Journal of Neurochemistry

Neale, Joseph H; Bzdega, Tomasz; Wroblewska, Barbara

doi: 10.1046/j.1471-4159.2000.0750443.xpmid: 10899918

In the progress of science, as in life, timing is important. The acidic dipeptide, N‐acetylaspartylglutamate (NAAG), was discovered in the mammalian nervous system in 1965, but initially was not considered to be a neurotransmitter candidate. In the mid‐1980s, a few laboratories revisited the question of NAAG's role in the nervous system and pursued hypotheses regarding its function that ranged from a precursor for the transmitter pool of glutamate to a direct role as a peptide transmitter. Since that time, NAAG has been tested against nearly all of the established criteria for identification of a neurotransmitter. It successfully meets each of these tests, including a concentrated presence in neurons and synaptic vesicles, release from axon endings in a calcium‐dependent manner following initiation of action potentials, and extracellular hydrolysis by membrane‐bound peptidase activity. NAAG is the most prevalent and widely distributed neuropeptide in the mammalian nervous system. NAAG activates NMDA receptors with a low potency that may vary among receptor subtypes, and it is a highly selective agonist at the type 3 metabotropic glutamate receptor (mGluR3). Acting through this receptor, NAAG reduces cyclic AMP levels, decreases voltage‐dependent calcium conductance, suppresses excitotoxicity, influences long‐term potentiation and depression, regulates GABAA receptor subunit expression, and inhibits synaptic release of GABA from cortical neurons. Cloning of peptidase activities against NAAG provides opportunities to study the cellular and molecular mechanisms by which synaptic NAAG peptidase activity is controlled. Given the codistribution of this peptide with a spectrum of traditional transmitters and its ability to activate mGluR3, we speculate that one role for NAAG following synaptic release is the activation of metabotropic autoreceptors that inhibit subsequent transmitter release. A second role is the production of extracellular glutamate following NAAG hydrolysis.

Baslow, Morris H

doi: 10.1046/j.1471-4159.2000.0750453.xpmid: 10899919

N‐Acetyl‐l‐aspartate (NAA) and its derivative N‐acetylaspartylglutamate (NAAG) are major osmolytes present in the vertebrate brain. Although they are synthesized primarily in neurons, their function in these cells is unclear. In the brain, these substances undergo intercompartmental cycles in which they are released by neurons in a regulated fashion and are then rapidly hydrolyzed by catabolic enzymes associated with glial cells. Recently, the catabolic enzyme for NAA hydrolysis has been found to be expressed only in oligodendrocytes, and the catabolic enzyme for NAAG expressed only in astrocytes. These results indicate an unusual tricellular metabolic sequence for the synthesis and hydrolysis of NAAG wherein it is synthesized in neurons from NAA and l‐glutamate, hydrolyzed to NAA and l‐glutamate by astrocytes, and further hydrolyzed to l‐aspartate and acetate by oligodendrocytes. Since the discovery that the NAA and NAAG anabolic products of neurons are specifically targeted to oligodendrocytes and astrocytes, respectively, this unique metabolic compartmentalization also suggests that these substances may play an important role in cell‐specific glial signaling. In this review, it is hypothesized that a key function of NAA and NAAG in the vertebrate brain is in cell signaling and that these substances are important in the regulation of interactions of brain cells and in the establishment and maintenance of the nervous system.

García‐Fernández, Luis F; Iñiguez, Miguel Angel; Eguchi, Naomi; Fresno, Manuel; Urade, Yoshihiro; Muñoz, Alberto

doi: 10.1046/j.1471-4159.2000.0750460.xpmid: 10899920

Lipocalin‐type prostaglandin (PG) D synthase (L‐PGDS) is responsible for the production of PGD2, the main PG in the CNS. PGD2 is an endogenous sleep inducer, and it is involved in the control of odor and pain responses and body temperature. In addition, PGD synthase transports lipophilic molecules in the subarachnoid space and CSF. By northern and western assays we show that the synthetic glucocorticoid dexamethasone, an inhibitor of PG production in most tissues, induces L‐PGDS mRNA and protein in a dose‐ and time‐dependent fashion in mouse neuronal GT1‐7 cells. Accordingly, dexamethasone increases cellular L‐PGDS enzymatic activity. Dexamethasone induced L‐PGDS gene transcription in run‐on assays and activated the mouse L‐PGDS gene promoter in transiently transfected cells. It is interesting that the tumor promoter 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA), which induces the synthesis of PGs in many tissues, inhibited the increase in L‐PGDS expression induced by dexamethasone. In contrast, neither dexamethasone nor TPA affected the expression of cyclooxygenases‐1 and ‐2. Our data demonstrate that dexamethasone induces L‐PGDS gene transcription in neuronal cells.

Waagepetersen, Helle S; Sonnewald, Ursula; Larsson, Orla M; Schousboe, Arne

doi: 10.1046/j.1471-4159.2000.0750471.xpmid: 10899921

The metabolism of [U‐13C]lactate (1 mM) in the presence of unlabeled glucose (2.5 mM) was investigated in glutamatergic cerebellar granule cells, cerebellar astrocytes, and corresponding co‐cultures. It was evident that lactate is primarily a neuronal substrate and that lactate produced glycolytically from glucose in astrocytes serves as a substrate in neurons. Alanine was highly enriched with 13C in the neurons, whereas this was not the case in the astrocytes. Moreover, the cellular content and the amount of alanine released into the medium were higher in neurons than astrocytes. On incubation of the different cell types in medium containing alanine (1 mM), the astrocytes exhibited the highest level of accumulation. Altogether, these results indicate a preferential synthesis and release of alanine in glutamatergic neurons and uptake in cerebellar astrocytes. A new functional role of alanine may be suggested as a carrier of nitrogen from glutamatergic neurons to astrocytes, a transport that may operate to provide ammonia for glutamine synthesis in astrocytes and dispose of ammonia generated by the glutaminase reaction in glutamatergic neurons. Hence, a model of a glutamate‐glutamine/lactate‐alanine shuttle is presented. To elucidate if this hypothesis is compatible with the pattern of alanine metabolism observed in the astrocytes and neurons from cerebellum, the cells were incubated in a medium containing [15N]alanine (1 mM) and [5‐15N]glutamine (0.5 mM), respectively. Additionally, neurons were incubated with [U‐13C]glutamine to estimate the magnitude of glutamine conversion to glutamate. Alanine was labeled from [5‐15N]glutamine to 3.3% and [U‐13C]glutamate generated from [U‐13C]glutamine was labeled to 16%. In spite of the modest labeling in alanine, it is clear that nitrogen from ammonia is transferred to alanine via transamination with glutamate formed by reductive amination of α‐ketoglutarate. With regard to the astrocytic part of the shuttle, glutamine was labeled to 22% in one nitrogen atom whereas 3.2% was labeled in two when astrocytes were incubated in [15N]alanine. Moreover, in co‐cultures, [U‐13C]alanine labeled glutamate and glutamine equally, whereas [U‐13C]lactate preferentially labeled glutamate. Altogether, these results support the role proposed above of alanine as a possible ammonia nitrogen carrier between glutamatergic neurons and surrounding astrocytes and they show that lactate is preferentially metabolized in neurons and alanine in astrocytes.

Bouzier, Anne‐Karine; Thiaudiere, Eric; Biran, Marc; Rouland, Richard; Canioni, Paul; Merle, Michel

doi: 10.1046/j.1471-4159.2000.0750480.xpmid: 10899922

Lactate metabolism in the adult rat brain was investigated in relation with the concept of lactate trafficking between astrocytes and neurons. Wistar rats were infused intravenously with a solution containing either [3‐13C]lactate (534 mM) or both glucose (750 mM) and [3‐13C]lactate (534 mM). The time courses of both the concentration and 13C enrichment of blood glucose and lactate were determined. The data indicated the occurrence of [3‐13C]lactate recycling through liver gluconeogenesis. The yield of glucose labeling was, however, reduced when using the glucose‐containing infusate. After a 20‐min or 1‐h infusion, perchloric acid extracts of the brain tissue were prepared and subsequently analyzed by 13C‐ and 1H‐observed/13C‐edited NMR spectroscopy. The 13C labeling of amino acids indicated that [3‐13C]lactate was metabolized in the brain. Based on the alanine C3 enrichment, lactate contribution to brain metabolism amounted to 35% under the most favorable conditions used. By contrast with what happens with [1‐13C]glucose metabolism, no difference in glutamine C2 and C3 labeling was evidenced, indicating that lactate was metabolized in a compartment deprived of pyruvate carboxylase activity. This result confirms, for the first time from an in vivo study, that lactate is more specifically a neuronal substrate.

Berkeley, Jennifer L; Levey, Allan I

doi: 10.1046/j.1471-4159.2000.0750487.xpmid: 10899923

Muscarinic acetylcholine receptors (mAChRs) activate many downstream signaling pathways, some of which can lead to mitogen‐activated protein kinase (MAPK) phosphorylation and activation. MAPKs play roles in regulating cell growth, differentiation, and synaptic plasticity. Here, the activation of MAPK was examined in PC12 cells endogenously expressing mAChRs. Western blot analysis using a phosphospecific MAPK antibody revealed a dose‐dependent and atropine‐sensitive increase in MAPK phosphorylation in cells stimulated with carbachol (CCh). The maximal response occurred after 5 min and was rapidly reduced to baseline. To investigate the receptors responsible for CCh activation of MAPK in PC12 cells, the mAChR subtypes present were determined using RT‐PCR and immunoprecipitation. RT‐PCR was used to amplify fragments of the appropriate sizes for m1, m4, and m5, and the identities of the bands were confirmed with restriction digests. Immunoprecipitation using subtype‐specific antibodies showed that ~95% of the expressed receptors were m4, whereas the remaining ~5% were m1 and m5. A highly specific m1 toxin completely blocked MAPK phosphorylation in response to CCh stimulation. The mAChR‐induced MAPK activation was abolished by protein kinase C down‐regulation and partially inhibited by pertussis toxin. Although m1 represents a small proportion of the total mAChR population, pharmacological evidence suggests that m1 is responsible for MAPK activation in PC12 cells.

Ishikawa, Yasuyuki; Ikeuchi, Toshihiko; Hatanaka, Hiroshi

doi: 10.1046/j.1471-4159.2000.0750494.xpmid: 10899924

Brain‐derived neurotrophic factor (BDNF) is known to have important functions in neuronal survival, differentiation, and plasticity. In addition to its role as a survival‐promoting factor, BDNF reportedly can enhance neuronal cell death in some cases, for example, the death caused by excitotoxicity or glucose deprivation. The cellular mechanism of the death‐enhancing effect of BDNF remains unknown, in contrast to that of its survival‐promoting effect. In this work, we found that BDNF markedly accelerated the nitric oxide (NO) donor‐induced death of cultured embryonic cortical neurons. BDNF increased the number of cells with nuclear condensation and DNA fragmentation 24 h after treatment with the NO donor, but it did not change the number of those cells 36 h after the treatment. The BDNF‐accelerated death of cortical neurons was inhibited by the addition of actinomycin D or cycloheximide. These results suggest that BDNF can accelerate apoptotic cell death elicited by NO donor. TrkB‐IgG and K252a blocked the BDNF‐induced acceleration of the death, indicating that the death‐accelerating effect by BDNF is mediated by TrkB. In addition, the BDNF‐accelerated apoptosis was inhibited by the addition of SB202190 and SB203580, specific inhibitors of p38 mitogen‐activated protein kinase (MAPK), and U0126, a specific inhibitor of MAPK/ERK kinase 1, indicating that the activation of both p38 MAPK and ERK is involved in the signaling cascade of the BDNF‐accelerated, NO donor‐induced apoptosis.

Riboni, Laura; Viani, Paola; Bassi, Rosaria; Giussani, Paola; Tettamanti, Guido

doi: 10.1046/j.1471-4159.2000.0750503.xpmid: 10899925

Sphingosine metabolism was studied in primary cultures of differentiated cerebellar granule cells and astrocytes. After a 2‐h pulse with [C3‐3H]sphingosine at different doses (0.1‐200 nmol/mg of cell protein), both cell types efficiently incorporated the long chain base ; the percentage of cellular [3H]sphingosine over total label incorporation was extremely low at sphingosine doses of <10 nmol/mg of cell protein and increased at higher doses. Most of the [3H]sphingosine taken up underwent metabolic processing by N‐acylation, 1‐phosphorylation, and degradation (assessed as 3H2O released in the medium). The metabolic processing of exogenous sphingosine was extremely efficient in both cells, granule cells and astrocytes being able to metabolize, respectively, an amount of sphingosine up to 80‐ and 300‐fold the cellular content of this long chain base in 2 h. At the different doses, the prevailing metabolic route of sphingosine was different. At lower doses and in a wide dose range, the major metabolic fate of sphingosine was N‐acylation. With increasing doses, there was first increased sphingosine degradation and then increased levels of sphingosine‐1‐phosphate. The data demonstrate that, in neurons and astrocytes, the metabolic machinery devoted to sphingosine processing is different, astrocytes possessing an overall higher capacity to synthesize the bioactive compounds ceramide and sphingosine‐1‐phosphate.

Seitz, Gabriele; Stegmann, Hartmut B; Jäger, Heidrun H; Schlude, Hans M; Wolburg, Hartwig; Roginsky, Vitaly A; Niethammer, Dietrich; Bruchelt, Gernot

doi: 10.1046/j.1471-4159.2000.0750511.xpmid: 10899926

6‐Hydroxydopamine (6‐OHDA) has been used for lesioning catecholaminergic neurons and attempted purging of neuroblastoma cells from hematopoietic stem cells in autologous bone marrow transplantation (ABMT). Neurotoxicity is mediated primarily by reactive oxygen species. In ABMT, 6‐OHDA, as a purging agent, has been unsuccessful. At physiological pH it autooxidizes before targeted uptake, resulting in nonspecific cytotoxicity of nontarget cells. A catecholamine analogue, similar to 6‐OHDA but with a lower rate of autooxidation enabling uptake by target cells, is thus required. Electron paramagnetic resonance spectra in this study show that 6‐fluorodopamine (6‐FDA) hydrolyzes slowly to 6‐OHDA at physiological pH. Oxygen consumption, H2O2, and quinone production are found to be intermediate between those of 6‐OHDA and dopamine (DA). Relative neurotoxicity of these compounds was assessed by cell viability and DNA damage in the human neuroblastoma lines SH‐SY5Y and SK‐N‐LO, which express and lack the noradrenaline transporter, respectively. Specific uptake of DA and 6‐FDA by SH‐SY5Y cells was demonstrated by competitive m‐[131I]iodobenzylguanidine uptake inhibition. The competition by 6‐OHDA was low owing to rapid autooxidation during incubation with equal toxicity toward both cell types. 6‐FDA toxicity was preferential for SH‐SY5Y cells and reduced in the presence of desipramine, a catecholamine uptake inhibitor. We demonstrate that 6‐FDA cytotoxicity is more specific for cells expressing catecholamine reuptake systems than is 6‐OHDA cytotoxicity.

Lee, Chung Soo; Han, Eun Sook; Jang, Yoon Young; Han, Jeong Ho; Ha, Hyun Wook; Kim, Doo Eung

doi: 10.1046/j.1471-4159.2000.0750521.xpmid: 10899927

The present study elucidated the protective effect of β‐carbolines (harmaline, harmalol, and harmine) on oxidative neuronal damage. MPTP treatment increased activities of total superoxide dismutase, catalase, and glutathione peroxidase and levels of malondialdehyde and carbonyls in the basal ganglia, diencephalon plus midbrain of brain compared with control mouse brain. Coadministration of harmalol (48 mg/kg) attenuated the MPTP effect on the enzyme activities and formation of tissue peroxidation products. Harmaline, harmalol, and harmine attenuated both the 500 μM MPP+‐induced inhibition of electron flow and membrane potential formation and the 100 μM dopamine‐induced thiol oxidation and carbonyl formation in mitochondria. The scavenging action of β‐carbolines on hydroxyl radicals was represented by inhibition of 2‐deoxy‐d‐ribose degradation. Harmaline and harmalol (100 μM) attenuated 200 μM dopamine‐induced viability loss in PC12 cells. The β‐carbolines (50 μM) attenuated 50 μM dopamine‐induced apoptosis in PC12 cells. The compounds alone did not exhibit significant cytotoxic effects. The results indicate that β‐carbolines attenuate brain damage in mice treated with MPTP and MPP+‐induced mitochondrial damage. The compounds may prevent dopamine‐induced mitochondrial damage and PC12 cell death through a scavenging action on reactive oxygen species and inhibition of monoamine oxidase and thiol oxidation.