Structurally different neuronal nicotinic acetylcholine receptor subtypes purified and characterized using monoclonal antibodies

Acetylcholine receptors that bind nicotine with high affinity but do not bind alpha-bungarotoxin have recently been immunoaffinity purified from brains of chickens and rats (Whiting and Lindstrom, 1986a, b; Whiting and Lindstrom, 1987a). Antisera to these receptors bind to the nicotinic receptors that regulate cation channel opening on chick ciliary ganglion neurons (Stollberg et al., 1986) and rat PC12 cells (Whiting et al., 1987c). Here we report the preparation and characterization of monoclonal antibodies to chicken brain acetylcholine receptors. These monoclonal antibodies are used to identify 2 nicotinic receptor subtypes in the chicken brain. The 2 subtypes have very similar affinities for nicotine and other cholinergic agonists and antagonists. However, they are structurally distinct, having very similar or identical alpha subunits (Mr 49,000), but different beta subunits (Mr 59,000, or for beta' subunit, Mr 75,000). Evidence is presented that suggests that the subunit stoichiometry of these neuronal nicotinic acetylcholine receptors is alpha n = 2 - 3 beta n = 2 - 3. Different levels of receptor subtype expression were detected in embryonic, compared to adult, chicken brain.     

Pathogenic autoantibodies to neuronal proteins in neurological disorders

Autoantibodies to acetylcholine receptors and to voltage-gated calcium and potassium channels are thought to be pathogenic in three peripheral neurological disorders: myasthenia gravis, the Lambert Eaton syndrome and acquired neuromyotonia. However, evidence for the role of antibodies in conditions involving the central nervous system, is scanty or unclear. This review describes the ways in which the roles of autoantibodies have been defined in the peripheral diseases, and discusses the more controversial evidence for involvement of autoantibodies in some central disorders such as multiple sclerosis.     

The role of impaired neuronal communication in neurological disorders

PURPOSE OF REVIEW: Basic and translational neuroscience findings indicate that normal brain function depends on activity synchronization within distributed brain networks. This conclusion suggests a view of how brain injury causes behavioral deficits that differs from traditional localizationist views. RECENT FINDINGS: Novel functional neuroimaging methods demonstrate coherent activity in large-scale networks not only during task performance but also, surprisingly, at rest (i.e. in the absence of stimuli, tasks, or overt responses). Furthermore, breakdown of activity coherence at rest, even in regions of the brain that are structurally intact, correlates with behavioral deficits and their recovery after injury. Breakdown of functional connectivity appears to occur not just after local injury but also in other conditions that affect large-scale neural communication. SUMMARY: A network perspective is fundamental to appreciating the pathophysiology of brain injury at the systems level and the underlying mechanisms of recovery, and for developing novel strategies of rehabilitation.     

Antibodies and neuronal autoimmune disorders of the CNS

We review the neuronal antibodies described in CNS disorders in order to clarify their diagnostic value, emphasize potentials pitfalls and limitations in the diagnosis of paraneoplastic neurological syndromes (PNS), and examine the current evidence for a possible pathogenic role. We propose to classify the neuronal antibodies associated with syndromes resulting from CNS neuronal dysfunction into two groups according to the location of the antigen: inside the neuron or in the cell membrane. Group I includes antibodies which target intracellular antigens and probably are not pathogenic. They are further subdivided into three groups. Group Ia comprises well-characterized onconeural antibodies (Hu (ANNA1), Yo (PCA1), Ri (ANNA2), CV2 (CRMP5), amphiphysin, Ma2) that are useful for the diagnosis of PNS. Group Ib antibodies (SOX and ZIC) are cancer-specific but there is no evidence that the immune response is in any way pathogenically related to the PNS. Antibodies in group Ic (glutamic acid decarboxylase (GAD), adenylate kinase 5 and Homer 3) identify non-PNS: stiff-person syndrome (SPS), cerebellar ataxia, and limbic encephalitis (LE). Group II antibodies recognize neuronal surface antigens. Antibodies in group IIa associate with characteristic CNS syndromes but their detection does not indicate that the disorder is paraneoplastic. Antibodies to potassium channels, AMPA and GABA_B receptors are associated with LE, NMDA receptor antibodies identify a well-defined encephalitis, and antibodies against glycine receptors associate with SPS with encephalitis. A pathogenic role of the antibodies is suggested by the response of symptoms to immunotherapy and the correlation between antibody titers and neurological outcome. Lastly, Group IIb includes antibodies that are found in patients with paraneoplastic cerebellar ataxia associated with lung cancer (P/Q type calcium channels antibodies) or Hodgkin disease (metabotropic glutamate receptor type 1 antibodies).     

Neuroprotection by nicotine against hypoxia-induced apoptosis in cortical cultures involves activation of multiple nicotinic acetylcholine receptor subtypes

Activation of neuronal nicotinic acetylcholine receptors (nAChR) by nicotine has been suggested to protect neurons against a hypoxic insult. The objective of this study was to examine the nature of cell death induced by acute hypoxia in rat primary cortical cultures and the neuroprotective potential of nicotine in ameliorating these processes. Neuronal cell death induced by a 4-h exposure to hypoxia (0.1% O(2)) was apoptotic, as shown by TUNEL staining and assays monitoring DNA strand breaks and caspase-3/7 activity. The presence of nicotine (10 microM) during the hypoxic insult protected a subpopulation of susceptible neurones against DNA damage and apoptosis induced by oxygen deprivation. This protective effect of nicotine was prevented by a 30-min pre-incubation with either 100 nM alpha-bungarotoxin or 1 microM dihydro-beta-erythroidine, but not 1 microM atropine, suggesting that activation of at least two subtypes of nAChR, alpha7 and beta2* nAChR, is involved in mediating nicotine neuroprotection.     

Regulation of neuronal nicotinic receptor traffic and expression

Neuronal nicotinic acetylcholine receptors (nAChRs) are a family of cation channels widely distributed in the brain, whose subunit composition and biophysical properties vary depending on the subtype and the area of the brain in which they are found. Brain nAChRs are also the target of nicotine, the most widespread drug of abuse. Chronic nicotine exposure differentially affects the number, subunit composition, stoichiometry and functional state of some nAChR subtypes, leaving others substantially unaffected. In this review, we will summarise recent data concerning the nAChR subtypes expressed in the CNS, and how they are regulated by means of chronic nicotine and/or nicotinic drugs. We will particularly focus on the possible mechanisms involved in the up-regulation of nAChRs.     

Pro-inflammatory cytokines modify neuronal nicotinic acetylcholine receptor assembly

We have examined the impact of the inflammatory cytokines interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) on assembly of nAChRs from subunit mixtures of nAChRalpha4, beta2 and beta4 transiently transfected into 293 cells. In control transfections approximately 55% of alpha4 associated preferentially with beta4, but less than 15% complexed with beta2 and the remainder was associated with both beta subunits. These relative ratios were modified by pro-inflammatory cytokines. IL-1beta strongly enhanced alpha4/beta2 association and decreased alpha4/beta4, whereas TNFalpha promoted mixed alpha4/beta2/beta4 interactions. These results show that the emerging rules governing assembly of nAChRs are subject to modification by the pro-inflammatory cytokine environment.     

Antibodies against glutamic acid decarboxylase: prevalence in neurological diseases

High prevalence of autoantibodies against glutamic acid decarboxylase (GAD-Ab) in stiff man syndrome (SMS) not only helps diagnosis, but also suggests immune mediated impairment of GABAergic functions. However, the presence of GAD-Ab has also been reported in other neurological syndromes. Therefore the prevalence of GAD-Ab was investigated in SMS, progressive encephalomyelitis with rigidity and myoclonus (PERM), and in other neurological diseases (OND).Serum antibodies against the GAD isoforms, GAD65 and GAD67, were investigated with radioimmunoassays in 13 patients with SMS, nine with PERM, 279 consecutive patients with OND, and in 100 normal controls. RESULTS: Prevalence of GAD65Ab was around 80% in patients with SMS/PERM compared with 5% in patients with OND and 1% in normal controls. Prevalence of GAD67Ab was 60% in SMS/PERM, 2% in patients with OND, and 1% in normal controls. Raised GAD-Ab clustered in an OND subgroup with sporadic progressive ataxia, but not in OND subgroups with recognised neuroimmunological diseases. In conclusion, increased GAD-Ab is neither a non-specific epiphenomenon of neuronal damage nor a common feature of recognised neuroimmunological disorders. In neurological diseases, GAD-Ab may be a pathogenetic agent or a marker for an ongoing autoimmune process, or both.     

Expression of nicotinic acetylcholine receptor subtypes in brain and retina.

Neuronal nicotinic acetylcholine receptors (AChRs) are composed of two types of subunits: ACh-binding (termed alpha 2, alpha 3, alpha 4 ...) and structural (termed beta 2, beta 3, beta 4 ...). AChR subtypes composed of combinations of subunits of these two types encoded by several related genes are expressed in different parts of the nervous system, where they presumably serve different functional roles. Here we identify the ACh-binding subunit of the most prominent chicken brain AChR subtype by N-terminal amino acid sequence and show that it corresponds to the alpha 4 gene. Previously we identified the structural subunit for this AChR subtype from chicken brain as beta 2 by N-terminal amino acid sequence. Thus, this identifies both genes which encode subunits of the major nicotinic AChR subtype in avian brains. By immunoprecipitation, immunohistochemistry, and northern blot analysis we show that alpha 3 (or a very closely related sequence) is expressed at low levels in the brain and relatively high levels in the retina, while alpha 4 is expressed at high levels in the brain and lower levels in the retina. This differential expression indicates that alpha 3-containing 'ganglionic-type' AChRs may be an important AChR subtype in avian retina.     

Influence of development and aging on nicotinic receptor subtypes in rodent brain

The influence of development and aging on nicotinic receptor subtypes in rodent brain was investigated. ³H-nicotine and ³H-acetylcholine (³H-ACh) were used as receptor ligands. Specific binding sites for ³H-nicotine and ³H-ACh were detected in mouse brain during the late prenatal period. A drop in the number of ³H-nicotine and ³H-ACh binding sites was measured shortly after birth. The ³H-nicotine and ³H-ACh binding sites showed different time courses during prenatal development. Competition experiments using unlabelled (−)nicotine and³H-nicotine revealed one population of high affinity nicotinic binding sites in the cortex of 1-day and 5-day-old mice whereas both a set of high and low affinity binding sites were found in adult mice. The proportion of cortical high and low affinity nicotinic binding sites did not change with aging although the absolute amount of high affinity nicotinic binding sites decreased. The ³H-nicotine binding showed different temperature dependence in rat brain of different ages. The results illustrate dynamic changes in nicotinic receptor properties during life span of rodents.     

Metabotropic glutamate receptor subtypes independently modulate neuronal intracellular calcium

Metabotropic glutamate receptors (mGluRs) modulate several G-protein-related signal transduction pathways including intracellular calcium (iCa(2+)) that control both neuronal development and demise. As an initial investigation, we characterized the ability of specific mGluR subtypes to modulate iCa(2+) by using Fura-2 microfluorometry in primary hippocampal neurons. Activation rather than inhibition of the metabotropic system with the group I and group II mGluR agonist 1S, 3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), the specific group I agonist (S)-3,5-dihydroxyphenylglycine (DHPG), and the specific group II agonist (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (LCCG-I) increased iCa(2+) with increasing concentrations. In contrast, the group III mGluR agonist, L(+)-2-amino-4-phosphonobutyric acid (L-AP4) produced no significant increase in iCa(2+). Through the pharmacological modulation of individual mGluR subtypes, we further examined the role of iCa(2+) release by the mGluR system. Release of iCa(2+) by both 1S,3R-ACPD and LCCG-I was prevented only through the administration of the antagonists (2S)-alpha-ethylglutamic acid (EGlu; mGluR2 and mGluR3) and (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-IV; mGluR2), suggesting that the mGluR2 subtype was responsible for the release of iCa(2+). As a control, the group I antagonists, L(+)-2-amino-3-phosphonopropionic acid (L-AP3) and (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), prevented DHPG release of iCa(2+) but were ineffective against iCa(2+) release by 1S,3R-ACPD. Although extracellular calcium influx did not significantly contribute to the release of iCa(2+) by the mGluR system, pharmacological inhibition of calcium-induced calcium-release-sensitive calcium pools played a critical role in the release of iCa(2+). Further characterization of the cellular calcium pools modulated by the mGluR subtypes may provide greater insight into the mechanisms that mediate neuronal function.     

N-methyl-D-aspartate receptor subtypes: multiple roles in excitotoxicity and neurological disease.

N-methyl-D-aspartate (NMDA) receptors are the major mediator of excitotoxicity. Although physiological activation of the NMDA receptor is necessary for cell survival, overactivation is a signal for cell death. Several pathways are activated through NMDA receptor stimulation, most of which can contribute to excitotoxicity. These include events leading to mitochondrial dysfunction, activation of calcium-dependent enzymes, and activation of mitogen-activated protein kinase pathways. Understanding the role of these mechanisms is important in developing agents that block excitotoxicity without inhibiting functions necessary for survival. NMDA receptor subtypes may be responsible for mediating separate pathways, and subtype-specific inhibition has shown promising results in some neurological models. This review examines the roles of NMDA receptor subtypes in excitotoxicity and neurological disorders.     

Lead modulation of the neuronal nicotinic acetylcholine receptor in PC12 cells

Lead is known to modulate several ligand- and voltage-gated ion channels, including the nicotinic acetylcholine receptor (AChR) channel. We examined the effects of lead on the nicotinic AChR in rat clonal phaeochromocytoma PC12 cells using whole-cell and single-channel patch-clamp techniques to clarify the detailed mechanism of action. Lead suppressed acetylcholine-induced currents in a dose-dependent manner with an EC₅₀ value of 37 μM and a Hill coefficient of 0.82. At the single-channel level, 1–10 μM lead shortened the opening and burst durations, and increased the duration of mean closed time. The open probability was significantly decreased by lead. These changes of single-channel kinetics result in a significant decrease in the total charge carried through the open AChR channels explaining the suppressive effect of lead on acetylcholine-induced whole-cell currents. © 1997 Elsevier Science B.V. All rights reserved.     

Calcium permeability of neuronal nicotinic acetylcholine receptor channels in PC12 cells

The conductance properties of neuronal nicotinic acetylcholine receptors (neuronal nAChR channels) in PC12 cells were studied using single channel and whole cell gigaohm seal voltage clamp techniques. Acetylcholine receptor agonists were applied using external pipettes. Neuronal nAChR channels were selective for monovalent cations, and were impermeable to anions. Based on measurements of shifts in reversal potential with changes in external Na and K ion concentrations and evaluation of the GHK constant field relation,P_K/P_Na was 1.45 (ignoring Ca permeability).P_Ca/P_K was 1.75 based on the shift in reversal potential seen when raising the external Ca concentration from 2 to 50 mM (holding the external Na ion concentration constant), and determining the best fit to the extended GHK constant field relation using ionic activities. This value is larger than that determined for the muscle-type nAChR channel. Evaluation of the extended GHK current equations using these permeability coefficients indicated that at subthreshold voltages approximately 5% of total current through neuronal nAChR channels will be carried by Ca ions. Thus, these channels may be important mediators of activity-dependent Ca influx in the nervous system.     

Characterization of nicotinic receptors in chick retina using a snake venom neurotoxin that blocks neuronal nicotinic receptor function

Nicotinic receptor function has been described in the retinas of a variety of vertebrate species. Neuronal bungarotoxin (NBT, also known as bungarotoxin 3.1, toxin F, or kappa-bungarotoxin) blocks nicotinic receptors in several neuronal preparations, while the neuromuscular antagonist alpha-bungarotoxin (BGT) fails to block most of these receptors. NBT (100 nM), but not BGT (10 microM), substantially blocks nicotinic function on ganglion cells in intact chick retina. 125I-NBT binds to 2 sites in homogenates of chick retina; one site that is shared with BGT (Kd = 5-7 nM, Bmax approximately 500 fmol/retina) and one which is not (Kd = 2-3 nM, Bmax approximately 100 fmol/retina). 125I-NBT binding to the NBT-specific site (binding in the presence of 1 microM unlabeled BGT) is localized to 2 bands in the inner plexiform layer, corresponding to regions richly innervated by neurons containing immunoreactivity for choline acetyltransferase. Furthermore, this binding is blocked by competitive nicotinic agonists and antagonists, but nicotine or other nicotinic agonists do not displace 125I-NBT binding with very high affinity relative to the displacement of 3H-nicotine reported by others in brain. Thus, of the 2 NBT binding sites, the site not recognized by BGT most likely represents functional nicotinic receptors in the chick retina, but these receptors have relatively low affinity for nicotinic agonists, similar to nicotinic receptors found in autonomic ganglia.     

Direct actions of anticholinesterases on the neuronal nicotinic acetylcholine receptor channels

Recent studies have suggested that anticholinesterases including organophosphates and carbamates act directly on the nicotinic acetylcholine receptor (AChR) channel. We performed whole-cell and single-channel patch-clamp experiments to elucidate the mechanism of action of anticholinesterases on the nicotinic AChR in rat clonal phaeochromocytoma (PC12) cells. Neostigmine and carbaryl showed a biphasic effect; enhancement and suppression of carbachol-induced whole-cell currents. The currents induced by 100 μM carbachol was enhanced by the first co-application with 10 or 100 μM neostigmine, and the current was eventually suppressed below the control level during repeated co-applications. The decay phase of current was accelerated by neostigmine. Carbaryl at 0.1 μM greatly potentiated the carbachol-induced current, and at higher concentrations (0.3–3 μM), current was suppressed. In single-channel experiments, these compounds increased the short closures or gaps during channel opening without changing the single-channel conductance. Mean open time and burst duration were decreased in the presence of neostigmine and carbaryl. These results indicate that neostigmine and carbaryl directly block the nicotinic AChR channel.     

Antibodies to simian virus 5 in patients with multiple sclerosis and other neurological disorders

Antibodies to the paramyxovirus simian virus 5 were measured in cerebrospinal fluid samples using an enzyme-linked immunosorbent assay. Six of the 13 clinically definite MS patients had elevated levels of antibodies compared with other neurological disease and orthopaedic controls. None of the samples from MS patients classed as probable or possible had increased amounts of SV5 antibodies. Simian virus 5 antibodies and measles antibodies showed a weak correlation and it is suggested that the elevated levels of the former are a manifestation of the increased antiviral response found in some MS patients.     

Association study of a nicotinic receptor variant with schizophrenic disorders

Nicotine acetylcholine receptors (nAChRs) are implicated in the pathogenesis of schizophrenia because the prevalence of smoking among schizophrenic patients is extraordinarily high, and nicotine has been demonstrated to improve some psychophysiological dysfunction in schizophrenics. In addition, recent studies have suggested linkage of the alpha(7) nAChR gene region in families of schizophrenics. In a population-based association study, we tested the hypothesis that the allelic variant, with a 2-bp deletion, of the human alpha(7) nAChR gene confers susceptibility to schizophrenic disorders. We genotyped alpha(7) nAChR in 146 patients with schizophrenic disorders and 151 controls. The results showed no significant difference in genotype or allele frequencies between schizophrenic patients and control subjects. This suggests that alpha(7) nAChR 2-bp deletion plays no major role in the pathogenesis of schizophrenic disorders. Other nAChR variants in schizophrenic disorders may need further investigation.