Immunolocalization of iron regulatory protein expression in the murine central nervous system.

We examined the expression of the iron regulatory proteins 1 and 2 (IRP1 and IRP2) in the brains of adult (4-6 months) CBA/J mice. Anti-IRP1 immunoreactivity was localized to cell bodies, including putative neurons and oligodendrocytes. In contrast, anti-IRP2 staining was prevalent throughout the neuropil of regions of the brain consistent with the central autonomic network (CAN) and mossy fibers emanating from hippocampal dentate granule cells. Essentially no staining for IRP2 was observed in the cerebellum in contrast to strong IRP1 immunoreactivity in Purkinje cells. Notably, cells within one vestibular nucleus exhibited staining by both IRP1 and IRP2. Our results suggest distinct roles for IRP1 and IRP2 in the regulation of iron homeostasis in the mammalian nervous system where IRP1 may provide a maintenance function in contrast to IRP2 that could participate in modulating proper CAN functions, including cardiopulmonary, gustatory as well as fine motor control.

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.

Neuronal nicotinic acetylcholine receptor expression by O2A/oligodendrocyte progenitor cells.

Oligodendrocyte precursor cells (O2A/OPC, A2B5(+)) were examined for expression of neuronal nicotinic acetylcholine receptors (nAChR). RT-PCR analysis and immunocytochemistry of O2A/OPCs purified from the rat corpus collusum revealed the expression of nAChR subunits alpha3, alpha4, alpha5, alpha7, beta2, and beta4. Immunoreactivity toward nAChR subunits was not detected in cells induced to differentiate into either oligodendrocytes or astrocytes. Approximately 65% of O2A/OPCs loaded with the calcium-responsive dye FURA-2 increased their intracellular free calcium in response to nicotine application. This response was sensitive to the nAChRalpha4/beta2 antagonist, dihydro-beta-erythroidine (DHbetaE), and the voltage-gated calcium channel antagonist, nifedipine. A subset of nicotine-responsive cells (37%) established DHbetaE or nifedipine-sensitive intracellular free calcium oscillations that continued in the presence of nicotine. Typical oscillations occurred at intervals of 20 to 30 s with progressively diminished amplitudes over a period of 2 to 3 min. In rare cases, oscillations persisted for as long as 10 min. O2A/OPCs exposed to carbachol or AMPA produced no oscillations despite robust increases in intracellular free calcium. The expression of nAChRs in non-neuronal glial precursor cells suggests an expanded role for this receptor system in the development of the mammalian brain. GLIA 33:306-313, 2001. Published 2001 Wiley-Liss, Inc.

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.

Age-related changes in neuronal nicotinic acetylcholine receptor subunit alpha4 expression are modified by long-term nicotine administration.

The distribution of the neuronal nicotinic acetylcholine receptor subunit alpha4 (nAChRalpha4) in the brains of young (2-4 months) or aged (24-28 months) CBA/J mice was examined using immunohistochemical staining. Anti-nAChRalpha4 immunoreactivity corresponded with nAChRalpha4 RNA expression and high-affinity [3H]nicotine binding. Immunostaining in aged mice relative to that in young animals was diminished in the medial septum and diagonal band but was unchanged in the globus pallidus and substantia nigra. The staining of neurons was almost completely absent in the hippocampus of aged animals. The oral administration of nicotine to aged animals for 6 weeks did not alter nAChRalpha4 expression relative to that in aged controls. However, the long-term delivery of nicotine (11 months) to 14-month-old animals corresponded with the highly specific preservation of nAChRalpha4 expression in some neurons of the dentate gyrus region and in neurite processes of remaining neurons of the hippocampal CA1 region. These results support the conclusion that the loss of nAChRalpha4 expression occurs in key cholinergic regions during normal aging. Furthermore, sustained long-term nicotine delivery may promote highly region-specific retention of nAChR expression, but only if initiated before normal age-related receptor decline.

Glutamate receptor GluR1 expression is altered selectively by chronic audiogenic seizures in the Frings mouse brain.

The audiogenic seizure-susceptible mouse, Frings, is genetically susceptible to sound-induced seizures and provides a reliable model of reflex epilepsy that lasts throughout the life span of the animal. We used immunohistochemistry to examine if the expression of the non-N-methyl-D-aspartate glutamate receptor (GluR) subunits GluR1, GluR2, or GluR3 were altered subsequent to multiple seizures. Following a regimen of one seizure per day for 3 weeks, GluR1 immunoreactivity, but not GluR2 or GluR3, was substantially elevated in the outer shell of the nucleus accumbens in 21 of 31 chronically seized Frings mice. No other brain regions such as the hippocampus exhibited any qualitative changes in expression of these subunits. In 9 of the 21 Frings mice exhibiting increased GluR1, but in none of the controls, bilateral structural lesions were observed in the lateral hypothalamus. These results support a model where highly localized changes in the expression of GluR1 occur in response to repeated audiogenic seizure.

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.

Autoantibodies to glutamate receptor subunit GluR2 in nonfamilial olivopontocerebellar degeneration.

We describe a 63-year-old man with a 5-year history of progressive sporadic olivopontocerebellar atrophy (OPCA) who exhibits high serum titers of IgM autoantibodies to the neuronal glutamate receptor subunit GluR2. Immunohistochemistry revealed intense staining of mouse cerebellar Purkinje cells and cells in the pontine nuclei and olivary complex. Glutamate receptor currents were activated in a subset of cultured mouse neurons by an anti-GluR2 IgM fraction, and they were blocked by the competitive AMPA-type glutamate receptor antagonist CNQX and by a synthetic peptide to a specific epitope region of GluR2 (AA 369-393). The patient was treated with nine courses of plasmapheresis with little improvement of symptomatology. However, IgM titers to GluR2 decreased approximately 8-fold and the serum functional activity decreased proportionally. These findings may suggest a role for autoimmunity to glutamate receptors in the pathophysiology of certain forms of progressive nervous system degeneration.

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.

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.

Kainic acid induced excitotoxicity and cfos expression in fibroblasts transfected with glutamate receptor subunit, GluR1.

Glutamate receptors participate in the majority of fast excitatory neurotransmission in the mammalian brain. Excessive excitation of these receptors has been linked to neuronal dysfunction and death through a process termed excitotoxicity. In this study we demonstrate that transfection of a single non-NMDA glutamate receptor subunit, GluR1, into cultured fibroblasts is sufficient to confer kainic acid mediated excitotoxicity similar to that seen in neuronal cells. Death of transfected cells requires at least 24 h of continuous exposure to kainic acid and can be blocked with a glutamate receptor antagonist. Also, the induction of protooncogene cfos transcripts occurs 30 min following kainic acid administration, and Fos protein accumulated in the nucleus within 90 min. These observations suggest that the signaling system(s) required to initiate gene expression and kainic acid excitotoxicity from a neuronal ionotropic receptor to the nucleus is present in these nonneuronal cells. Finally, antibodies prepared to amino acids 185-449 of GluR1 are demonstrated to be useful for fluorescence-activated sorting of live cells transfected with a GluR1 expression vector. This supports the conclusion that this region of the protein is located extracellularly.

Nicotinic receptor subunits alpha 3, alpha 4, and beta 2 and high affinity nicotine binding sites are expressed by P19 embryonal cells.

Controlled exposure to retinoic acid (RA) induces the murine embryonal carcinoma cell line P19S18O1A1 (P19) to differentiate into a variety of cell types. One of the cell types exhibits neuronal-like morphology and expresses neuronal markers including neurofilament proteins, glutamate receptors, and the cholinergic enzymes choline acetyl-transferase and acetylcholinesterase. In this study we use Northern blot analysis, double-label immunocytochemistry, and single cell RNA analysis using polymerase chain reaction to show that RA-treated P19 cells with neuronal-like morphology also express neuronal nicotinic acetylcholine receptor (nAChR) subunits alpha 3, alpha 4, and beta 2. Greater than 80% of RA-treated P19 cells with a neuronal-like phenotype express nAChR alpha 4 subunit transcripts and both alpha 4 and beta 2 protein. The RA-induced expression of alpha 3 transcripts accounts for a comparably small number of nAChR-containing cells (< 20%) of which half coexpress alpha 4 transcripts. Expression of high-levels of alpha 4 RNA is dependent upon both cell-cell contact and RA exposure. The appearance of nAChR subunits also coincides with RA-induced expression of high affinity [3H]-nicotine binding receptors. The P19 cell line offers an inducible neuronal cell system to study mammalian neuronal nicotinic receptor expression and the development of high affinity nicotinic binding sites similar to those expressed in the mammalian central nervous system.

The role of autoimmunity to glutamate receptors in neurological disease.

Understanding and experimentally approaching the processes that underlie the origin and progression of many severe neurological disorders presents a challenge to both clinical and basic researchers. We have found that the origin of some neurological diseases, including a rare form of childhood epilepsy and a neurodegenerative disease associated with peripheral cancers, may be related to production of physiologically active autoantibodies that are directed towards excitatory ionotropic glutamate receptors of the brain. This suggests that some neurological diseases may result from dysfunction of the immune system.

Glutamate receptor antibodies activate a subset of receptors and reveal an agonist binding site.

Two rabbits immunized with a portion of glutamate receptor (GluR) subunit GluR3 (amino acids 245-457) exhibited seizure-like behaviors, suggesting that antibodies to GluR3 may modulate neuronal excitability. Using whole-cell recording, rabbit GluR3 antisera were tested on cultured fetal mouse cortical neurons. In a subset of kainate-responsive neurons, miniperfusion of antisera and IgG evoked currents that were blocked by CNQX. Immunoreactivity to synthetic peptides prepared to subregions GluR3A (amino acids 245-274) and GluR3B (amino acids 372-395) was present in both rabbit sera. Peptide GluR3B, but not GluR3A, specifically blocked antisera- and IgG-evoked currents. Similar receptor activation and anti-GluR3 reactivity was present in sera from patients with active Rasmussen's encephalitis, an intractable pediatric epilepsy. Thus, antibodies to GluR3 define a region involved in agonist binding and specific receptor activation. These data suggest that antibodies to neuronal receptors can function as agonists and that autoantibodies to GluRs may be highly specific neurotoxicants in some neurological diseases.