a critical role in many CNS and retinal diseases. Accordingly, glutamate excitotoxicity has been used as a model to study neuronal diseases. Immune proteins, such as major histocompatibility complex (MHC) class I molecules and their receptors, play important roles in many neuronal diseases, while T-cell receptors (TCR) are the primary receptors of MHCI. We previously showed that a critical component of TCR, CD3ζ, is expressed by mouse retinal ganglion cells (RGCs). The mutation of CD3ζ or MHCI molecules compromises the development of RGC structure and function. In this study, we investigated whether CD3ζ-mediated molecular signaling regulates RGC death in glutamate excitotoxicity. We show that mutation of CD3ζ significantly increased RGC survival in NMDA-induced excitotoxicity. In addition, we found that several downstream molecules of TCR, including Src (proto-oncogene tyrosine-protein kinase) family kinases (SFKs) and spleen tyrosine kinase (Syk), are expressed by RGCs. Selective inhibition of an SFK member, Hck, or Syk members, Syk or Zap70, significantly increased RGC survival in NMDA-induced excitotoxicity. These results provide direct evidence to reveal the underlying molecular mechanisms that control RGC death under disease conditions.