Targeting renal purinergic signalling for the treatment of lithium-induced nephrogenic diabetes insipidus.

Lithium still retains its critical position in the treatment of bipolar disorder by virtue of its ability to prevent suicidal tendencies. However, chronic use of lithium is often limited by the development of nephrogenic diabetes insipidus (NDI), a debilitating condition. Lithium-induced NDI is due to resistance of the kidney to arginine vasopressin (AVP), leading to polyuria, natriuresis and kaliuresis. Purinergic signalling mediated by extracellular nucleotides (ATP/UTP), acting via P2Y receptors, opposes the action of AVP on renal collecting duct (CD) by decreasing the cellular cAMP and thus AQP2 protein levels. Taking a cue from this phenomenon, we discovered the potential involvement of ATP/UTP-activated P2Y2 receptor in lithium-induced NDI in rats and showed that P2Y2 receptor knockout mice are significantly resistant to Li-induced polyuria, natriuresis and kaliuresis. Extension of these studies revealed that ADP-activated P2Y12 receptor is expressed in the kidney, and its irreversible blockade by the administration of clopidogrel bisulphate (Plavix(®)) ameliorates Li-induced NDI in rodents. Parallel in vitro studies showed that P2Y12 receptor blockade by the reversible antagonist PSB-0739 sensitizes CD to the action of AVP. Thus, our studies unravelled the potential beneficial effects of targeting P2Y2 or P2Y12 receptors to counter AVP resistance in lithium-induced NDI. If established in further studies, our findings may pave the way for the development of better and safer methods for the treatment of NDI by bringing a paradigm shift in the approach from the current therapies that predominantly counter the anti-AVP effects to those that enhance the sensitivity of the kidney to AVP action.

Daclizumab phase II trial in relapsing and remitting multiple sclerosis: MRI and clinical results.

OBJECTIVE: Daclizumab is an interleukin 2 receptor alpha chain specific humanized monoclonal antibody that has shown promising therapeutic effects in multiple sclerosis (MS). Daclizumab treatment in patients with relapsing and remitting MS was administered to determine effects on MRI and clinical outcomes. METHODS: Patients with MS on interferon (IFN) therapy but with continuing relapses and contrast enhancing lesions (CEL) were selected. Patients were evaluated with monthly MRI scans and clinical rating scales starting 3 months prior to treatment and then at 0.5 to 27.5 months during treatment. Daclizumab (1 mg/kg IV) was administered twice in the first month (initiated and administered again in 2 weeks), followed by treatments every 4 weeks. IFN was continued until 5.5 months after daclizumab was initiated. Patients were then placed on daclizumab monotherapy. Patients with recurrent CEL were restarted on IFN with daclizumab therapy at (1.5 mg/kg IV) every 28 days. RESULTS: Nine patients qualified for inclusion and completed the trial. Efficacy measured by both total CEL and new CEL (p < 0.001), relapses, timed ambulation, Expanded Disability Status Scale, and Neurologic Rating Scale (p < 0.05 to p < 0.01) was observed. CONCLUSION: Daclizumab was effective in reducing contrast enhancing lesions and improving clinical scores in patients with relapsing and remitting multiple sclerosis with active disease not controlled by interferon therapy. These results provide evidence for long-term efficacy and support further clinical development of daclizumab.

Identification of the amino acids on a neuronal glutamate receptor recognized by an autoantibody from a patient with paraneoplastic syndrome.

Autoantibodies from a patient with paraneoplastic disease were identified previously to bind to the glutamate receptor (GluR) subunit GluR5 and to function as potential allosteric modulators of receptor activity (Gahring et al. [1995] Mol Med 1:245-253). In the present study we have used deletion mapping and mutagenesis to define the residues in GluR5 bound by this autoreactivity. The autoantibody contact residues include residues K497, N508, K510, E512, and to a lesser extent Q507. Residues 507-512 confer autoantibody specificity of the autoreactivity to GluR5. These residues have been shown in crystallographic studies (Armstrong et al. [1998] Nature 395:913-917) to participate in a loop structure, whereas residue K497 is located on a beta-strand. Notably, this binding spans tyrosine 504, a residue important in forming the agonist-binding site. We propose that autoantibody binding of essential residues in this GluR5 autoantigenic region defines a subunit-specific allosteric regulatory site on neuronal glutamate receptors and suggests how receptor dysfunction and region-specific neuronal death in the brain can progress in certain autoimmune neurological diseases.

Granzyme B proteolysis of a neuronal glutamate receptor generates an autoantigen and is modulated by glycosylation.

Autoimmune processes are initiated when tolerance to self-proteins fails to be established or maintained and immune cells are stimulated by self-Ags. Although intracellular autoantigens are common, the origin of extracellular autoantigens is poorly defined and possibly more dangerous. In this study, we considered a mechanism for the origin of an extracellular autoantigen from the neuronal glutamate receptor subunit 3 (GluR3) in Rasmussen's encephalitis, a severe form of pediatric epilepsy. We demonstrate that specific cleavage of GluR3 by granzyme B (GB), a serine protease released by activated immune cells, can generate the GluR3B autoantigenic peptide, but only if an internal N:-linked glycosylation sequon within the GluR3-GB recognition sequence (ISND*S) is not glycosylated. However, this N:-glycon sequon while glycosylated normally is inefficiently used and glycosylation can fail. These results suggest that GB/N:-glycon sites may escape normal tolerance mechanisms and contribute to autoantibody-mediated immune diseases.

RNA editing (Q/R site) and flop/flip splicing of AMPA receptor transcripts in young and old brains.

The effects of aging on the efficiency of RNA processing of AMPA glutamate receptor (GluR) subunits GluR1, GluR2, and GluR 3 was examined for RNA editing at the Q/R site of GluR2 and for alternative splicing of the flip or flop exons for GluR1-3. RNA isolated from six young (3 months old) and old (22-23 months old) animals was reverse-transcribed for PCR and restriction endonuclease analyses to distinguish between edited forms of GluR2 and flip/flop isoforms of GluR1-3. Unedited transcripts of GluR2 at the Q/R site (which controls calcium permeability) were not detected (at the limit of detection of >/= 2.5%) from the corticies and hippocampi of young and old animals. Distribution of flop/flip isoforms in the cortex, hippocampus, hypothalamus, and striatum varied between GluR subunits and brain region, with GluR2 showing the greatest differences. However, no differences in alternative splicing of GluRs 1-3 were observed between young and old animals, suggesting that the fidelity of GluR transcript processing remains intact in the brains of aged animals.

Alcohol blocks TNFalpha but not other cytokine-mediated neuroprotection to NMDA.

BACKGROUND: We have investigated the effects of ethanol/alcohol (ETOH) on the pro-inflammatory CNS cytokine network that mediates neuroprotection to an excitotoxic challenge with the glutamate receptor agonist N-methyl-D-aspartic acid (NMDA). METHODS: Cultured murine cortical neurons were incubated with either TNFalpha, IL-1alpha, IL-1beta, or IL-6 in the presence or absence of 20 mM ETOH, maintained in an alcohol equilibrated humidified chamber, and the effects of these cytokines on neuronal survival after a chronic 20 hr exposure to NMDA was quantified. RESULTS: Neuroprotection induced by TNFalpha, but not IL-1alpha, IL-1beta, or IL-6, was inhibited by a concentration of alcohol (20 mM) that alone did not neuroprotect. Alcohol also affected the paracrine/autocrine induction of cytokine transcripts in neuronal cell cultures, which included enhancing the ability of TNFalpha to stimulate IL-6 transcripts. This result supports distinct cytokine-modulated neuroprotective pathways of which only TNFalpha is sensitive to low alcohol concentrations. We have shown previously that nicotine, acting through an alpha-bungarotoxin sensitive receptor, is also neuroprotective, but it too specifically abolishes TNFalpha-mediated neuroprotection. However, alcohol did not affect nicotine-induced neuroprotection. CONCLUSIONS: We suggest that the effects of low concentrations of alcohol on neuronal cytokine networks proceed through antagonism of neuroprotective pathway(s) unique to TNFalpha.

Inflammatory cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha impart neuroprotection to an excitotoxin through distinct pathways.

The proinflammatory cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha are produced within the CNS, and, similar to the periphery, they have pleotrophic and overlapping functions. We have shown previously that TNF-alpha increases neuronal survival to a toxic influx of calcium mediated through neuronal N-methyl-d -aspartic acid (NMDA) glutamate-gated ion channels. This process, termed excitotoxicity, is a major contributor to neuronal death following ischemia or stroke. Neuroprotection by this cytokine requires both activation of the p55/TNF receptor type I and the release of TNF-alpha from neurons, and it is inhibited by the plant alkaloid nicotine. Here, we report that other inflammatory cytokines (IL-1 alpha, IL-1 beta, and IL-6) are also neuroprotective to excessive NMDA challenge in our system. Neuroprotection provided by IL-1 is distinct from TNF-alpha because it is inhibited by IL-1 receptor antagonist; it is not antagonized by nicotine, but it is inhibited by a neutralizing Ab to nerve growth factor (NGF). Similar to IL-1, IL-6-mediated neuroprotection is also antagonized by pretreatment with IL-1 receptor antagonist and it is not affected by nicotine. However, neutralizing anti-NGF only partially blocks IL-6-mediated protection. These studies support an important role for distinct but overlapping neuroprotective cytokine effects in the CNS.

A model for a glutamate receptor agonist antibody-binding site.

A combination of mutagenesis, computer modeling and immunoreactivity has been used to develop a structural model of a segment of the glutamate receptor (GluR), termed GluR3B, which is bound by receptor-activating autoantibodies. In this model, the GluR3B epitope is located in a reverse hairpin loop that places key residues important for antibody recognition and receptor activation in a linear arrangement on the solvent-exposed surface. The conformation of the loop is stabilized by a hydrophobic core which is critical for functional integrity of the epitope. The proximity of the amino- and carboxy-terminal residues suggested that the GluR3B peptide could be cyclized without diminishing immunoreactivity through replacement of these residues with cysteines and formation of a disulfide bond. This prediction was confirmed experimentally since the cyclized peptide retained full immunoreactivity. The model provides insight into GluR subunit-specific functional diversity and the role of autoantibodies to this region in neurological disease.

Nicotine blocks TNF-alpha-mediated neuroprotection to NMDA by an alpha-bungarotoxin-sensitive pathway.

Excitotoxic neuronal death mediated by N-methyl-D-aspartate (NMDA) glutamate receptors can contribute to the extended brain damage that often accompanies trauma or disease. Both the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and nicotine have been identified as possible neuroprotective agents to NMDA assault. We find that TNF-alpha protection of a subpopulation of cultured cortical neurons to chronic NMDA-mediated excitotoxic death requires both the activation of the p55/TNFRI, but not p75/TNFRII, and the release of endogenous TNF-alpha. Nicotine protection to NMDA was mediated through an alpha-bungarotoxin-sensitive receptor. When coapplied, neuroprotection to NMDA by either TNF-alpha or nicotine was abolished but could be recovered with alpha-bungarotoxin. These results suggest that the cytokine TNF-alpha and alpha-bungarotoxin-sensitive nicotinic neurotransmitter receptors confer neuroprotection through potentially antagonistic pathways.

Antibodies prepared to neuronal glutamate receptor subunit3 bind IFNalpha-receptors: implications for an autoimmune process.

Anti-glutamate receptor (GluR) agonist-like antibodies prepared in rabbits that are similar to autoantibodies found in Rasmussen's encephalitis (RE) were found to exhibit greater immunoreactivity towards human interferon alpha receptor (IFNAR-1) than to GluR3. Since antibodies prepared to a defined region of GluR3 can react preferentially to an unrelated sequence in the human IFNAR-1, we propose that the IFNAR-1 may be an heteroclitic antigen of GluR3. These results suggest that differential reactivity toward heteroclitic antigens may contribute to variable clinical characteristics of certain autoimmune diseases.

Identification of amino acids in the glutamate receptor, GluR3, important for antibody-binding and receptor-specific activation.

We reported (Twyman, R. E., Gahring, L. C., Speiss, J., and Rogers, S. W. (1995) Neuron 14, 755-762) that antibodies to a subregion of the glutamate receptor (GluR) subunit GluR3 termed GluR3B (amino acids 372-395), act as highly specific GluR agonists. In this study we produced additional rabbit anti-GluR3B-specific antibodies, ranked them according to their ability to function as GluR agonists and characterized the immunoreactivity using deletion and alanine substitution mutagenesis. These anti-GluR3B antibodies bound to a subset of the residues in GluR3B (amino acids 372-386), of which glutamate 375, valine 378, proline 379, and phenylalanine (Phe) 380 were preferred. The level of GluR activation correlated with the binding of antibody to Phe-380, which suggests that immunoreactivity directed toward Phe-380 is an index for the anti-GluR agonist potential. Since the identity of this residue varies between respective GluR subunits, this suggested that this residue may be important for imparting antibody subunit specificity. To test this possibility, the alanine in GluR1 was converted to a phenylalanine, which extended the subunit specificity from GluR3 to the modified GluR1. We conclude that antibody contacts with key residues in the GluR3B region define a novel GluR subunit-specific agonist binding site and impart subunit-specific immunoreactivity.

Interleukin-1alpha in the brain is induced by audiogenic seizure.

We examined the expression of the sleep-inducing cytokine interleukin-1alpha (IL-1alpha) in the brains of audiogenic seizure-susceptible mice subsequent to the induction of sound-induced seizure. Animal models of epilepsy often require lesioning or trauma that may nonspecifically alter IL-1alpha expression. To avoid this, we employed the Frings mouse strain; a model of auditory-evoked reflex epilepsy. Frings mice were exposed to a high-intensity sound stimulus to induce a tonic extension seizure, and the expression of IL-1alpha transcripts in different brain regions was measured thereafter. Compared to control animals, IL-1alpha transcripts were elevated 6 to 8 h postseizure in the hypothalamus, but not hippocampus, by a dexamethasone-sensitive pathway. Similar results were obtained from the genetically distinct DBA/2J audiogenic seizure-susceptible mouse strain. These findings demonstrate that the expression of IL-1alpha is altered following generalized seizure activity, induced by noninvasive sensory stimulation, in a brain-region-specific manner.

Prostaglandin F2alpha is required for NMDA receptor-mediated induction of c-fos mRNA in dentate gyrus neurons.

Activation of NMDA receptors has been linked to a diversity of lasting physiological and pathological changes in the mammalian nervous system. The cellular and molecular mechanisms underlying permanent modifications of nervous system structure and function after brief episodes of neuronal activity are unknown. Immediate-early genes (IEGs) have been implicated in the conversion of short-term stimuli to long-term changes in cellular phenotype by regulation of gene expression. The intracellular signaling pathways coupling activation of receptors at the cell surface with induction of IEGs in the nucleus are incompletely understood. NMDA produces a striking increase in the IEG c-fos in dentate gyrus (DG) neurons in vitro; this induction is dependent, in part, on the arachidonic acid cascade. Here we show that NMDA receptor activation triggers the synthesis of the prostaglandins PGF2alpha and PGE2, but not PGD2, in rat cerebral cortical neurons in vitro. We further demonstrate that PGF2alpha, but not PGE2 or PGD2, is necessary but not sufficient for NMDA induction of c-fos mRNA in DG neurons. These findings provide insight into the molecular events coupling activation of the NMDA receptor with regulation of the IEG c-fos and identify the diffusable messenger PGF2alpha as obligatory for NMDA receptor-mediated transcription of a nuclear IEG.

Neuronal expression of tumor necrosis factor alpha in the murine brain.

We have examined the expression of the inflammatory cytokine, tumor necrosis factor alpha (TNF alpha) in the mouse brain. Using immunohistochemical methods developed, we found anti-TNF alpha immunoreactivity localized in the basal ganglia and other discrete brain structures. Constitutive immunoreactivity, present in normal, unstimulated brain, was observed in glial and microglial-like cells, but it was predominant in neuronal-like cells. Intravenous administration of lipopolysaccharide (LPS) increased TNF alpha transcript levels detected by RT-PCR in specific brain subregions in which contaminating blood cells were removed. The maximal increase occurred within 2 h of LPS injection; transcripts diminished to near control levels in the next 4 h. Immunocytochemical analysis and single-cell RT-PCR analysis of primary cultures of cortical neuronal cells confirmed expression of TNF alpha in cells that also express neuronal-specific enolase RNA. Addition of LPS or recombinant TNF alpha protein to neuronal cultures enhanced expression of TNF alpha transcripts. Our results indicate that in addition to glial and microglial cells, a well-defined subset of neuronal cells also express TNF alpha constitutively; this expression can be altered by both extrinsic (LPS) and intrinsic (TNF alpha itself) factors.

N-methyl-D-aspartate receptors activate transcription of c-fos and NGFI-A by distinct phospholipase A2-requiring intracellular signaling pathways.

Activation of N-methyl-D-aspartate (NMDA) receptors is required for induction of some lasting changes in nervous system structure and function. The cellular mechanisms involved in transducing receptor stimulation into long-lasting changes in cellular activity are unknown. Immediate-early genes (IEGs) have been implicated in the conversion of short term stimuli to long term changes in cellular phenotype, by regulation of gene expression. Activation of NMDA receptors on dentate gyrus neurons triggers the transcriptional activation of several IEGs. To determine whether the same intracellular pathways transduce the signal from this ligand-gated ion channel to the nucleus, we compared NMDA induction of two IEGs. NMDA was sufficient to produce a striking increase in both c-fos and NGFI-A mRNAs in dentate granule neurons, in a calcium-dependent manner. The induction of both IEGs was blocked by structurally distinct inhibitors of phospholipase A2, an enzyme that catalyzes phospholipid degradation and formation of arachidonic acid. Arachidonic acid itself is catalyzed to biologically active metabolites by multiple enzymes, including cyclooxygenase and lipoxygenase. Selective inhibitors of cyclooxygenase attenuated NMDA induction of c-fos but not NGFI-A. Conversely, structurally distinct inhibitors of lipoxygenase blocked NMDA induction of NGFI-A but not c-fos. The signaling pathways linking NMDA receptors to the transcriptional activation of c-fos and NGFI-A are related but distinct. We suggest that phospholipase A2 and the arachidonic acid cascade play a pivotal role in NMDA receptor regulation of gene expression.

Cooperative DNA-protein interactions. Effects of changing the spacing between adjacent binding sites.

Cooperative binding of specific DNA-binding proteins plays crucial roles in gene regulatory circuitry, and is a model system for interactions between proteins bound to DNA. We have studied coliphage HK022 repressor, which binds to two adjacent operators with a cooperativity parameter of approximately 2000. We examined the effect of changing the spacing between these two operators on cooperativity and on the conformation of the complex. Maximum cooperativity was seen with the wild-type spacing; considerable cooperativity was retained for most spacing variants, but was abolished when the operators lay on opposite faces of the DNA helix. Most spacing variants conferred changes in the conformation of the DNA-protein complex. Our data indicate that the pairwise cooperativity observed with the wild-type spacing results from a conformation that prevents protein-protein contacts with flanking bound dimers. We conclude that protein-DNA complexes involving the same specific binding sites and the same protein molecules can adopt many different conformations, depending on the spacing between the binding sites. This conclusion may be broadly applicable to protein-DNA interactions in other systems.

A novel antivirulence element in the temperate bacteriophage HK022.

Lysogens of the temperate lambdoid phage HK022 are immune to superinfection by HK022. Superinfection immunity is conferred in part by the action of the HK022 CI repressor at the O.R operators. In this work, we have identified an additional regulatory element involved in immunity. This site, termed OFR (operator far right), is located just downstream of the cro gene, more than 250 nucleotides distant from OR. The behavior of phage containing a mutation in OFR suggests that the wild-type site functions as an antivirulence element. HK022 OFR- mutants were able to form turbid plaques indistinguishable from those of the wild type. However, they gave rise to virulent derivatives at a far higher frequency than the wild type (approximately 10(-5) for OFR- versus about 10(-9) for the wild type). This frequency was so high that cultures of HK022 OFR- lysogens were rapidly overgrown by virulent derivatives. Whereas virulent mutants arising from a wild-type OFR+ background contained mutations in both OR1 and OR2, virulent derivatives of the OFR- mutant phage contained a single mutation in either OR1 or OR2. We conclude that the wild-type OFR site functions to prevent single mutations in OR from conferring virulence. The mechanism by which OFR acts is not yet clear. Both CI and Cro bound to OFR and repressed a very weak rightward promoter (PFR). It is unlikely that repression of PFR by CI or Cro binding to OFR can account in full for the antivirulence phenotype conferred by this element, since PFR is such a weak promoter. Other models for the possible action of OFR are discussed.

Highly cooperative DNA binding by the coliphage HK022 repressor.

The CI repressor protein from the temperate lambdoid phage HK022 was purified to near homogeneity and used in DNase I footprinting analyses to identify six binding sites in this phage. All these sites contained homologous 15 bp inverted repeats. Three of these 15 bp inverted repeats were located between the cI and cro (OR1 to OR3), and the other three were 3' to the cI gene (OL1 to OL3). Two of these sites were identified as operator sites for the repressor by DNA sequence analyses of virulent phage mutants. Almost all these mutations identified lay within the 15 bp inverted repeats comprising OR1 and OR2, and almost all were in the most highly conserved positions in the operators. The majority of virulent mutants contained mutations in both OR1 and OR2. Intrinsic affinities for individual operators were measured by DNase I footprinting analyses using DNA fragments which contained a single wild-type operator adjacent to two mutant operators. Comparison of these values with the affinity observed with these sites in the wild-type operator indicated that HK022 CI repressor bound cooperativity to OR1 and OR2 with a cooperativity parameter, omega, of almost 2000. Cooperative binding occurred in an alternative pairwise fashion, as previously seen with lambda CI repressor. In addition to cooperative binding between two adjacent operators, the repressor also increased the affinity for adjacent non-specific DNA sites, resulting in a periodic pattern of binding termed "phasing". This phasing pattern extended beyond regions predicted for pair-wise interaction, but was significantly decreased on a template with two adjacent operators, suggesting that pairwise cooperativity interfered with phasing.