A novel family of negative and positive allosteric modulators of NMDA receptors.

The N-methyl-D-aspartate (NMDA) receptor family regulates various central nervous system functions, such as synaptic plasticity. However, hypo- or hyperactivation of NMDA receptors is critically involved in many neurological and psychiatric conditions, such as pain, stroke, epilepsy, neurodegeneration, schizophrenia, and depression. Consequently, subtype-selective positive and negative modulators of NMDA receptor function have many potential therapeutic applications not addressed by currently available compounds. We have identified allosteric modulators with several novel patterns of NMDA receptor subtype selectivity that have a novel mechanism of action. In a series of carboxylated naphthalene and phenanthrene derivatives, compounds were identified that selectively potentiate responses at GluN1/GluN2A [e.g., 9-iodophenanthrene-3-carboxylic acid (UBP512)]; GluN1/GluN2A and GluN1/GluN2B [9-cyclopropylphenanthrene-3-carboxylic acid (UBP710)]; GluN1/GluN2D [3,5-dihydroxynaphthalene-2-carboxylic acid (UBP551)]; or GluN1/GluN2C and GluN1/GluN2D receptors [6-, 7-, 8-, and 9-nitro isomers of naphth[1,2-c][1,2,5]oxadiazole-5-sulfonic acid (NSC339614)] and have no effect or inhibit responses at the other NMDA receptors. Selective inhibition was also observed; UBP512 inhibits only GluN1/GluN2C and GluN1/GluN2D receptors, whereas 6-bromo-2-oxo-2H-chromene-3-carboxylic acid (UBP608) inhibits GluN1/GluN2A receptors with a 23-fold selectivity compared with GluN1/GluN2D receptors. The actions of these compounds were not competitive with the agonists L-glutamate or glycine and were not voltage-dependent. Whereas the N-terminal regulatory domain was not necessary for activity of either potentiators or inhibitors, segment 2 of the agonist ligand-binding domain was important for potentiating activity, whereas subtype-specific inhibitory activity was dependent upon segment 1. In terms of chemical structure, activity profile, and mechanism of action, these modulators represent a new class of pharmacological agents for the study of NMDA receptor subtype function and provide novel lead compounds for a variety of neurological disorders.

The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions.

Interactions of the three transported Na ions with the Na/K pump remain incompletely understood. Na/K pump crystal structures show that the extended C terminus of the Na,K-adenosine triphosphatase (ATPase) alpha subunit directly contacts transmembrane helices. Deletion of the last five residues (KETYY in almost all Na/K pumps) markedly lowered the apparent affinity for Na activation of pump phosphorylation from ATP, a reflection of cytoplasmic Na affinity for forming the occluded E1P(Na3) conformation. ATPase assays further suggested that C-terminal truncations also interfere with low affinity Na interactions, which are attributable to extracellular effects. Because extracellular Na ions traverse part of the membrane's electric field to reach their binding sites in the Na/K pump, their movements generate currents that can be monitored with high resolution. We report here electrical measurements to examine how Na/K pump interactions with extracellular Na ions are influenced by C-terminal truncations. We deleted the last two (YY) or five (KESYY) residues in Xenopus laevis alpha1 Na/K pumps made ouabain resistant by either of two kinds of point mutations and measured their currents as 10-mM ouabain-sensitive currents in Xenopus oocytes after silencing endogenous Xenopus Na/K pumps with 1 microM ouabain. We found the low affinity inhibitory influence of extracellular Na on outward Na/K pump current at negative voltages to be impaired in all of the C-terminally truncated pumps. Correspondingly, voltage jump-induced transient charge movements that reflect pump interactions with extracellular Na ions were strongly shifted to more negative potentials; this signals a several-fold reduction of the apparent affinity for extracellular Na in the truncated pumps. Parallel lowering of Na affinity on both sides of the membrane argues that the C-terminal contacts provide important stabilization of the occluded E1P(Na3) conformation, regardless of the route of Na ion entry into the binding pocket. Gating measurements of palytoxin-opened Na/K pump channels additionally imply that the C-terminal contacts also help stabilize pump conformations with occluded K ions.

Induction and selective accumulation of mutant ubiquitin in CA1 pyramidal neurons after transient global ischemia.

Accumulation of mutant ubiquitin-B (UBB(+1)) in neurons is considered the hallmark of proteasomal dysfunction in neurodegenerative disorders, however no such evidence in ischemic brain has been reported. We investigated the contribution of UBB(+1) in delayed neuronal death after transient global ischemia. Transient global ischemia was achieved by occlusion of bilateral common carotid arteries for 5 min and reperfusion in male Mongolian gerbils (n=6 per each time point). In the CA1 region, UBB(+1) immunoreactivity appeared in the cytoplasm of pyramidal cells at 30 min post-ischemia, and the density of these neurons increased at day 2 (P<0.001) and further increased at day 4 post-ischemia. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL)-positive (apoptotic) cells appeared selectively in the CA1 region at day 3 and their density increased further at day 4 post-ischemia (P<0.001). In contrast, UBB(+1) immunoreactivity was only transiently detected from 30 min to 1 day post-ischemia in CA3, dentate gyrus, and frontal cortex, but disappeared at day 2 post-ischemia. No TUNEL-positive cells were observed in these three regions. UBB(+1) mRNA was detected by reverse transcription-polymerase chain reaction in every region of the hippocampus and frontal cortex of ischemic gerbils and even in the non-ischemic control animals, and its expression level was independent of brain region and time after ischemia. Our results indicate induction and selective accumulation of UBB(+1) protein in dying neurons of the CA1 region and suggest that UBB(+1) expression may be induced by proteasomal dysfunction after transient global ischemia.

Silencing primary dystonia: lentiviral-mediated RNA interference therapy for DYT1 dystonia.

DYT1 is the most common inherited dystonia. Currently, there are no preventive or curative therapies for this dominantly inherited disease. DYT1 dystonia is caused by a common three-nucleotide deletion in the TOR1A gene that eliminates a glutamic acid residue from the protein torsinA. Recent studies suggest that torsinA carrying the disease-linked mutation, torsinA(DeltaE) acts through a dominant-negative effect by recruiting wild-type torsinA [torsinA(wt)] into oligomeric structures in the nuclear envelope. Therefore, suppressing torsinA(DeltaE) expression through RNA interference (RNAi) could restore the normal function of torsinA(wt), representing a potentially effective therapy regardless of the biological role of torsinA. Here, we have generated short hairpin RNAs (shRNAs) that mediate allele-specific suppression of torsinA(DeltaE) and rescue cells from its dominant-negative effect, restoring the normal distribution of torsinA(wt). In addition, delivery of this shRNA by a recombinant feline immunodeficiency virus effectively silenced torsinA(DeltaE) in a neural model of the disease. We further establish the feasibility of this viral-mediated RNAi approach by demonstrating significant suppression of endogenous torsinA in mammalian neurons. Finally, this silencing of torsinA is achieved without triggering an interferon response. These results support the potential use of viral-mediated RNAi as a therapy for DYT1 dystonia and establish the basis for preclinical testing in animal models of the disease.

Human CuZn superoxide dismutase enzymatic activity in cells is regulated by the length of the mRNA.

Single functional human CuZnSOD gene encodes two species of mRNA differing in size by 200 nucleotides in the 3'-untranslated region (UTR). We studied the expression of the CuZnSOD cDNA with different 3'- and 5'-UTR. Deletion in the 5'-end does not affect the expression of the enzyme, however, deletion in the 3'-UTR decreases the level of expression of CuZnSOD. The plasmids containing the long CuZnSOD cDNA with all polyadenylation signal sequences utilize primarily the last polyadenylation site and give a long mRNA, which produces three times more enzyme than the short mRNA lacking the last polyadenylation site and the AU-rich region.

Functional interactions between YY1 and adenovirus E1A.

YY1 is a C2H2-type zinc finger transcription factor that is a member of the human GLl-Kruppel family of proteins. YY1 represses transcription when bound upstream of transcription initiation sites. The repression can be relieved by adenovirus E1A and activation of target genes occurs. We have mapped the repression domain of YY1 to the C-terminal region, overlapping its DNA binding domain. We have also identified an activation domain within the first 69 amino acids of YY1. The YY1 C-terminal region is involved in physical interactions with E1A and is functionally necessary for YY1 to respond to E1A. This suggests that relief of YY1 repression by E1A involves YY1-E1A physical interactions. Although not involved in interactions with E1A, the N-terminal activation domain is also necessary for YY1 to respond to E1A. Presumably, under repressing conditions, the activation domain is masked by the conformation of YY1, but is released upon binding of E1A and is required to subsequently activate transcription. Consistent with this hypothesis, an ATF-2-YY1 chimeric protein containing the activation domain of ATF-2 and the C-terminal two-thirds of YY1 is still a potent repressor. Unlike the mutant YY1 lacking its own N-terminal activation domain, the chimeric protein is fully responsive to E1A.

Jasmonate-inducible expression of a potato cathepsin D inhibitor-GUS gene fusion in tobacco cells.

A potato gene encoding cathepsin D inhibitor (CDI) is expressed constitutively in tubers and flower buds and it is inducible in leaves upon wounding of the tissue or by treatment with methyl jasmonate (MJA). A fusion gene (CDI:GUS) in which the 2.4 kb long promoter of the CDI gene was translationaly fused with the coding sequence for beta-glucuronidase (GUS) showed MJA-inducible expression in transformed tobacco cells in suspension. The maximum level of induction by MJA was obtained in the absence of auxin and repression of MJA-inducible expression of the fusion gene by auxin was released by aphidicolin, the results suggesting that MJA-inducible expression is repressed during active cell division. JA and MJA showed similar activities in inducing the expression of the fusion gene, while other JA-related compounds such as cucurbic acid, tuberonic acid and dihydrojasmonic acid neither induced expression of the fusion gene nor inhibited the MJA-inducible expression of the fusion gene. Methyl dihydrojasmonate specifically stimulated the MJA-inducible expression of the fusion gene. The MJA-inducible expression of the fusion gene was observed even with a 100 bp long promoter of the CDI gene albeit with significantly decreased level of expression compared to the 2.4 kb long promoter. The 100 bp long CDI promoter did not contain a G-box or hexamer motif that had been implicated in the MJA-responsive expression of several other plant genes. Further mutagenesis of the 100 bp long promoter by deletion or oligonucleotide insertion suggested that although a sequence between -100 and -82 is required for the MJA-responsive expression, the presence of this sequence alone does not confer the MJA-responsive expression.

Abl protein-tyrosine kinase selects the Crk adapter as a substrate using SH3-binding sites.

To understand the normal and oncogenic functions of the protein-tyrosine kinase Abl, the yeast two-hybrid system has been used for identifying proteins that interact with it. One interacting protein is Crk-I, an SH3/SH2-containing adapter protein that was originally identified as the oncogenic element in the avian sarcoma virus CT10. Direct interaction between the Crk-I SH3 and Abl at novel, approximately 10 amino acid sites just carboxy-terminal to the Abl kinase domain occurs in vitro and in mammalian cells. There is a nearby site specific for binding another adapter, Nck, and these sites also bind Grb-2. When bound to Abl, Crk-I was phosphorylated on tyrosine. Thus, the SH3-binding sites on Abl serve as substrate recognition sites for the relatively nonspecific kinase of Abl. In Crk-I-transformed cells, Crk-I associates with endogenous c-Abl and is phosphorylated on tyrosine. The association of Crk and Abl suggests that Abl could play a role in v-Crk and Crk-I transformation and that normal Abl function may be partly mediated through bound adapter molecules.