Role In Glutamate Metabolism Research Articles

The role of glutamate metabolism in neuronal excitotoxicity

The role of glutamate metabolism in neuronal excitotoxicity

Abstract 287: Novel p62 interaction suggests novel role in glutamate metabolism

Abstract Prostate cancer (PCa) is the second most common cause of cancer-related death among American men. Androgen Receptor (AR) transcriptional activity is required for PCa tumor growth. Thus, androgen deprivation therapy (ADT) is a first line PCa therapy. However, 10-20% of patients will inevitably develop lethal castration-resistant PCa (CRPC). Thus, we must uncover the mechanisms that drive CPRC. Chronic inflammation is a known hallmark of cancer initiation and progression. Using the LNCaP human PCa cell line, we have shown that chronic exposure to the inflammatory cytokine, interleukin-1 (IL-1) can transform castration-sensitive PCa cells into castration-resistant PCa cells. IL-1 is elevated in PCa patient tissue and serum and is associated with disease progression and metastasis. IL-1 is clinically relevant, but the role of IL-1 in CRPC development is not fully elucidated. To mimic ADT, we serum-starved LNCaP and the chronic IL-1 sublines, LNas1 and LNbs1, and performed mass spectrometry to identify chronic IL-1-induced changes in protein-protein interactions. Specifically, we looked at p62 protein interactors. p62 is a multi-domain, multifunctional pro-survival protein that mediates autophagic flux in response to cellular stress and serum starvation, promotes NF-κB inflammatory signaling and induces NRF2 antioxidant signaling. We discovered that p62 forms a constitutive interaction with KEAP1 under serum starved and normal growth conditions in the chronic IL-1 sublines. KEAP1 inhibits NRF2 nuclear translocation. In response to antioxidant stress, p62 binds and sequesters KEAP1 from NRF2 to activate NFR2 signaling. Surprisingly, we do not observe constitutive canonical NRF2 signaling in the chronic IL-1 sublines. However, in response to serum starvation, we do see both high basal levels and upregulation, specifically, of the NRF2-induced gene, GCLC. GCLC is a glutamate-cysteine ligase that synthesizes the antioxidant, glutathione. Therefore, using mass spectrometry and immunoprecipitation western blotting, we looked at p62 interactions under serum starvation conditions. While we did not find p62 interaction with GCLC in the chronic IL-1 sublines, we do find that p62 interacts with GLUL. GLUL is a glutamate-ammonia ligase that plays a role in cellular detoxification. These data suggests that chronic IL-1 exposure induces novel p62 interactions that have a role in glutamate metabolism. Citation Format: Haley Christine Dahl, Anisha R. Konakalla, Preethi Kanumuri, Basir Mansoor, Anastasia Kelley, Riya Shipurkar, Nikki A. Delk. Novel p62 interaction suggests novel role in glutamate metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 287.

Axon-terminals expressing EAAT2 (GLT-1; Slc1a2) are common in the forebrain and not limited to the hippocampus

The excitatory amino acid transporter type 2 (EAAT2) represents the major mechanism for removal of extracellular glutamate. In the hippocampus, there is some EAAT2 in axon-terminals, whereas most of the protein is found in astroglia. The functional importance of the neuronal EAAT2 is unknown, and it is debated whether EAAT2-expressing nerve terminals are present in other parts of the brain. Here we selectively deleted the EAAT2 gene in neurons (by crossing EAAT2-flox mice with synapsin 1-Cre mice in the C57B6 background). To reduce interference from astroglial EAAT2, we measured glutamate accumulation in crude tissue homogenates. EAAT2 proteins levels were measured by immunoblotting. Although synapsin 1-Cre mediated gene deletion only reduced the forebrain tissue content of EAAT2 protein to 95.5 ± 3.4% of wild-type (littermate) controls, the glutamate accumulation in homogenates of neocortex, hippocampus, striatum and thalamus were nevertheless diminished to, respectively, 54 ± 4, 46 ± 3, 46 ± 2 and 65 ± 7% of controls (average ± SEM, n = 3 pairs of littermates). GABA uptake was unaffected. After injection of U-13C-glucose, lack of neuronal EAAT2 resulted in higher 13C-labeling of glutamine and GABA in the hippocampus suggesting that neuronal EAAT2 is partly short-circuiting the glutamate-glutamine cycle in wild-type mice. Crossing synapsin 1-Cre mice with Ai9 reporter mice revealed that Cre-mediated excision occurred efficiently in hippocampus CA3, but less efficiently in other regions and hardly at all in the cerebellum. Conclusions: (1) EAAT2 is expressed in nerve terminals in multiple brain regions. (2) The uptake catalyzed by neuronal EAAT2 plays a role in glutamate metabolism, at least in the hippocampus. (3) Synapsin 1-Cre does not delete floxed genes in all neurons, and the contribution of neuronal EAAT2 is therefore likely to be larger than revealed in the present study.

Commentary: Systemic, Local, and Imaging Biomarkers of Brain Injury: More Needed, and Better Use of Those Already Established?

Commentary: Systemic, Local, and Imaging Biomarkers of Brain Injury: More Needed, and Better Use of Those Already Established?

Re-programming CHO cell metabolism using miR-23 tips the balance towards a highly productive phenotype.

microRNA engineering of CHO cells has already proved successful in enhancing various industrially relevant phenotypes and producing various recombinant products. A single miRNA's ability to interact with multiple mRNA targets allows their regulatory capacity to extend to processes such as cellular metabolism. Various metabolic states have previously been associated with particular CHO cell phenotypes such as glycolytic or oxidative metabolism accommodating growth and productivity, respectively. miR-23 has previously been demonstrated to play a role in glutamate metabolism resulting in enhanced oxidative phosphorylation through the TCA cycle. Re-programming cellular bioenergetics through miR-23 could tip the balance, forcing mammalian production cells to be more productive by favoring metabolic channelling into oxidative metabolism. CHO clones depleted of miR-23 using a miR-sponge decoy demonstrated an average ∼three-fold enhanced specific productivity with no impact on cell growth. Using a cell respirometer, mitochondrial activity was found to be enhanced by ∼30% at Complex I and II of the electron transport system. Additionally, label-free proteomic analysis uncovered various potential novel targets of miR-23 including LE1 and IDH1, both implicated in oxidative metabolism and mitochondrial activity. These results demonstrate miRNA-based engineering as a route to re-programming cellular metabolism resulting in increased productivity, without affecting growth.

New insights into the role of the branched-chain aminotransferase proteins in the human brain.

The human cytosolic branched-chain aminotransferase (hBCATc) enzyme is strategically located in glutamatergic neurons, where it is thought to provide approximately 30% of de novo nitrogen for brain glutamate synthesis. In health, glutamate plays a dominant role in facilitating learning and memory. However, in patients with Alzheimer's disease (AD), synaptic levels of glutamate become toxic, resulting in a direct increase in postsynaptic neuronal calcium, causing a cascade of events that contributes to the destruction of neuronal integrity and cell death, pathological features of AD. Our group is the first to map the hBCAT proteins to the human brain, where cell-specific compartmentation indicates key roles for these proteins in regulating glutamate homeostasis. Moreover, increased expression of hBCAT was observed in the brains of patients with AD relative to matched controls. We reflect on the importance of the redox-active CXXC motif, which confers novel roles for the hBCAT proteins, particularly with respect to substrate channeling and protein folding. This implies that, in addition to their role in glutamate metabolism, these proteins have additional functional roles that might impact redox cell signaling. This review discusses how these proteins behave as potential neuroprotectors during periods of oxidative stress. These findings are particularly important because an increase in misfolded proteins, linked to increased oxidative stress, occurs in several neurodegenerative conditions. Together, these studies give an overview of the diverse role that these proteins play in brain metabolism, in which a dysregulation of their expression may contribute to neurodegenerative conditions such as AD.

Time-dependent effects of haloperidol on glutamine and GABA homeostasis and astrocyte activity in the rat brain

Schizophrenia is a severe, persistent, and fairly common mental illness. Haloperidol is widely used and is effective against the symptoms of psychosis seen in schizophrenia. Chronic oral haloperidol administration decreased the number of astrocytes in the parietal cortex of macaque monkeys (Konopaske et al., Biol Psych 63:759-765, 2008). Since astrocytes play a key role in glutamate metabolism, chronic haloperidol administration was hypothesized to modulate astrocyte metabolic function and glutamate homeostasis. This study investigated the effects of chronic haloperidol administration on astrocyte metabolic activity and glutamate, glutamine, and GABA homeostasis. We used ex vivo ¹³C magnetic resonance spectroscopy along with high-performance liquid chromatography after [1-¹³C]glucose and [1,2-¹³C]acetate administration to analyze forebrain tissue from rats administered oral haloperidol for 1 or 6 months. Administration of haloperidol for 1 month produced no changes in ¹³C labeling of glutamate, glutamine, or GABA, or in their total levels. However, a 6-month haloperidol administration increased ¹³C labeling of glutamine by [1,2-¹³C]acetate. Moreover, total GABA levels were also increased. Haloperidol administration also increased the acetate/glucose utilization ratio for glutamine in the 6-month cohort. Chronic haloperidol administration in rats appears to increase forebrain GABA production along with astrocyte metabolic activity. Studies exploring these processes in subjects with schizophrenia should take into account the potential confounding effects of antipsychotic medication treatment.

The role of glia in late-life depression

Late-life depression (LLD) has a complex and multifactoral etiology. There is growing interest in elucidating how glia, acting alone or as part of a glial-neuronal network, may contribute to the pathophysiology of depression. In this paper, we explore results from neuroimaging studies showing gray-matter volume loss in key frontal and subcortical structures implicated in LLD, and present the few histological studies that have examined neuronal and glial densities in these regions. Compared to results in younger people with depression, there appear to be age-dependent differences in neuronal pathology but the changes in glial pathology may be more subtle, perhaps reflecting a longer-term compensatory gliosis to earlier damage. We then consider the mechanisms by which both astrocytes and microglia may mediate and modulate neuronal dysfunction and possible degeneration in depression. These include a critical role in the response to peripheral inflammation and central microglial activation, as well as a key role in glutamate metabolism. Advances in our understanding of glia are highlighted, including the role of microglia as "electricians" of the brain and astrocytes as key communicating cells, an integral part of the tripartite synapse. Finally, implications for clinicians are discussed, including the consideration of glia as biomarkers for LLD and incorporation of glia into future therapeutic strategies.

The neuroprotective effect of two statins: simvastatin and pravastatin on a streptozotocin-induced model of Alzheimer’s disease in rats

Astrocytes play a fundamental role in glutamate metabolism by regulating the extracellular levels of glutamate and intracellular levels of glutamine. They also participate in antioxidant defenses, due to the synthesis of glutathione, coupled to glutamate metabolism. Although the cause of Alzheimer's disease (AD) remains elusive, some changes in neurochemical parameters, such as glutamate uptake, glutamine synthetase activity and glutathione have been investigated in this disease. A possible neuroprotective effect of two statins, simvastatin and pravastatin (administered p.o.), was evaluated using a model of dementia, based on the intracerebroventricular (ICV) administration of streptozotocin (STZ), and astrocyte parameters were determined. We confirmed a cognitive deficit in rats submitted to ICV-STZ, and a prevention of this deficit by statin administration. Moreover, both statins were able to prevent the decrease in glutathione content and glutamine synthetase activity in this model of AD. Interestingly, simvastatin increased per se glutamate uptake activity, while both statins increased glutamine synthetase activity per se. These results support the idea that these drugs could be effective for the prevention of alterations observed in the STZ dementia model and may contribute to reduce the cognitive impairment and brain damage observed in AD patients.

Distribution of radiolabeled l-glutamate and d-aspartate from blood into peripheral tissues in naive rats: Significance for brain neuroprotection

Excess l-glutamate (glutamate) levels in brain interstitial and cerebrospinal fluids (ISF and CSF, respectively) are the hallmark of several neurodegenerative conditions such as stroke, traumatic brain injury or amyotrophic lateral sclerosis. Its removal could prevent the glutamate excitotoxicity that causes long-lasting neurological deficits. As in previous studies, we have established the role of blood glutamate levels in brain neuroprotection, we have now investigated the contribution of the peripheral organs to the homeostasis of glutamate in blood. We have administered naive rats with intravenous injections of either l-[1- 14C] Glutamic acid ( l-[1- 14C] Glu), l-[G- 3H] Glutamic acid ( l-[G- 3H] Glu) or d-[2,3- 3H] Aspartic acid ( d-[2,3- 3H] Asp), a non-metabolized analog of glutamate, and have followed their distribution into peripheral organs. We have observed that the decay of the radioactivity associated with l-[1- 14C] Glu and l-[G- 3H] Glu was faster than that associated with glutamate non-metabolized analog, d-[2,3- 3H] Asp. l-[1- 14C] Glu was subjected in blood to a rapid decarboxylation with the loss of 14CO 2. The three major sequestrating organs, serving as depots for the eliminated glutamate and/or its metabolites were skeletal muscle, liver and gut, contributing together 92% or 87% of total l-[U- 14C] Glu or d-[2,3- 3H] Asp radioactivity capture. l-[U- 14C] Glu and d-[2,3- 3H] Asp showed a different organ sequestration pattern. We conclude that glutamate is rapidly eliminated from the blood into peripheral tissues, mainly in non-metabolized form. The liver plays a central role in glutamate metabolism and serves as an origin for glutamate metabolites that redistribute into skeletal muscle and gut. The findings of this study suggest now that pharmacological manipulations that reduce the liver glutamate release rate or cause a boosting of the skeletal muscle glutamate pumping rate are likely to cause brain neuroprotection.

1, 2‐13C]‐Acetate metabolism in glutamate dehydrogenase mutants of Saccharomyces cerevisiae

Glutamate dehydrogenases (GDH) interconvert 2‐oxoglutarate and glutamate. In yeast, NADP‐dependent enzymes, encoded by GDH1 and GDH3, are reported to synthesize glutamate from 2‐oxoglutarate, while an NAD‐dependent enzyme, encoded by GDH2, catalyzes the reverse. Cells were grown in nonfermentable acetate/raffinose (Ace/Raf) to examine the role(s) of these enzymes during aerobic metabolism. The doubling time of wt, gdh2Δ, and gdh3Δ cells was comparable at ~ 4 hours. NADP‐dependent GDH activity (Gdh1p + Gdh3p) in wt, gdh2Δ, and gdh3Δ was decreased ~80% and NAD‐dependent activity (Gdh2p) in wt and gdh3Δ was increased ~20‐fold in Ace/Raf as compared to glucose. Cells carrying the gdh1Δ allele did not grow in Ace/Raf, yet both the NADP‐dependent (Gdh3p) and NAD‐dependent (Gdh2p) GDH activity was ~3‐fold higher. Metabolism of [1, 2‐13C]‐acetate and analysis of carbon NMR spectra was used to examine glutamate metabolism. Analysis of 13C‐isotopmers and enrichment of glutamate carbons indicate a decreased rate of glutamate biosynthesis from acetate in gdh2Δ and gdh3Δ strains as compared to wt. Enrichment of 13C of glutamate was nearly undetectable in gdh1Δ cells, reflecting a GDH activity at < 15% of wt. These data suggest that Gdh1p is the primary GDH enzyme, even under nonfermentative conditions, and Gdh2p and Gdh3p play a noticeable role in glutamate metabolism. (Supported by NIH grant GM069372 [P. J. T.])

Relationships between glutamine, glutamate, and GABA in nerve endings under Pb-toxicity conditions

Glutamine (Gln), glutamate (Glu) and γ-amino butyric acid (GABA) are essential amino acids for brain metabolism and function. Astrocytic-derived glutamine is the precursor of the two most important neurotransmitters: glutamate, an excitatory neurotransmitter, and GABA, an inhibitory neurotransmitter. In addition to their roles in neurotransmission these neurotransmitters act as alternative metabolic substrates that enable metabolic coupling between astrocytes and neurons. The relationships between Gln, Glu and GABA were studied under lead (Pb) toxicity conditions using synaptosomal fractions obtained from adult rat brains to investigate the cause of Pb neurotoxicity-induced seizures. We have found that diminished transport of [ 14C]GABA occurs after Pb treatment. Both uptake and depolarization-evoked release decrease by 40% and 30%, respectively, relative to controls. Lower expression of glutamate decarboxylase (GAD), the GABA synthesizing enzyme, is also observed. In contrast to impaired synaptosomal GABA function, the GABA transporter GAT-1 protein is overexpressed (possibly as a compensative mechanism). Furthermore, similar decreases in synaptosomal uptake of radioactive glutamine and glutamate are observed. However, the K +-evoked release of Glu increases by 20% over control values and the quantity of neuronal EAAC1 transporter for glutamate reaches remarkably higher levels after Pb treatment. In addition, Pb induces decreased activity of phosphate-activated glutaminase (PAG), which plays a role in glutamate metabolism. Most noteworthy is that the overexpression and reversed action of the EAAC1 transporter may be the cause of the elevated extracellular glutamate levels. In addition to the impairment of synaptosomal processes of glutamatergic and GABAergic transport, the results indicate perturbed relationships between Gln, Glu and GABA that may be the cause of altered neuronal–astrocytic interactions under conditions of Pb neurotoxicity.