[Stem cell-based in vitro models as alternative methods for toxicity and efficacy tests in animals]. / Stammzellbasierte In-vitro-Modelle als Ersatz für Tiermodelle bei Toxizitäts- und Wirksamkeitsprüfungen.

Regarding toxicity and efficacy tests of pharmacological and chemical substances (REACH legislation in Europe), there is a strong need to develop alternative methods for animal in vivo studies, in particular for human in vitro models. Here we present results from early phases of projects exploring the potential of embryonic stem cell models, with a special emphasis on embryo toxicity and neuronal stress.We have been able to demonstrate key functional read-outs of neural hESC models, in addition to representing mechanistic aspects which are characteristic for ischemia or excitotoxicity. There is agreement that these mechanisms underlie a variety of human neurodegenerative diseases. We discuss the possibilities to develop more precise endpoints on the molecular level and present an example of a protein biomarker signature emerging from a European FP6 project about embryo toxicity (www.reprotect.eu), employing murine and human embryonic stem cell models.

Synthesis, dopamine D2 receptor binding studies and docking analysis of 5-[3-(4-arylpiperazin-1-yl)propyl]-1H-benzimidazole, 5-[2-(4-arylpiperazin-1-yl)ethoxy]-1H-benzimidazole and their analogs.

5-[3-(4-Arylpiperazin-1-yl)propyl]-1H-benzimidazoles and 5-[2-(4-arylpiperazin-1-yl)ethoxy]-1H-benzimidazoles were synthesized and their affinity for the D1, D2 and 5-HT1A receptors examined. They expressed a rather high affinity for the D2 dopamine receptor. The main features of ligand-D2 receptor interactions revealed by docking analyses were: salt bridge between piperazine ring protonated N1 and Asp 86, hydrogen bonds of ligand bezimidazole part with Ser 141, Ser 122 and His 189, edge-to-face interactions of arylpiperazine aromatic ring with Phe 178, Tyr 216 and Trp 182 and hydrogen bond between ethereal oxygen in ethylenoxy ligands and hydrogen of Phe 185 or Trp 115. The most active 5-[2-[4-(2-methoxyphenyl)-piperazin-1-yl]ethoxy]-1,3-dihydro-2H-benzimidazole-2-thione (27) has a maximal number of attractive interactions. A satisfactory correlation between docking of the compounds into the D2 receptor and competition binding results was observed.

Differential and quantitative molecular analysis of ischemia complexity reduction by isotopic labeling of proteins using a neural embryonic stem cell model.

The analysis of rapid changes of protein expression in living systems in response to insults requires rigorous methods of complexity reduction. To control dynamic pattern of hundreds or even thousands of protein isoforms, we applied a novel method of differential molecular analysis to a cellular model which is suited to study ischemia. Neural derivatives of murine embryonic stem cells were exposed to chemical ischemia. The model was used to obtain starting material for a quantitative differential proteomics analysis. Fractionation of phosphoproteins from these samples and subsequent identification by mass spectrometry of differential proteins provide proof of principle of how novel molecular analytical tools provide new insight into the network of neuroprotective molecular events during specific situations of neuronal stress and related pharmaceutical intervention. Our results indicate a particular role of an isoform of the acidic calcium-independent phospholipase A2 in this type of insult.

Allosterically potentiating ligands of nicotinic receptors as a treatment strategy for Alzheimer's disease.

One of the most prominent cholinergic deficit in Alzheimer's disease (AD) is the reduced number of nicotinic acetylcholine receptors (nAChR) in the hippocampus and cortex of AD patients, as compared to age-matched controls. This deficit results in reduced nicotinic cholinergic excitation which may not only impair postsynaptic depolarization but also presynaptic neurotransmitter release and Ca2+-dependent intracellular signaling, including transcriptional activity. Presently, the most common approach to correct the nicotinic cholinergic deficit in AD is the application of cholinesterase inhibitors. Due to the resulting increase in synaptic acetylcholine levels, both in concentration and time, additional nAChR molecules, e.g. those more distant from the ACh release sites, could be activated. As an obvious disadvantage, this approach affects cholinergic neurotransmission as a whole, including muscarinic neurotransmission. As a novel and alternative approach, a treatment strategy which exclusively targets nicotinic receptors is suggested. The strategy is based on a group of modulating ligands of nicotinic receptors, named allosterically potentiating ligands (APL), which increase the probability of channel opening induced by ACh and nicotinic agonists, and in addition decrease receptor desensitization. The action of APL on nicotinic receptors is reminiscent of that of benzodiazepines on GABA(A) receptors and of that of glycine on the NMDA-subtype of glutamate receptor. Representative nicotinic APL are the plant alkaloids physostigmine, galanthamine and codeine, and the neurotransmitter serotonin (5HT). The potentiating effect of APL on nicotinic neurotransmission has been shown by whole-cell patch-clamp studies in natural murine and human neurons, and in murine and human cell lines expressing various subtypes of neuronal nAChR.

Expression and renaturation of the N-terminal extracellular domain of torpedo nicotinic acetylcholine receptor alpha-subunit.

The N-terminal extracellular region (amino acids 1-209) of the alpha-subunit of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue was expressed as inclusion bodies in Escherichia coli using the pET 3a vector. Employing a novel protocol of unfolding and refolding, in the absence of detergent, a water-soluble globular protein of 25 kDa was obtained displaying approximately 15% alpha-helical and 45% beta-structure. The fragment bound alpha-[3H]bungarotoxin in 1:1 stoichiometry with a KD value of 0.5 nM as determined from kinetic measurements (4 nM from equilibrium binding). The kinetics of association of toxin and fragment were of second order, with a similar rate constant (8.2 x 10(5) M-1 s-1) as observed previously for the membrane-bound heteropentameric nAChR. Binding of small ligands was demonstrated by competition with alpha-[3H]bungarotoxin yielding the following KI values: acetylcholine, 69 microM; nicotine, 0.42 microM; anatoxin-a, 3 miroM; tubocurarine, 400 microM; and methyllycaconitine, 0.12 microM. The results demonstrate that the N-terminal extracellular region of the nAChR alpha-subunit forms a self-assembling domain that functionally expresses major elements of the ligand binding sites of the receptor.

Neuronal nicotinic receptors in the locust Locusta migratoria. Cloning and expression.

We have identified five cDNA clones that encode nicotinic acetylcholine receptor (nAChR) subunits expressed in the nervous system of the locust Locusta migratoria. Four of the subunits are ligand-binding alpha subunits, and the other is a structural beta subunit. The existence of at least one more nAChR gene, probably encoding a beta subunit, is indicated. Based on Northern analysis and in situ hybridization, the five subunit genes are expressed. localpha1, localpha3, and locbeta1 are the most abundant subunits and are expressed in similar areas of the head ganglia and retina of the adult locust. Because Loc

Mapping of a binding site for ATP within the extracellular region of the Torpedo nicotinic acetylcholine receptor beta-subunit.

Using 2,8,5'-[3H]ATP as a direct photoaffinity label for membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata, we have identified a binding site for ATP in the extracellular region of the beta-subunit of the receptor. Photolabeling was completely inhibited in the presence of saturating concentrations of nonradioactive ATP, whereas neither the purinoreceptor antagonists suramin, theophyllin, and caffeine nor the nAChR antagonists alpha-bungarotoxin and d-tubocurarine affected the labeling reaction. Competitive and noncompetitive nicotinic agonists and Ca2+ increased the yield of the photoreaction by up to 50%, suggesting that the respective binding sites are allosterically linked with the ATP site. The dissociation constant KD of binding of ATP to the identified site on the nAChR was of the order of 10(-4) M. Sites of labeling were found in the sequence regions Leu11-Pro17 and Asp152-His163 of the nAChR beta-subunit. These regions may represent parts of a single binding site for ATP, which is discontinuously distributed within the primary structure of the N-terminal extracellular domain. The existence of an extracellular binding site for ATP confirms, on the molecular level, that this nucleotide can directly act on nicotinic receptors, as has been suggested from previous electrophysiological and biochemical studies.

Allosteric modulation of Torpedo nicotinic acetylcholine receptor ion channel activity by noncompetitive agonists.

Similar to other neuroreceptors of the vertebrate central nervous system, the nicotinic acetylcholine receptor (nAChR) is subject to modulatory control by allosterically acting ligands. Of particular interest in this regard are allosteric ligands that enhance the sensitivity of the receptor to its natural agonist acetylcholine (ACh), as such ligands could be useful as drugs in diseases associated with impaired nicotinic neurotransmission. Here we discuss the action of a novel class of nAChR ligands which act as allosterically potentiating ligands (APL) on the nicotinic responses induced by ACh and competitive agonists. In addition, APLs also act as noncompetitive agonists of very low efficacy, and as direct blockers of ACh-activated channels. These actions are observed with nAChRs from brain, muscle and electric tissue, and they depend on the structure of the APL and the concentration range applied. We focus here on Torpedo nAChR because (i) the unusual pharmacology of these ligands was first discovered with this system, and (ii) large quantities of this receptor are readily available for biochemical studies.

Nicotinic acetylcholine receptors on hippocampal neurons: distribution on the neuronal surface and modulation of receptor activity.

The recent development of a technique that uses infrared microscopy for the visualization of well-defined areas on the surface of neurons, and a computerized system of micromanipulators led to the discovery that functional nicotinic acetylcholine receptors (nAChRs) are expressed at higher density on the dendrites than on the soma of rat hippocampal neurons. The finding that the expression of alpha-bungarotoxin-sensitive, alpha 7-bearing, nAChRs and dihydro-beta-erythroidine-sensitive, alpha 4 beta 2 nAChRs tends to increase along the dendritic length suggests that these receptors may be highly involved in the integration of synaptic functions in hippocampal neurons. The present report also discusses the finding that ligands such as the anticholinesterase galanthamine can modulate the nAChR activity by binding to a novel receptor site, and that 5-hydroxytryptamine (5-HT) may serve as an endogenous ligand for this site. The ability of 5-HT to modulate the nAChR function in vivo supports the concept that the overall CNS function is determined not only by the neuronal network established by the neuronal wiring, but also by a chemical network established by the ability of a single substance to act as the primary neurotransmitter in one system and as a co-transmitter in another system.

Stable expression in HEK-293 cells of the rat alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor.

The alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor (nAChR) was stably expressed in human embryonic kidney (HEK) 293 cells that co-expressed a voltage-gated Ca2+ channel. alpha3/beta4-nAChR-expressing clones were identified using the fura-2 Ca2+ imaging technique, and were further characterised by single-cell and whole-cell patch-clamp studies. Acetylcholine (ACh) induced fast activating currents which showed desensitisation and inward rectification. The conductance of the ACh-activated channel was 29 pS. The order of potency of the nicotinic agonists tested was cytisine approximately = nicotine > acetylcholine. The EC50 value for ACh was 145 microM; the Hill coefficient was close to 2. The currents elicited by ACh were effectively blocked by nicotinic antagonists, but not by the muscarinic antagonist atropine. These properties are comparable to the pharmacological and physiological profile of ganglionic nicotinic receptors and type III currents of cultured hippocampal neurons.

Agonist responses of neuronal nicotinic acetylcholine receptors are potentiated by a novel class of allosterically acting ligands.

Similar to the gamma-aminobutyric acidA receptor and the N-methyl-D-aspartate subtype of glutamate receptor, neuronal nicotinic acetylcholine receptors are subject to positive modulatory control by allosterically acting ligands. Exogenous ligands such as galanthamine and the neurotransmitter 5-hydroxytryptamine, when applied in submicromolar concentrations with nicotinic agonists, significantly increase the frequency of opening of nicotinic receptor channels and potentiate agonist-activated currents. Because these effects have been shown to be blocked by the monoclonal antibody FK1, they are mediated by binding sites that are located on alpha subunits of nicotinic receptors and distinct from those for acetylcholine and acetylcholine-competitive ligands. At higher concentrations, the potentiating effect of these ligands decreases and is eventually overcome by an inhibition of the agonist-induced response. The sensitizing actions of galanthamine, 5-hydroxytryptamine, and related compounds, at submicromolar concentrations, may reflect the existence of cross-talk between adjacent neuroreceptors and synapses in the central nervous system and thus suggests the formation of transiently active chemical networks in the vertebrate brain.

Membrane protein subunit fractionation by means of inverse pore gradient elution polyacrylamide gel electrophoresis.

We report here the preparative scale isolation of the four subunits of the nicotinic acetylcholine receptor (nAChR) applying short inverse pore gradient SDS gels on an elution-PAGE apparatus. The nAChR subunits are of similar molecular weights (alpha, 50.2 kDa; beta, 53.7 kDa; gamma, 56.3 kDa; delta, 57.6 kDa) and isoelectric point (approx 5.5) and share the typical properties of amphiphatic membrane proteins that are difficult to separate by chromatographic procedures. Preparative PAGE, which has proved to be the method of choice for nAChR-subunit fractionation, however, is time-consuming and achieves only moderate resolutions yielding dilute fractions. We present here the fractionation of a membrane preparation of Torpedo electric organ (2 mg of total protein) on short gels (2 cm) with linear or concave inverse pore gradients. All of the four subunits are isolated in homogeneous fractions of approx 70 micrograms/ml within only 4 h. Compared to the standard method, this means a 50% reduction in separation times with threefold higher concentrated protein fractions. We also give the theoretical explanation and experimental validation of the improved features resulting from short inverse-gradient polyacrylamide gels.

Physostigmine, galanthamine and codeine act as 'noncompetitive nicotinic receptor agonists' on clonal rat pheochromocytoma cells.

The acetylcholine esterase inhibitor (-)-physostigmine has been shown to act as agonist on nicotinic acetylcholine receptors from muscle and brain, by binding to sites on the alpha-polypeptide that are distinct from those for the natural transmitter acetylcholine (Schröder et al., 1994). In the present report we show that (-)-physostigmine, galanthamine, and the morphine derivative codeine activate single-channel currents in outside-out patches excised from clonal rat pheochromocytoma (PC12) cells. Although several lines of evidence demonstrate that the three alkaloids act on the same channels as acetylcholine, the competitive nicotinic antagonist methyllycaconitine only inhibited channel activation by acetylcholine but not by (-)-physostigmine, galanthamine or codeine. In contrast, the monoclonal antibody FK1, which competitively inhibits (-)-physostigmine binding to nicotinic acetylcholine receptors, did not affect channel activation by acetylcholine but inhibited activation by (-)-physostigmine, galanthamine and codeine. The three alkaloids therefore act via binding sites distinct from those for acetylcholine, in a 'noncompetitive' fashion. The potency of (-)-physostigmine and related compounds to act as a noncompetitive agonist is unrelated to the level of acetylcholine esterase inhibition induced by these drugs. (-)-Physostigmine, galanthamine and codeine do not evoke sizable whole-cell currents, which is due to the combined effects of low open-channel probability, slow onset and slow inactivation of response. In contrast, they sensitize PC12 cell nicotinic receptors in their submaximal response to acetylcholine. While the abundance of nicotinic acetylcholine receptor isoforms expressed in PC12 cells excludes identification of specific nicotinic acetylcholine receptor subtypes that interact with noncompetitive agonists, the identical patterns of single-channel current amplitudes observed with acetylcholine and with noncompetitive agonists suggested that all PC12 cell nicotinic acetylcholine receptor subtypes that respond to acetylcholine also respond to noncompetitive agonist. The action of noncompetitive agonists therefore seems to be highly conserved between nicotinic acetylcholine receptor subtypes, in agreement with the high level of structural conservation in the sequence region harboring major elements of this site.

Noncompetitive agonism at nicotinic acetylcholine receptors; functional significance for CNS signal transduction.

The alkaloids (-)physostigmine (Phy), galanthamine (Gal) and codeine (Cod), and several derivatives and homologous compounds, can act as noncompetitive agonists (NCA) of nicotinic acetylcholine receptors (nAChR) from Torpedo electrocytes, frog and mammalian muscle cells, clonal rat pheochromocytoma cells, cultured hippocampal neurons and several ectopic expression systems, by interacting with a binding site on the alpha-subunits of these nAChRs that is insensitive to the natural transmitter, acetylcholine (ACh), and ACh-competitive agonists and antagonists. Several endogenous ligands, including opioid-type compounds, can also act via this site, albeit at higher concentrations than is typical for the interaction with their cognate receptors. The NCA-evoked responses can be observed at the single-channel level but they do not summate to significant macroscopic currents, suggesting that the major role of NCAs is to act as "co-agonists", thereby potentiating nAChR channel activation by the natural transmitter. In more general terms, noncompetitive agonists may constitute part of a "chemical network", by which intercellular messengers, in addition to serving their cognate receptors, could modulate the sensitivity of other neuroreceptors to their archetypic ligands. Such a mode of action would make centrally acting NCAs interesting candidate drugs in the treatment of neuro-degenerative diseases.

Identification of purine binding sites on Torpedo acetylcholine receptor.

Electrophysiological studies from this and other laboratories have suggested a direct action of ATP on nicotinic acetylcholine receptors (nAChR). To determine the site of binding of this purine derivative, we have covalently modified the nAChR from Torpedo marmorata electrocytes employing 2-[3H]-8-azido-ATP as a photoactivable affinity label. Covalently attached radioactivity was predominantly found in the beta-polypeptide of the receptor. Based on the results of protection studies with several nAChR ligands whose target sites at the receptor are known, we conclude that the purine site(s) differ from those of acetylcholine and of physostigmine, galanthamine and related ligands, and those of local anesthetics.

Monoclonal antibodies FK1 and WF6 define two neighboring ligand binding sites on Torpedo acetylcholine receptor alpha-polypeptide.

Previous studies have identified the sequence region flanking the invariant vicinal cysteinyl residues at positions 192 and 193 of the nicotinic acetylcholine receptor alpha-subunit as containing major elements of the binding site for acetylcholine and its agonists and antagonists, including antibody WF6 (Conti-Tronconi, B. M., Diethelm, B. M., Wu, X., Tang, F., Bertazzon, T., Schröder, B., Reinhardt-Maelicke, A., and Maelicke, A. (1991) Biochemistry 30, 2575-2584). Recently we have shown that the sequence region flanking lysine alpha 125 contains elements of the binding site for physostigmine and related ligands, including antibody FK1 (Schrattenholz, A., Godovac-Zimmerman, J., Schäfer, H.-J., Albuquerque, E. X., and Maelicke, A. (1993) Eur. J. Biochem. 216, 671-677). Here we report the identification by enzyme-linked immunosorbent assay techniques, employing fragments of the Torpedo nicotinic acetylcholine receptor alpha-subunit N-terminal region and a panel of synthetic peptides matching in sequence preselected portions of this subunit, of the sequence regions alpha 118-145 and alpha 181-216 as contributing to the FK1 epitope. Of the synthetic peptides employed, alpha 118-137 displayed the highest affinity of FK1 binding. Binding of FK1 and WF6 to single residue-substituted analogs of the sequence alpha 181-200 indicated that the two antibodies have different attachment point patterns within this sequence region. These results, and those of ligand competition studies, suggest that the binding sites for FK1 and physostigmine, and those of WF6 and acetylcholine, are within the same general region of the receptor's three-dimensional structure. The sites neighbor each other, with limited overlap in the case of occupation by high molecular weight ligands.

Photoaffinity labeling of Torpedo acetylcholine receptor by physostigmine.

The plant alkaloid physostigmine, an established anti-cholinesterase agent of the carbamate type, has recently been shown to bind to the nicotinic acetylcholine receptor from Torpedo marmorata electrocytes [Okonjo, K. O., Kuhlmann, J. & Maelicke, A. (1991) Eur. J. Biochem. 200, 671-677]. Pharmacological studies of physostigmine-induced ion flux into nicotinic-acetylcholine-receptor-rich membrane vesicles, indicated distinct binding sites for physostigmine and acetylcholine. As shown in this study by photoaffinity labeling with [phenyl-(n)-3H](-)physostigmine, the physostigmine-binding site is located within the same subunit (alpha polypeptide) of the receptor as the acetylcholine-binding site. Using a variety of proteolytic cleavage conditions for the purified alpha polypeptide, several [3H]physostigmine-labeled peptides were isolated and sequenced. From the radioactivity released in the course of the Edman degradations of the labeled peptides, it was found that the label was associated in all cases with Lys125. These results identify a novel ligand-binding site for the Torpedo nicotinic acetylcholine receptor that is different in location from binding sites identified previously for acetylcholine, its established agonists and antagonists, and direct channel blockers.

Identification and functional characterization of a new agonist site on nicotinic acetylcholine receptors of cultured hippocampal neurons.

Electrophysiological and biochemical techniques were used to demonstrate that alpha-bungarotoxin-, methyllycaconitine-sensitive neuronal nicotinic acetylcholine receptors (nAChRs) can be activated via a novel agonist site(s). The residue proposed to be essential to this site is the amino acid Lys-125 of the receptor alpha subunit. In outside-out patches excised from cultured hippocampal neurons, physostigmine (PHY) and 1-methyl-PHY activated single channels whose main conductances were 46 and 23 pS. This action was insensitive to DL-2-amino-5-phosphonovaleric acid, atropine, tetrodotoxin and competitive nicotinic antagonists, but blocked by benzoquinonium or FK1, a nAChR-specific antibody raised against rat muscle nAChR alpha subunits that binds to the novel site. Indirect immunofluorescence staining demonstrated that FK1 binds to the hippocampal neurons, as would be expected based on the high degree of homology among nAChR alpha subunits from diverse sources in the region surrounding Lys-125. PHY prevented the binding of FK1, thus supporting that FK1 is a specific probe for the PHY site. High-affinity sites (KD approximately 35 nM) for 1-methyl-PHY were identified in hippocampal neurons. Similar to PHY, benzoquinonium (0.1-10 microM) and galanthamine (1-10 microM) activated nicotinic single channels. The agonists benzoquinonium and PHY were also open-channel blockers at the neuronal nAChRs, whereas galanthamine was predominantly a desensitizing agent. In mouse fibroblasts transfected with cDNAs of alpha 4 and beta 2 neuronal nAChR subunits, PHY also activated single channels that were blocked by FK1. In these cells, dihydro-beta-erythroidine blocked single channels activated by (+)-anatoxin-a and did not affect those opened by PHY. Thus, the present results suggest that the novel agonist site located on the receptor alpha subunit is a common feature of neuronal nAChRs.

Physostigmine and neuromuscular transmission.

Single channel studies carried out in cultured rat myoballs and cultured hippocampal neurons, and ion flux studies performed on Torpedo electrocyte membrane vesicles, showed that physostigmine (Phy), a well-established acetylcholinesterase inhibitor, interacts directly with nicotinic acetylcholine receptors (nAChR). Low concentrations (0.1 microM) of Phy activate the receptor integral channel, whereas higher concentrations blocked the channel in its opened state. In contrast to channel activation by acetylcholine (ACh) and classical cholinergic agonists, however, Phy was capable of activating the nAChR channel even when the ACh binding sites were blocked by competitive antagonists, such as alpha-neurotoxins and d-tubocurarine, or when the nAChR was desensitized by preincubation with high concentrations of ACh. The binding site at which Phy binds and activates the nAChR was mapped. It was located within the N-terminal extracellular region of the alpha-polypeptide, in close proximity to the binding site of the natural transmitter. These data identify a novel binding site at nAChRs from many species and tissues that may be involved in receptor regulatory processes.