N-Methyl-d-aspartate antagonists and apoptotic cell death triggered by head trauma in developing rat brain

Morbidity and mortality from head trauma is highest among children. No animal model mimicking traumatic brain injury in children has yet been established, and the mechanisms of neuronal degeneration after traumatic injury to the developing brain are not understood. In infant rats subjected to percussion head trauma, two types of brain damage could be characterized. The first type or primary damage evolved within 4 hr and occurred by an excitotoxic mechanism. The second type or secondary damage evolved within 6–24 hr and occurred by an apoptotic mechanism. Primary damage remained localized to the parietal cortex at the site of impact. Secondary damage affected distant sites such as the cingulate/retrosplenial cortex, subiculum, frontal cortex, thalamus and striatum. Secondary apoptotic damage was more severe than primary excitotoxic damage. Morphometric analysis demonstrated that the N-methyl-d-aspartate receptor antagonists 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonate and dizocilpine protected against primary excitotoxic damage but increased severity of secondary apoptotic damage. 2-Sulfo-α-phenyl-N-tert-butyl-nitrone, a free radical scavenger, did not affect primary excitotoxic damage but mitigated apoptotic damage. These observations demonstrate that apoptosis and not excitotoxicity determine neuropathologic outcome after traumatic injury to the developing brain. Whereas free radical scavengers may prove useful in therapy of head trauma in children, N-methyl-d-aspartate antagonists should be avoided because of their propensity to increase severity of apoptotic damage.

Biosynthesis of terpenoids: 4-Diphosphocytidyl-2-C-methyl-d-erythritol kinase from tomato

The putative catalytic domain (residues 81–401) of a predicted tomato protein with similarity to 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase of Escherichia coli was expressed in a recombinant E. coli strain. The protein was purified to homogeneity and was shown to catalyze the phosphorylation of the position 2 hydroxy group of 4-diphosphocytidyl-2-C-methyl-d-erythritol at a rate of 33 μmol⋅mg−1⋅min−1. The structure of the reaction product, 4-diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate, was established by NMR spectroscopy. Divalent metal ions, preferably Mg2+, are required for activity. Neither the tomato enzyme nor the E. coli ortholog catalyzes the phosphorylation of isopentenyl monophosphate.

m5C RNA and m5C DNA methyl transferases use different cysteine residues as catalysts

A family of RNA m5C methyl transferases (MTases) containing over 55 members in eight subfamilies has been identified recently by an iterative search of the genomic sequence databases by using the known 16S rRNA m5C 967 MTase, Fmu, as an initial probe. The RNA m5C MTase family contained sequence motifs that were highly homologous to motifs in the DNA m5C MTases, including the ProCys sequence that contains the essential Cys catalyst of the functionally similar DNA-modifying enzymes; it was reasonable to assign the Cys nucleophile to be that in the conserved ProCys. The family also contained an additional conserved Cys residue that aligns with the nucleophilic catalyst in m5U54 tRNA MTase. Surprisingly, the mutant of the putative Cys catalyst in the ProCys sequence was active and formed a covalent complex with 5-fluorocytosine-containing RNA, whereas the mutant at the other conserved Cys was inactive and unable to form the complex. Thus, notwithstanding the highly homologous sequences and similar functions, the RNA m5C MTase uses a different Cys as a catalytic nucleophile than the DNA m5C MTases. The catalytic Cys seems to be determined, not by the target base that is modified, but by whether the substrate is DNA or RNA. The function of the conserved ProCys sequence in the RNA m5C MTases remains unknown.

Molecular determinants of coordinated proton and zinc inhibition of N-methyl-d-aspartate NR1/NR2A receptors

Modulation of the N-methyl-d-aspartate (NMDA)-selective glutamate receptors by extracellular protons and Zn2+ may play important roles during ischemia in the brain and during seizures. Recombinant NR1/NR2A receptors exhibit a much higher apparent affinity for voltage-independent Zn2+ inhibition than receptors with other subunit combinations. Here, we show that the mechanism of this apparent high-affinity, voltage-independent Zn2+ inhibition for NR2A-containing receptors results from the enhancement of proton inhibition. We also show that the N-terminal leucine/isoleucine/valine binding protein (LIVBP)-like domain of the NR2A subunit contains critical determinants of the apparent high-affinity, voltage-independent Zn2+ inhibition. Mutations H42A, H44G, or H128A greatly increase the Zn2+ IC50 (by up to ≈700-fold) with no effect on the potencies of glutamate and glycine or on voltage-dependent block by Mg2+. Furthermore, the amino acid residue substitution H128A, which mediates the largest effect on the apparent high-affinity Zn2+ inhibition among all histidine substitutions we tested, is also critical to the pH-dependency of Zn2+ inhibition. Our data revealed a unique interaction between two important extracellular modulators of NMDA receptors.

Remodeling of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia

Transient global ischemia induces selective delayed cell death, primarily of principal neurons in the hippocampal CA1. However, the molecular mechanisms underlying ischemia-induced cell death are as yet unclear. The present study shows that global ischemia triggers a pronounced and cell-specific reduction in GluR2 [the subunit that limits Ca2+ permeability of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors] in vulnerable CA1 neurons, as evidenced by immunofluorescence of brain sections and Western blot analysis of microdissected hippocampal subfields. At 72 h after ischemia (a time before cell death), virtually all CA1 pyramidal neurons exhibited greatly reduced GluR2 immunolabeling throughout their somata and dendritic processes. GluR2 immunolabeling was unchanged in pyramidal cells of the CA3 and granule cells of the dentate gyrus, regions resistant to ischemia-induced damage. Immunolabeling of the AMPA receptor subunit GluR1 was unchanged in CA1, CA3, and dentate gyrus. Western analysis indicated that GluR2 subunit abundance was markedly reduced in CA1 at 60 and 72 h after the ischemic insult; GluR1 abundance was unchanged in all subfields at all times examined. These findings, together with the previous observation of enhanced AMPA-elicited Ca2+ influx in postischemic CA1 neurons, show that functional GluR2-lacking, Ca2+-permeable AMPA receptors are expressed in vulnerable neurons before cell death. Thus, the present study provides an important link in the postulated causal chain between global ischemia and delayed death of CA1 pyramidal neurons.

Identification of the gene (lgtG) encoding the lipooligosaccharide β chain synthesizing glucosyl transferase from Neisseria gonorrhoeae

The lipooligosaccharide from Neisseria gonorrhoeae (GC), consists of lipid A, an oligosaccharide core and three branches, α, β, and γ. We report the cloning of the gene (lgtG, lipooligosaccharide glycosyl transferase G) encoding the glucosyl transferase of GC that initiates the β chain which consists of a lactosyl moiety. This gene contains a homopolymeric tract of cytidine [poly(C)] and we demonstrate that changes in the number of Cs in poly(C) account for the variation of β chain expression in different GC strains. Biochemical analyses and mass spectrometry clearly attribute the reactivity of mAb 2C7 to the presence of the lactosyl β chain. In addition, we demonstrate that in the absence of the lactosyl group, a phosphoethanolamine is added to generate a new antigenic epitope as evidenced by the gain of reactivity to mAb 2-L1–8. These results show that, like the α chain, the β chain of lipooligosaccharide is subject to antigenic variation.

Characterization of a calmodulin kinase II inhibitor protein in brain

Ca2+/calmodulin-dependent protein kinase II (CaM-KII) regulates numerous physiological functions, including neuronal synaptic plasticity through the phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. To identify proteins that may interact with and modulate CaM-KII function, a yeast two-hybrid screen was performed by using a rat brain cDNA library. This screen identified a unique clone of 1.4 kb, which encoded a 79-aa brain-specific protein that bound the catalytic domain of CaM-KII α and β and potently inhibited kinase activity with an IC50 of 50 nM. The inhibitory protein (CaM-KIIN), and a 28-residue peptide derived from it (CaM-KIINtide), was highly selective for inhibition of CaM-KII with little effect on CaM-KI, CaM-KIV, CaM-KK, protein kinase A, or protein kinase C. CaM-KIIN interacted only with activated CaM-KII (i.e., in the presence of Ca2+/CaM or after autophosphorylation) by using glutathione S-transferase/CaM-KIIN precipitations as well as coimmunoprecipitations from rat brain extracts or from HEK293 cells cotransfected with both constructs. Colocalization of CaM-KIIN with activated CaM-KII was demonstrated in COS-7 cells transfected with green fluorescent protein fused to CaM-KIIN. In COS-7 cells phosphorylation of transfected α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors by CaM-KII, but not by protein kinase C, was blocked upon cotransfection with CaM-KIIN. These results characterize a potent and specific cellular inhibitor of CaM-KII that may have an important role in the physiological regulation of this key protein kinase.

Modulation of N-methyl-d-aspartate receptor function by glycine transport

The recent discovery of glycine transporters in both the central nervous system and the periphery suggests that glycine transport may be critical to N-methyl-d-aspartate receptor (NMDAR) function by controlling glycine concentration at the NMDAR modulatory glycine site. Data obtained from whole-cell patch–clamp recordings of hippocampal pyramidal neurons, in vitro, demonstrated that exogenous glycine and glycine transporter type 1 (GLYT1) antagonist selectively enhanced the amplitude of the NMDA component of a glutamatergic excitatory postsynaptic current. The effect was blocked by 2-amino-5-phosphonovaleric acid and 7-chloro-kynurenic acid but not by strychnine. Thus, the glycine-binding site was not saturated under the control conditions. Furthermore, GLYT1 antagonist enhanced NMDAR function during perfusion with medium containing 10 μM glycine, a concentration similar to that in the cerebrospinal fluid in vivo, thereby supporting the hypothesis that the GLYT1 maintains subsaturating concentration of glycine at synaptically activated NMDAR. The enhancement of NMDAR function by specific GLYT1 antagonism may be a feasible target for therapeutic agents directed toward diseases related to hypofunction of NMDAR.

The C9 methyl group of retinal interacts with glycine-121 in rhodopsin

The visual pigment rhodopsin is a prototypical G protein-coupled receptor. These receptors have seven transmembrane helices and are activated by specific receptor–ligand interactions. Rhodopsin is unusual in that its retinal prosthetic group serves as an antagonist in the dark in the 11-cis conformation but is rapidly converted to an agonist on photochemical cis to trans isomerization. Receptor–ligand interactions in rhodopsin were studied in the light and dark by regenerating site-directed opsin mutants with synthetic retinal analogues. A progressive decrease in light-dependent transducin activity was observed when a mutant opsin with a replacement of Gly121 was regenerated with 11-cis-retinal analogues bearing progressively larger R groups (methyl, ethyl, propyl) at the C9 position of the polyene chain. A progressive decrease in light activity was also observed as a function of increasing size of the residue at position 121 for both the 11-cis-9-ethyl- and the 11-cis-9-propylretinal pigments. In contrast, a striking increase of receptor activity in the dark—i.e., without chromophore isomerization—was observed when the molecular volume at either position 121 of opsin or C9 of retinal was increased. The ability of bulky replacements at either position to hinder ligand incorporation and to activate rhodopsin in the dark suggests a direct interaction between these two sites. A molecular model of the retinal-binding site of rhodopsin is proposed that illustrates the specific interaction between Gly121 and the C9 methyl group of 11-cis-retinal. Steric interactions in this region of rhodopsin are consistent with the proposal that movement of transmembrane helices 3 and 6 is concomitant with receptor activation.

Effect of 2′-O-methyl antisense ORNs on expression of thymidylate synthase in human colon cancer RKO cells

Translation of thymidylate synthase (TS) mRNA is controlled by its own protein end-product TS in a negative autoregulatory manner. Disruption of this regulation results in increased synthesis of TS and may lead to the development of cellular drug resistance to TS-directed anticancer agents. As a strategy to inhibit TS expression, antisense 2′-O-methyl RNA oligoribonucleotides (ORNs) were designed to directly target the 5′ upstream cis-acting regulatory element (nucleotides 80–109) of TS mRNA. A 30 nt ORN, HYB0432, inhibited TS expression in human colon cancer RKO cells in a dose-dependent manner but had no effect on the expression of β-actin, α-tubulin or topoisomerase I. TS expression was unaffected by treatment with control sense or mismatched ORNs. HYB0504, an 18 nt ORN targeting the same core sequence, also repressed expression of TS protein. However, further reduction in oligo size resulted in loss of antisense activity. Following HYB0432 treatment, TS protein levels were reduced by 60% within 6 h and were maximally reduced by 24 h. Expression of p53 protein was inversely related to that of TS, suggesting that p53 expression may be directly linked to intracellular levels of TS. Northern blot analysis demonstrated that TS mRNA was unaffected by HYB0432 treatment. The half-life of TS protein was unchanged after antisense treatment suggesting that the mechanism of action of antisense ORNs is mediated through a process of translational arrest. These findings demonstrate that an antisense ORN targeted at a critical cis-acting element on TS mRNA can specifically inhibit expression of TS protein in RKO cells.

Bax ablation prevents dopaminergic neurodegeneration in the 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damages dopaminergic neurons in the substantia nigra pars compacta (SNpc) as seen in Parkinson's disease. Here, we show that the pro-apoptotic protein Bax is highly expressed in the SNpc and that its ablation attenuates SNpc developmental neuronal apoptosis. In adult mice, there is an up-regulation of Bax in the SNpc after MPTP administration and a decrease in Bcl-2. These changes parallel MPTP-induced dopaminergic neurodegeneration. We also show that mutant mice lacking Bax are significantly more resistant to MPTP than their wild-type littermates. This study demonstrates that Bax plays a critical role in the MPTP neurotoxic process and suggests that targeting Bax may provide protective benefit in the treatment of Parkinson's disease.

An N-methyl-d-aspartate receptor channel blocker with neuroprotective activity

Excitotoxicity, resulting from sustained activation of glutamate receptors of the N-methyl-d-aspartate (NMDA) subtype, is considered to play a causative role in the etiology of ischemic stroke and several neurodegenerative diseases. The NMDA receptor is therefore a target for the development of neuroprotective agents. Here, we identify an N-benzylated triamine (denoted as NBTA) as a highly selective and potent NMDA-receptor channel blocker selected by screening a reduced dipeptidomimetic synthetic combinatorial library. NBTA blocks recombinant NMDA receptors expressed in Xenopus laevis oocytes with a mean IC50 of 80 nM; in contrast, it does not block GluR1, a glutamate receptor of the non-NMDA subtype. The blocking activity of NBTA on NMDA receptors exhibits the characteristics of an open-channel blocker: (i) no competition with agonists, (ii) voltage dependence, and (iii) use dependence. Significantly, NBTA protects rodent hippocampal neurons from NMDA receptor, but not kainate receptor-mediated excitotoxic cell death, in agreement with its selective action on the corresponding recombinant receptors. Mutagenesis data indicate that the N site, a key asparagine on the M2 transmembrane segment of the NR1 subunit, is the main determinant of the blocker action. The results highlight the potential of this compound as a neuroprotectant.

CA1-specific N-methyl-d-aspartate receptor knockout mice are deficient in solving a nonspatial transverse patterning task

In both humans and animals, the hippocampus is critical to memory across modalities of information (e.g., spatial and nonspatial memory) and plays a critical role in the organization and flexible expression of memories. Recent studies have advanced our understanding of cellular basis of hippocampal function, showing that N-methyl-d-aspartate (NMDA) receptors in area CA1 are required in both the spatial and nonspatial domains of learning. Here we examined whether CA1 NMDA receptors are specifically required for the acquisition and flexible expression of nonspatial memory. Mice lacking CA1 NMDA receptors were impaired in solving a transverse patterning problem that required the simultaneous acquisition of three overlapping odor discriminations, and their impairment was related to an abnormal strategy by which they failed to adequately sample and compare the critical odor stimuli. By contrast, they performed normally, and used normal stimulus sampling strategies, in the concurrent learning of three nonoverlapping concurrent odor discriminations. These results suggest that CA1 NMDA receptors play a crucial role in the encoding and flexible expression of stimulus relations in nonspatial memory.

Insulin promotes rapid delivery of N-methyl-d- aspartate receptors to the cell surface by exocytosis

Insulin potentiates N-methyl-d-aspartate receptors (NMDARs) in neurons and Xenopus oocytes expressing recombinant NMDARs. The present study shows that insulin induced (i) an increase in channel number times open probability (nPo) in outside-out patches excised from Xenopus oocytes, with no change in mean open time, unitary conductance, or reversal potential, indicating an increase in n and/or Po; (ii) an increase in charge transfer during block of NMDA-elicited currents by the open channel blocker MK-801, indicating increased number of functional NMDARs in the cell membrane with no change in Po; and (iii) increased NR1 surface expression, as indicated by Western blot analysis of surface proteins. Botulinum neurotoxin A greatly reduced insulin potentiation, indicating that insertion of new receptors occurs via SNARE-dependent exocytosis. Thus, insulin potentiation occurs via delivery of new channels to the plasma membrane. NMDARs assembled from mutant subunits lacking all known sites of tyrosine and serine/threonine phosphorylation in their carboxyl-terminal tails exhibited robust insulin potentiation, suggesting that insulin potentiation does not require direct phosphorylation of NMDAR subunits. Because insulin and insulin receptors are localized to glutamatergic synapses in the hippocampus, insulin-regulated trafficking of NMDARs may play a role in synaptic transmission and plasticity, including long-term potentiation.

Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase

V(D)J recombination generates a remarkably diverse repertoire of antigen receptors through the rearrangement of germline DNA. Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds random nucleotides (N regions) to recombination junctions, is a key enzyme contributing to this diversity. The current model is that TdT adds N regions during V(D)J recombination by random collision with the DNA ends, without a dependence on other cellular factors. We previously demonstrated, however, that V(D)J junctions from Ku80-deficient mice unexpectedly lack N regions, although the mechanism responsible for this effect remains undefined in the mouse system. One possibility is that junctions are formed in these mice during a stage in development when TdT is not expressed. Alternatively, Ku80 may be required for the expression, nuclear localization or enzymatic activity of TdT. Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts are devoid of N regions, as were junctions in Ku80-deficient mice. In these cells TdT protein is abundant at the time of recombination, localizes properly to the nucleus and is enzymatically active. Based on these data, we propose that TdT does not add to recombination junctions through random collision but is actively recruited to the V(D)J recombinase complex by Ku80.