Glutamate and Schizophrenia: Beyond the Dopamine Hypothesis

1. After 50 years of antipsychotic drug development focused on the dopamine D2 receptor, schizophrenia remains a chronic, disabling disorder for most affected individuals.2. Studies over the last decade demonstrate that administration of low doses of NMDA receptor antagonists can cause in normal subjects the negative symptoms, cognitive impairments and physiologic disturbances observed in schizophrenia.3. Furthermore, a number of recently identified risk genes for schizophrenia affect NMDA receptor function or glutamatergic neurotransmission.4. Placebo-controlled trials with agents that directly or indirectly activate the glycine modulatory site on the NMDA receptor have shown reduction in negative symptoms, improvement in cognition and in some cases reduction in positive symptoms in schizophrenic patients receiving concurrent antipsychotic medications.5. Thus, hypofunction of the NMDA receptor, possibly on critical GABAergic inter-neurons, may contribute to the pathophysiology of schizophrenia.     

Differential in-gel electrophoresis (DIGE) analysis of CHO cells under hyperosmotic pressure: osmoprotective effect of glycine betaine addition

The use of glycine betaine combined with hyperosmolality is known to be an efficient means for achieving high protein production in recombinant Chinese hamster ovary (rCHO) cells. In order to understand the intracellular events and identify the key factors in rCHO cells cultivated with glycine betaine under hyperosmotic conditions, two-dimensional differential in-gel electrophoresis (2D-DIGE) followed by mass spectrometric analysis was applied. Differentially expressed 19 protein spots were selected and 16 different kinds of proteins were successfully identified. The identified proteins were associated with cellular metabolism (PEPCK, GAPDH, and PK), cellular architecture (β-tubulin and β-actin), protein folding (GRP78 and OSP94), mRNA processing (Rbm34, ACF, and IPMK), and protein secretion (γ-COP). 2D-Western blot analysis of β-tubulin, GAPDH, Peroxidoxin-1, and GRP78 confirmed the proteomic findings. The proteins identified from this study, which are related to cell growth and antibody production, can be applied to cell engineering for maximizing the efficacy of the use of glycine betaine combined with hyperosmolality in rCHO cells.     

Glycine decarboxylase controls photosynthesis and plant growth

Photorespiration makes oxygenic photosynthesis possible by scavenging 2-phosphoglycolate. Hence, compromising photorespiration impairs photosynthesis. We examined whether facilitating photorespiratory carbon flow in turn accelerates photosynthesis and found that overexpression of the H-protein of glycine decarboxylase indeed considerably enhanced net-photosynthesis and growth of Arabidopsis thaliana. At the molecular level, lower glycine levels confirmed elevated GDC activity in vivo, and lower levels of the CO₂ acceptor ribulose 1,5-bisphosphate indicated higher drain from CO₂ fixation. Thus, the photorespiratory enzyme glycine decarboxylase appears as an important feed-back signaller that contributes to the control of the Calvin-Benson cycle and hence carbon flow through both photosynthesis and photorespiration.     

Probing the pharmacological properties of distinct subunit interfaces within heteromeric glycine receptors reveals a functional ββ agonist-binding site

Synaptic glycine receptors (GlyRs) are hetero-pentameric chloride channels composed of α and β subunits, which are activated by agonist binding at subunit interfaces. To examine the pharmacological properties of each potential agonist-binding site, we substituted residues of the GlyR α(1) subunit by the corresponding residues of the β subunit, as deduced from sequence alignment and homology modeling based on the recently published crystal structure of the glutamate-gated chloride channel GluCl. These exchange substitutions allowed us to reproduce the βα, αβ and ββ subunit interfaces present in synaptic heteromeric GlyRs by generating recombinant homomeric receptors. When the engineered α(1) GlyR mutants were expressed in Xenopus oocytes, all subunit interface combinations were found to form functional agonist-binding sites as revealed by voltage clamp recording. The ββ-binding site displayed the most distinct pharmacological profile towards a range of agonists and modulators tested, indicating that it might be selectively targeted to modulate the activity of synaptic GlyRs. The mutational approach described here should be generally applicable to heteromeric ligand-gated ion channels composed of homologous subunits and facilitate screening efforts aimed at targeting inter-subunit specific binding sites.     

Ammonium formation and assimilation in P(SARK)∷IPT tobacco transgenic plants under low N

Wild Type (WT) and transgenic tobacco plants expressing isopentenyltransferase (IPT), a gene encoding the enzyme regulating the rate-limiting step in cytokinins (CKs) synthesis, were grown under limited nitrogen (N) conditions. We analyzed nitrogen forms, nitrogen metabolism related-enzymes, amino acids and photorespiration related-enzymes in WT and P_SARK∷IPT tobacco plants. Our results indicate that the WT plants subjected to N deficiency displayed reduced nitrate (NO₃⁻) assimilation. However, an increase in the production of ammonium (NH₄⁺), by the degradation of proteins and photorespiration led to an increase in the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle in WT plants. In these plants, the amounts of amino acids decreased with N deficiency, although the relative amounts of glutamate and glutamine increased with N deficiency. Although the transgenic plants expressing P_SARK∷IPT and growing under suboptimal N conditions displayed a significant decline in the N forms in the leaf, they maintained the GS/GOGAT cycle at control levels. Our results suggest that, under N deficiency, CKs prevented the generation and assimilation of NH₄⁺ by increasing such processes as photorespiration, protein degradation, the GS/GOGAT cycle, and the formation of glutamine.     

The role of amino acid transporters in GSH synthesis in the blood-brain barrier and central nervous system

Glutathione (GSH) plays a critical role in protecting cells from oxidative stress and xenobiotics, as well as maintaining the thiol redox state, most notably in the central nervous system (CNS). GSH concentration and synthesis are highly regulated within the CNS and are limited by availability of the sulfhydryl amino acid (AA) l-cys, which is mainly transported from the blood, through the blood-brain barrier (BBB), and into neurons. Several antiporter transport systems (e.g., x(c)(-), x(-)(AG), and L) with clearly different luminal and abluminal distribution, Na(+), and pH dependency have been described in brain endothelial cells (BEC) of the BBB, as well as in neurons, astrocytes, microglia and oligodendrocytes from different brain structures. The purpose of this review is to summarize information regarding the different AA transport systems for l-cys and its oxidized form l-cys(2) in the CNS, such as expression and activity in blood-brain barrier endothelial cells, astrocytes and neurons and environmental factors that modulate transport kinetics.     

Differences in kainate receptor involvement in hippocampal mossy fibre long-term potentiation depending on slice orientation

Long-term potentiation (LTP) is a well-established experimental model used to investigate the synaptic basis of learning and memory. LTP at mossy fibre - CA3 synapses in the hippocampus is unusual because it is normally N-methyl-d-aspartate (NMDA) receptor-independent. Instead it seems that the trigger for mossy fibre LTP involves kainate receptors (KARs). Although it is generally accepted that pre-synaptic KARs play an essential role in frequency facilitation and LTP, their subunit composition remains a matter of significant controversy. We have reported previously that both frequency facilitation and LTP can be blocked by selective antagonism of GluK1 (formerly GluR5/Glu(K5))-containing KARs, but other groups have failed to reproduce this effect. Moreover, data from receptor knockout and mRNA expression studies argue against a major role of GluK1, supporting a more central role for GluK2 (formerly GluR6/Glu(K6)). A potential reason underlying the controversy in the pharmacological experiments may reside in differences in the preparations used. Here we show differences in pharmacological sensitivity of synaptic plasticity at mossy fibre - CA3 synapses depend critically on slice orientation. In transverse slices, LTP of fEPSPs was invariably resistant to GluK1-selective antagonists whereas in parasagittal slices LTP was consistently blocked by GluK1-selective antagonists. In addition, there were pronounced differences in the magnitude of frequency facilitation and the sensitivity to the mGlu2/3 receptor agonist DCG-IV. Using anterograde labelling of granule cells we show that slices of both orientations possess intact mossy fibres and both large and small presynaptic boutons. Transverse slices have denser fibre tracts but a smaller proportion of giant mossy fibre boutons. These results further demonstrate a considerable heterogeneity in the functional properties of the mossy fibre projection.     

Identification of congenital muscular dystonia 2 associated with an inherited GlyT2 defect in Belgian Blue cattle from the United Kingdom

Two newborn Belgian Blue calves from a farm in the United Kingdom exhibited lateral recumbency, low head carriage and transient muscle spasms following tactile or auditory stimulation. DNA sequence analysis indicated that both calves were homozygous for the recessive congenital muscular dystonia type 2 (CMD2) mutation (c.809T>C, p.Leu270Pro) in SLC6A5, encoding the neuronal glycine transporter GlyT2. Further testing of animals from the index farm and a sample of Belgian Blue sires revealed an unexpectedly high frequency of CMD2 carriers. This implies that linked quantitative trait loci may be influencing the prevalence of CMD2 in the estimated 55,000 Belgian Blue cattle in the United Kingdom. We have therefore developed new inexpensive tests for the CMD2 allele that can be used to confirm diagnosis, identify carriers and guide future breeding strategy, thus avoiding animal distress/premature death and minimizing the future economic impact of this disorder.