NMDA Receptor Regulation by <Emphasis Type="SmallCaps">D</Emphasis>-serine: New Findings and Perspectives

The N-methyl-d-aspartate (NMDA) receptors play key roles in excitatory neurotransmission and are involved in several important processes, including learning, behavior, and synaptic plasticity. The regulation of NMDA receptor neurotransmission has been extensively studied, but many important questions still remain unsolved. One of the most debated aspects of the NMDA receptor regulation relates to the identity, role, and cellular origin of the NMDA coagonist(s). In addition to glutamate, the NMDA receptor activity was believed to be regulated by the coagonist glycine. More recently, d-serine has also been proposed to play a role as a key coagonist for NMDA receptor activity and neurotoxicity. A surprising unique biosynthetic pathway for d-serine has been demonstrated, indicating the conservation of d-amino acid metabolism in mammals. d-Serine was originally shown to be exclusively made in astrocytes, indicating a possible role as a gliotransmitter. Nevertheless, recent data indicate that d-serine has a neuronal origin as well, which raises several new questions on d-serine disposition. In this review, I discuss recent advances in the field and propose a novel model of d-serine signaling that includes a bidirectional flow of d-serine between astrocytes and neurons. This review is dedicated to the memory of Dr. Marcos Wolosker.     

Occurrence of two l-threonine (l-serine) dehydratases in the thermophile Chloroflexus aurantiacus

The thermophilic phototrophic prokaryote, Chloroflexus aurantiacus was shown to contain high constitutive l-threonine (l-serine) deaminating activity. Separation of cellular proteins by DE 52-cellulose chromatography and by polyacrylamide gel electrophoresis with subsequent activity staining of the gels yielded two bands, one representing an isoleucine-sensitive, the other one an isoleucine-insensitive form of l-threonine dehydratase. Both enzymes had a molecular weight of 120,000 but were distinguished by their different affinities to the two substrates, l-threonine and l-serine.Abbreviations SDH l-serine dehydratase - TDH l-threonine dehydratase     

New ubiquitous translocators: amino acid export by<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis>

Molecular access to amino acid excretion by Corynebacterium glutamicum and Escherichia coli led to the identification of structurally novel carriers and novel carrier functions. The exporters LysE, RhtB, ThrE and BrnFE each represent the protoype of new transporter families, which are in part distributed throughout all of the kingdoms of life. LysE of C. glutamicum catalytes the export of basic amino acids. The expression of the carrier gene is regulated by the cell-internal concentration of basic amino acids. This serves, for example, to maintain homoeostasis if an excess of l-lysine or l-arginine inside the cell should arise during growth on complex media. RhtB is one of five paralogous systems in E. coli, of which at least two are relevant for l-threonine production. A third system is relevant for l-cysteine production. It is speculated that the physiological function of these paralogues is related to quorum sensing. ThrE of C. glutamicum exports l-threonine and l-serine. However, a ThrE domain with a putative hydrolytic function points to an as yet unknown role of this exporter. BrnFE in C. glutamicum is a two-component permease exporting branched-chained amino acids from the cell, and an orthologue in B. subtilis exports 4-azaleucine.     

Up-regulation of d-serine Might Induce GABAergic Neuronal Degeneration in the Cerebral Cortex and Hippocampus in the Mouse Pilocarpine Model of Epilepsy

Epilepsy is a serious neurological disorder with neuronal loss and spontaneous recurrent seizures, but the neurochemical basis remains largely unclear. We hypothesize that d-serine, a newly identified endogenous co-agonist of N-methyl-d-aspartate (NMDA) receptor, may trigger excitotoxicity and neuronal damage in epileptogenesis. By using a mouse pilocarpine model, immunohistochemistry, Fluoro-Jade staining and double-labeling, the present study revealed up-regulation of d-serine expression in a proportion (41%) of neurons in the cerebral cortex and hippocampus. The d-serine-positive neurons occurred at 4 h, reached peak levels at 12–24 h, and gradually went down at 3–14 days. Moreover, most of d-serine-positive neurons were GABAergic (98%), underwent degenerating death (93%), and were accompanied enhancing phosphorylation of NMDA receptor subunit 1. This study has provided new evidence that up-regulation of d-serine production might induce GABAergic neuronal degeneration through excitotoxic mechanism in the pilocarpine model and may be involved in early pathogenesis and recurrent seizure of chronic epilepsy. Ms. L. Wang is on leave from Department of Neurology, Kunming General Hospital of Chengdu Military Region, China.     

Metabotropic Glutamate Receptors in Glial Cells

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS) and exerts its actions via a number of ionotropic glutamate receptors/channels and metabotropic glutamate (mGlu) receptors. In addition to being expressed in neurons, glutamate receptors are expressed in different types of glial cells including astrocytes, oligodendrocytes, and microglia. Astrocytes are now recognized as dynamic signaling elements actively integrating neuronal inputs. Synaptic activity can evoke calcium signals in astrocytes, resulting in the release of gliotransmitters, such as glutamate, ATP, and d-serine, which in turn modulate neuronal excitability and synaptic transmission. In addition, astrocytes, and microglia may play an important role in pathology such as brain trauma and neurodegeneration, limiting or amplifying the pathologic process leading to neuronal death. The present review will focus on recent advances on the role of mGlu receptors expressed in glial cells under physiologic and pathologic conditions. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

The human endogenous retrovirus envelope glycoprotein, syncytin-1, regulates neuroinflammation and its receptor expression in multiple sclerosis: a role for endoplasmic reticulum chaperones in astrocytes

Retroviral envelopes are pathogenic glycoproteins which cause neuroinflammation, neurodegeneration, and endoplasmic reticulum stress responses. The human endogenous retrovirus (HERV-W) envelope protein, Syncytin-1, is highly expressed in CNS glia of individuals with multiple sclerosis (MS). In this study, we investigated the mechanisms by which Syncytin-1 mediated neuroimmune activation and oligodendrocytes damage. In brain tissue from individuals with MS, ASCT1, a receptor for Syncytin-1 and a neutral amino acid transporter, was selectively suppressed in astrocytes (p < 0.05). Syncytin-1 induced the expression of the endoplasmic reticulum stress sensor, old astrocyte specifically induced substance (OASIS), in cultured astrocytes, similar to findings in MS brains. Overexpression of OASIS in astrocytes increased inducible NO synthase expression but concurrently down-regulated ASCT1 (p < 0.01). Treatment of astrocytes with a NO donor enhanced expression of early growth response 1, with an ensuing reduction in ASCT1 expression (p < 0.05). Small-interfering RNA molecules targeting Syncytin-1 selectively down-regulated its expression, preventing the suppression of ASCT1 and the release of oligodendrocyte cytotoxins by astrocytes. A Syncytin-1-transgenic mouse expressing Syncytin-1 under the glial fibrillary acidic protein promoter demonstrated neuroinflammation, ASCT1 suppression, and diminished levels of myelin proteins in the corpus callosum, consistent with observations in CNS tissues from MS patients together with neurobehavioral abnormalities compared with wild-type littermates (p < 0.05). Thus, Syncytin-1 initiated an OASIS-mediated suppression of ASCT1 in astrocytes through the induction of inducible NO synthase with ensuing oligodendrocyte injury. These studies provide new insights into the role of HERV-mediated neuroinflammation and its contribution to an autoimmune disease.     

Engineering of an <Emphasis Type="SmallCaps">l</Emphasis>-arabinose metabolic pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar l-arabinose, a product of the degradation of lignocellulosic biomass. The resultant CRA1 recombinant strain expressed the Escherichia coli genes araA, araB, and araD encoding l-arabinose isomerase, l-ribulokinase, and l-ribulose-5-phosphate 4-epimerase, respectively, under the control of a constitutive promoter. Unlike the wild-type strain, CRA1 was able to grow on mineral salts medium containing l-arabinose as the sole carbon and energy source. The three cloned genes were expressed to the same levels whether cells were cultured in the presence of d-glucose or l-arabinose. Under oxygen deprivation and with l-arabinose as the sole carbon and energy source, strain CRA1 carbon flow was redirected to produce up to 40, 37, and 11%, respectively, of the theoretical yields of succinic, lactic, and acetic acids. Using a sugar mixture containing 5% d-glucose and 1% l-arabinose under oxygen deprivation, CRA1 cells metabolized l-arabinose at a constant rate, resulting in combined organic acids yield based on the amount of sugar mixture consumed after d-glucose depletion (83%) that was comparable to that before d-glucose depletion (89%). Strain CRA1 is, therefore, able to utilize l-arabinose as a substrate for organic acid production even in the presence of d-glucose.     

Cell Selective Conditional Null Mutations of Serine Racemase Demonstrate a Predominate Localization in Cortical Glutamatergic Neurons

d-Serine, which is synthesized by the enzyme serine racemase (SR), is a co-agonist at the N-methyl-d-aspartate receptor (NMDAR). Crucial to an understanding of the signaling functions of d-serine is defining the sites responsible for its synthesis and release. In order to quantify the contributions of astrocytes and neurons to SR and d-serine localization, we used recombinant DNA techniques to effect cell type selective suppression of SR expression in astrocytes (aSRCKO) and in forebrain glutamatergic neurons (nSRCKO). The majority of SR is expressed in neurons: SR expression was reduced by ~65% in nSRCKO cerebral cortex and hippocampus, but only ~15% in aSRCKO as quantified by western blots. In contrast, nSRCKO is associated with only modest decreases in d-serine levels as quantified by HPLC, whereas d-serine levels were unaffected in aSRCKO mice. Liver expression of SR was increased by 35% in the nSRCKO, suggesting a role for peripheral SR in the maintenance of brain d-serine. Electrophysiologic studies of long-term potentiation (LTP) at the Schaffer collateral–CA1 pyramidal neuron synapse revealed no alterations in the aSRCKO mice versus wild-type. LTP induced by a single tetanic stimulus was reduced by nearly 70% in the nSRCKO mice. Furthermore, the mini-excitatory post-synaptic currents mediated by NMDA receptors but not by AMPA receptors were significantly reduced in nSRCKO mice. Our findings indicate that in forebrain, where d-serine appears to be the endogenous co-agonist at NMDA receptors, SR is predominantly expressed in glutamatergic neurons, and co-release of glutamate and d-serine is required for optimal activation of post-synaptic NMDA receptors.     

Origin ofd-serine present in urine of mutant mice lackingd-amino-acid oxidase activity

Summary Urine of ddY/DAO mice lackingd-amino-acid oxidase contained 5.7 times more serine than that of normal ddY/DAO⁺ mice. Most of the serine wasd-isomer. The origin of this^d-serine was examined. Oral administration of 0.02% amoxicillin and 0.004% minocycline to the ddY/ DAO^- mice for 7 days did not reduce the urinaryd-serine, indicating that thed-serine was not of intestinal bacterial origin. When the mouse diet was changed to one with different compositions, the urinaryd-serine was considerably reduced. Furthermore, starvation of the ddY/DAO^- mice for 24 hours reduced the urinaryd-serine to 33% of the original level. These results indicate that most of the urinaryd-serine comes from the diet. However, the urine of the starved ddY/DAO^- mice still contained 4.6 times mored-serine than that of the ddY/DAO⁺ mice, suggesting a part of the D-serine have an endogenous origin.     

Spectroscopic,theoretical and structural characterization of hydrogensquarates of <Emphasis Type="SmallCaps">l</Emphasis>-threonyl-<Emphasis Type="SmallCaps">l</Emphasis>-serine and <Emphasis Type="SmallCaps">l</Emphasis>-serine

Summary. Hydrogensquarates of dipeptide l-threonyl-l-serine (H-Thr-Ser-OH) and l-serine (HSq × Ser) have been synthesized, isolated and spectroscopic characterized by solid-state linear-polarized IR-spectroscopy, ¹H- and ¹³C-NMR, ESI-MS and HPLC with tandem masspectrometry (MS-MS) methods. The structures of the salts and neutral dipeptide have been predicted theoretically by ab initio calculations. In the case of H-Thr-Ser-OH the theoretical data are supported by IR-LD ones. The hydrogensquarates consist in positive charged dipeptide or amino acid moiety and negative hydrogensquarate anion (HSq) stabilizing by strong intermolecular hydrogen bonds. The data about the l-serine hydrogensquarate are compared with known crystallographic data thus indicating a good correlation between the theoretical predicted structures and experimentally obtained by single crystal X-ray diffraction.     

<Emphasis Type="SmallCaps">l</Emphasis>-Aspartate-immunoreactive neurons in the rat enteric nervous system

l-Aspartate (l-Asp) is an excitatory neurotransmitter in the central nervous system. In the present study, we demonstrate, for the first time, the presence of l-Asp in a particular neuronal cell class in the enteric nervous system (ENS). Scattered l-Asp-immunoreactive neuronal cell bodies and nerve fibers were found extensively in both the myenteric and submucosal plexus throughout the small and large intestines. Many l-Asp-immunoreactive nerve fibers, which originated from intrinsic nerve cell bodies, were found in the ganglia and interconnecting nerve bundles. Electron microscopy revealed that l-Asp-immunoreactive terminals frequently formed synaptic contacts with intrinsic nerve cells, suggesting that some l-Asp-immunoreactive neurons might function as interneurons. These results suggest that l-Asp-immunoreactive neurons play a significant role within the ENS to control intestinal functions. The presence of enteric l-Asp-immunoreactive neurons provides strong support for the proposal that l-Asp is a neuromodulator in the rat ENS.     

<Emphasis Type="SmallCaps">L</Emphasis>-Tyrosine production by deregulated strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The excretion of the aromatic amino acid l-tyrosine was achieved by manipulating three gene targets in the wild-type Escherichia coli K12: The feedback-inhibition-resistant (fbr) derivatives of aroG and tyrA were expressed on a low-copy-number vector, and the TyrR-mediated regulation of the aromatic amino acid biosynthesis was eliminated by deleting the tyrR gene. The generation of this l-tyrosine producer, strain T1, was based only on the deregulation of the aromatic amino acid biosynthesis pathway, but no structural genes in the genome were affected. A second tyrosine over-producing strain, E. coli T2, was generated considering the possible limitation of precursor substrates. To enhance the availability of the two precursor substrates phosphoenolpyruvate and erythrose-4-phosphate, the ppsA and the tktA genes were over-expressed in the strain T1 background, increasing l-tyrosine production by 80% in 50-ml batch cultures. Fed-batch fermentations revealed that l-tyrosine production was tightly correlated with cell growth, exhibiting the maximum productivity at the end of the exponential growth phase. The final l-tyrosine concentrations were 3.8 g/l for E. coli T1 and 9.7 g/l for E. coli T2 with a yield of l-tyrosine per glucose of 0.037 g/g (T1) and 0.102 g/g (T2), respectively.     

Whole body autoradiographic study on the distribution of14C-d-serine administered intravenously to rats

Summary The distribution of radioactivities in rats following intravenous administration of¹⁴C-d- or -l-serine was investigated by whole body autoradiography. The radioactivities were distributed throughout the whole body in both cases with the greatest amount being found in the pancreas. D- andl- Serine levels in the pancreas were determined by high-performance liquid chromatography with a chiral column which revealed, for the first time, the existence ofd-serine in the rat pancreas (12.6 ± 7.90 nmol/g wet tissue) together with a much higher concentration (924 ± 116 nmol/g) ofl-serine. The results suggested that exogenous D-serine of dietary origin contributed at least in part to the D-serine levels found in mammalian tissues.The accumulation of radioactivity in the kidney, especially in the corticomedullary area, even at 24 hr after administration of¹⁴C-l-serine suggested a possible link between acute necrosis of the renal proximal tubules and the administration of a large dose of D-serine [Am J Pathol 77: 269–282 (1974)].     

Metabolic engineering and flux analysis of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for L-serine production

l-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of l-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and attenuating the conversion of l-serine to pyruvate and glycine, releasing the feedback inhibition by l-serine to 3-phosphoglycerate dehydrogenase (PGDH), in combination with the co-expression of 3-phosphoglycerate kinase (PGK) and feedback-resistant PGDH (PGDH^r). The resulting strain, SER-8, exhibited a lower specific growth rate and significant differences in l-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular l-serine pool reached (14.22±1.41) μmol g_CDM ⁻¹, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway (PPP) towards the glycolysis pathway (EMP), and the expression of PGDH^r improved the l-serine biosynthesis pathway. In addition, the flux from l-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR resulted in down-regulation of serine hydroxymethyltransferase (SHMT) expression. Taken together, our findings imply that l-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C₁ units generation by introducing the glycine cleavage system (GCV) to degrade the excessive glycine is a promising target for l-serine production in C. glutamicum.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under oxygen deprivation

In mineral salts medium under oxygen deprivation, Corynebacterium glutamicum exhibits high productivity of l-lactic acid accompanied with succinic and acetic acids. In taking advantage of this elevated productivity, C. glutamicum was genetically modified to produce d-lactic acid. The modification involved expression of fermentative d-lactate dehydrogenase (d-LDH)-encoding genes from Escherichia coli and Lactobacillus delbrueckii in l-lactate dehydrogenase (l-LDH)-encoding ldhA-null C. glutamicum mutants to yield strains C. glutamicum ΔldhA/pCRB201 and C. glutamicum ΔldhA/pCRB204, respectively. The productivity of C. glutamicum ΔldhA/pCRB204 was fivefold higher than that of C. glutamicum ΔldhA/pCRB201. By using C. glutamicum ΔldhA/pCRB204 cells packed to a high density in mineral salts medium, up to 1,336 mM (120 g l⁻¹) of d-lactic acid of greater than 99.9% optical purity was produced within 30 h.     

Regulation of metabolite production by precursors and elicitors in liquid cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis>

Four precursors (l-phenylalanine, l-tryptophan, cinnamic acid and emodin) and one signal elicitor (methyl jasmonate, MeJA) were added to liquid cultures of Hypericum perforatum L. to study their effect on production of hyperforin and hypericins (pseudohypericin and hypericin). The addition of l-phenylalanine (75 to 100 mg l⁻¹) enhanced production of hypericins, but hyperforin levels were decreased. Hypericin, pseudohypericin and hyperforin concentrations were all decreased when l-tryptophan (25 to 100 mg l⁻¹) was added to the medium. However, addition of l-tryptophan (50 mg l⁻¹) with MeJA (100 μM) stimulated hyperforin production significantly (1.81-fold) and resulted in an increased biomass. Cinnamic acid (25, 50 mg l⁻¹) and emodin (1.0 to 10.0 mg l⁻¹) each enhanced hyperforin accumulation in H. perforatum, but did not affect accumulation of hypericins.     

Effect of pyruvate dehydrogenase complex deficiency on <Emphasis Type="SmallCaps">l</Emphasis>-lysine production with <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Intracellular precursor supply is a critical factor for amino acid productivity of Corynebacterium glutamicum. To test for the effect of improved pyruvate availability on l-lysine production, we deleted the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex (PDHC) in the l-lysine-producer C. glutamicum DM1729 and characterised the resulting strain DM1729-BB1 for growth and l-lysine production. Compared to the host strain, C. glutamicum DM1729-BB1 showed no PDHC activity, was acetate auxotrophic and, after complete consumption of the available carbon sources glucose and acetate, showed a more than 50% lower substrate-specific biomass yield (0.14 vs 0.33 mol C/mol C), an about fourfold higher biomass-specific l-lysine yield (5.27 vs 1.23 mmol/g cell dry weight) and a more than 40% higher substrate-specific l-lysine yield (0.13 vs 0.09 mol C/mol C). Overexpression of the pyruvate carboxylase or diaminopimelate dehydrogenase genes in C. glutamicum DM1729-BB1 resulted in a further increase in the biomass-specific l-lysine yield by 6 and 56%, respectively. In addition to l-lysine, significant amounts of pyruvate, l-alanine and l-valine were produced by C. glutamicum DM1729-BB1 and its derivatives, suggesting a surplus of precursor availability and a further potential to improve l-lysine production by engineering the l-lysine biosynthetic pathway. This study is dedicated to Prof. Dr. Hermann Sahm on the occasion of his 65th birthday.     

Characterization of ribose-5-phosphate isomerase of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-allose from <Emphasis Type="SmallCaps">d</Emphasis>-psicose

The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h, respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l⁻¹ was produced without by-products from 500 g d-psicose l⁻¹ after 6 h.     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.