Title: Acetylcholine and regulation of gene expression in developing systems Augusti-Tocco G, Biagioni S, Tata AM Ref: Journal of Molecular Neuroscience, 30:45, 2006 : PubMed
One of the major questions related to nervous system development is the identification of signals directing neuronal populations to specific phenotypes (e.g., cholinergic, adrenergic, or peptidergic neurons) and involved in cell-to-cell interactions. Although neurotrophins have long been known for their function in development, the neurotransmitter role as modulator of gene expression and differentiation has been recognized only recently. Evidence for the ability of various neurotransmitter molecules to influence various cellular events during neuron differentiation has been reported in several systems (Lauder and Schambra, 1999). We have focused our interest on acetylcholine (ACh) and its possible role in the regulation of neuron-specific gene expression, using different experimental systems: (1) neuroblastoma cell lines, as a model of cholinergic neuron differentiation; (2) dorsal root ganglia (DRG) sensory neurons, which activate the expression of a cholinergic system early in development, in spite of their peptidergic or aminoacidergic neurotransmission; and (3) primary cultures of Schwann cells. Data obtained on each system will be described briefly.
        
Title: Therapeutic potential of novel selective drugs targeting nicotinic acetylcholine receptors Bencherif M, Hauser TA, Jordan KG, Gatto GJ Ref: Journal of Molecular Neuroscience, 30:17, 2006 : PubMed
The potential therapeutic benefit of nicotinic ligands in a variety of neurodegenerative pathologies involving the CNS has energized research efforts to develop nicotinic acetylcholine receptor (nAChR) subtype-selective ligands (Bencherif and Schmitt, 2005). In particular, there has been a concerted effort to develop nicotinic compounds with selectivity for CNS nAChRs as potential pharmaceutical tools in the management of these disorders. Clinical and experimental data demonstrate a central role for alpha7 and alpha4beta2 nAChRs in cognitive function, sensory processing, mood, and neuroprotection (Bencherif and Schmitt, 2005; Buccafusco et al., 2005). The development of safe alpha7-selective ligands has been hampered by their lack of discrimination with hERG channels and 5-HT3 receptors. We have developed a number of compounds that display nanomolar affinity to the alpha7 and/or the alpha4beta2 receptor. Investigation of alpha7 functional activity showed a full range of activities from antagonists to full agonists without any significant activity at the human 5-HT3 receptor, P450 isozymes, hERG channels, or in the AMES test. Our findings demonstrate that potent and highly selective nAChR ligands can be designed.
        
Title: Nicotinic signal transduction machinery Berg DK, Conroy WG, Liu Z, Zago WM Ref: Journal of Molecular Neuroscience, 30:149, 2006 : PubMed
Nicotinic synapses employ acetylcholine to activate ligand-gated ion channels that are cation-selective in vertebrates. Although the resulting nicotinic cholinergic transmission is famously excitatory at the neuromuscular junction, it plays many additional roles in the CNS. Most prevalent is that of modulation, usually involving calcium and signal transduction. Because of this, it is becoming increasingly important not only to understand the mechanisms that guide nicotinic receptors to appropriate locations but also to identify the postsynaptic machinery making possible the requisite signal transduction. Clearly, the kinds of components tethered in the vicinity of the receptor will assume a major role in determining the consequences of receptor activation. One of the most abundant and interesting nicotinic receptors in this respect is the species comprised of the alpha7 gene product (Broide and Leslie, 1999). These alpha7 homopentameric nicotinic acetylcholine receptors (alpha7 nAChRs) have a high relative permeability to calcium, rivaling that of NMDA receptors. But unlike NMDA receptors, alpha7 nAChRs promote calcium influx without requiring a coincident event such as membrane depolarization. As a result, the receptors are well equipped to regulate calcium-dependent events in neurons, particularly when depolarization might be occluded.
        
Title: Phosphorylation and function of alpha4beta2 receptor Bermudez I, Moroni M Ref: Journal of Molecular Neuroscience, 30:97, 2006 : PubMed
The neuronal nicotinic acetylcholine receptor (nAChR) alpha4 and beta2 subunits expressed in heterologous expression systems assemble into high- and low-affinity receptors (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001; Nelson et al., 2003), which reflects the assembly of two distinct subunit stoichiometries of alpha4beta2 receptor (Nelson et al., 2003). The high-affinity receptor ([alpha4]2[beta2]3) is about 100-fold more sensitive to ACh than the low-affinity receptor ([alpha4]3[beta2]2) (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001; Nelson et al., 2003). Recent evidence implicated 14-3-3 proteins as modulators of the relative abundance of nAChR subunits in the endoplasmic reticulum (ER), where ligand-gated ion channels assemble. The 14-3-3 proteins influence ER-to-plasma membrane trafficking of multimeric cell-surface proteins (O'Kelly et al., 2002). 14-3-3 proteins bind components of these multimeric proteins, and this interaction overrides dibasic COP1 retention signal to permit forward transport of the protein (O'Kelly et al., 2002). In the case of alpha4beta2 nAChRs, 14-3-3 binds the alpha4 subunit, and this association is dependent on phosphorylation of a serine residue within a protein kinase A(PKA) consensus sequence in the large cytoplasmic domain of the alpha4 subunit, which is also a binding motif recognized by 14-3-3 (Jeancloss et al., 2001; O'Kelly et al., 2002). The interplay among PKA, alpha4 subunits, and 14-3-3 proteins increases cell-surface expression of alpha4beta2 nAChRs by increasing steady-state levels of the alpha4 subunit available for assembly with beta2 subunits (Jeancloss et al., 2001). Because it is not known how 14-3-3-dependent changes in the steady-state levels of the alpha4 subunit might affect the functional type of alpha4beta2 receptors, we have investigated the effects of mutations of the 14-3-3 binding motif in the alpha4 subunit on alpha4beta2 nAChR function.
Many peptidic toxins from animal venoms target neuronal or peripheral synaptic receptors with high affinities and specificities. Hence, these toxins are not only potent natural weapons but also precise molecular tools for pharmacological studies of their receptors. Although they belong to various structural and/or functional subfamilies, they often share similar molecular features, such as a highly reticulated scaffold presenting specific binding determinants.
Previous studies in other laboratories have shown that alpha4beta2 nicotinic acetylcholine receptor (nAChR) exhibits a biphasic concentration-response relationship for ACh with low and high EC50 components, and that the low EC50 component can be augmented by decreasing the alpha4:beta2 message ratio or incubating overnight in nicotine or at low temperature (Zwart and Vijverberg, 1998; Covernton and Connolly, 2000; Buisson and Bertrand, 2001; Nelson et al., 2003; Zhou et al., 2003). In the process of cloning ferret nAChR subunits, we found alpha4 and beta2 messages with long untranslated regions (UTRs), as well as those with no UTRs. Combinations of these messages revealed that the presence of UTRs influenced the ability to exclusively express high-sensitivity subforms of alpha4beta2 and alpha3beta2 nAChRs. Injection of oocytes with alpha4 and beta2 RNAs lacking UTRs (1:1 ratio) led to expression of a biphasic concentration-response relationship for ACh with EC50 values of 0.5 (high sensitivity) and 114 microM(low sensitivity). Decreasing the alpha4:beta2 message ratio to as much as 1:120 increased the high-sensitivity component slightly, but the ACh concentration response remained biphasic. In contrast, injection of messages with UTRs (1:1 ratio) led to expression of a monophasic concentration response to ACh and a high-sensitivity EC50 value of 2.3 microM, as shown in Fig. 1.
        
Title: Presynaptic muscarinic control of glutamatergic synaptic transmission Buno W, Cabezas C, Fernandez de Sevilla D Ref: Journal of Molecular Neuroscience, 30:161, 2006 : PubMed
The hippocampus receives cholinergic projections from the medial septal nucleus and Broca's diagonal band that terminate in the CA1, CA3, and dentate gyrus regions (Frotscher and Leranth, 1985). Glutamatergic synapses between CA3 and CA1 pyramidal neurons are presynaptically inhibited by acetylcholine (ACh), via activation of muscarinic ACh receptors (mAChRs) at the terminals of Schaffer collaterals (SCs) (Hounsgaard, 1978; Fernandez de Sevilla et al., 2002, 2003). There are two types of SC-CA1 pyramidal neuron synapses. One type, called functional synapse, shows postsynaptic alpha- amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-receptor mediated currents at resting potential (Vm) and both AMPA and N-methyl-D-aspartate receptor (NMDAR)-mediated currents when depolarized. The other type, termed silent synapse, only displays postsynaptic NMDAR-mediated currents at depolarized Vms, but does not respond at the resting Vm (Isaac et al., 1995). Using hippocampal slices obtained from young Wistar rats, we examined the effects of activation of cholinergic afferents at the stratum oriens/alveus on excitatory postsynaptic currents (EPSCs) evoked in CA1 pyramidal neurons by stimulation of SCs. We also tested the action of the nonhydrolyzable cholinergic agonist carbamylcholine chloride (CCh) on EPSCs evoked by minimal stimulation of SCs (which activates a single or very few synapses) in functional and silent synapses.
Neurotransmitter-gated receptors are assembled in the endoplasmic reticulum and transported to the cell surface through a process that might be of central importance to regulate the efficacy of synaptic transmission (Kneussel and Betz, 2000; Kittler and Moss, 2003). This process is relatively inefficient- what may be the consequence of tight quality controls that guarantee the functional competence of the final product. For this purpose, specific proteins involved in assembly and trafficking of receptors might be required (Keller and Taylor, 1999; Millar, 2003; Wanamaker et al., 2003). The RIC-3 protein could be one of them, as mutations in the ric-3 gene affect maturation of nicotinic acetylcholine receptors (nAChRs) in Caenorhabditis elegans (Halevi et al., 2002). Moreover, the human homolog hRIC-3 showed differential effects when coexpressed with several ligand-gated receptors (Halevi et al., 2003). Thus, it enhanced alpha7 nAChR expression while inhibiting expression of other nAChR subtypes (alpha4beta2 and alpha3beta4) and 5-HT3 serotonin receptors (5-HT3Rs). These opposite effects suggested that the RIC-3 protein might play a key role in the biogenesis of some ligand-gated receptors and prompted us to investigate how it performs its action. Here, we show that the RIC-3 protein acts as a barrier for some receptors like alpha4beta2 nAChRs and 5-HT3Rs, stopping the traffic of mature receptors to the membrane. In contrast, the inefficient transport of alpha7 nAChRs is enhanced by RIC-3 in a process in which certain amino acids at the amphipathic helix located at the C-terminal region of the large cytoplasmic domain are involved.
Transmissible spongiform encephalopaties are caused by an extracellular surface protein, the scrapie prion protein (PrPsc), which is an aberrant form of normal and functional cellular PrP (PrPc). The pathological hallmarks of these diseases are the accumulation and deposition of PrPsc in the form of amyloid fibrils in the central nervous system (Tateishi et al., 1988), similar to amyloid-beta (Abeta) protein in Alzheimer's disease (AD). In some patients, Abeta and prion pathology can coexist (Hainfellner et al., 1998), and a common spatial pattern of protein deposition has been described (Armstrong et al., 2001). In addition, it is well-known that acetylcholinesterase (AChE) colocalizes with Abeta deposits of brains in AD patients and accelerates assembly of Abeta peptides through the peripheral site of the enzyme (Inestrosa et al., 1996). The aim of the present study was to analyze time course and concentration dependence of the AChE proaggregating effect on synthetic peptide-spanning residues 106-126 of human PrP (PrP106-126) and the reversion of this effect by different AChE inhibitors (AChEIs).
        
Title: Vesicular calcium transport shapes rapid acetylcholine secretion Cordeiro JM, Dunant Y, Goncalves PP Ref: Journal of Molecular Neuroscience, 30:41, 2006 : PubMed
Rapid secretion relies on the occurrence of spike-like Ca2+ transients in active zones (Llinas et al., 1992; Yazejian et al., 2000; Dunant and Bloc, 2003). Presynaptic Ca2+ nanodomains are to be restricted both in time and in space as to assure rapid onset and termination of transmitter release (Llinas et al., 1992; Pozzan et al., 1994; Yazejian et al., 2000; Dunant and Bloc, 2003). A very fast Ca2+-buffering mechanism should allow Ca2+ rise above approximately 100 microM for less than approximately 250 micros and then rapid reduction of Ca2+ to subthreshold levels of release (Llinas et al., 1992; Pozzan et al., 1994; Yazejian et al., 2000; Dunant and Bloc, 2003). Swift Ca2+ clearance by vesicular Ca2+/H+ antiport as a low-affinity, high-capacity extrusion mechanism was postulated in the past (Pozzan et al., 1994; Dunant and Bloc, 2003). We demonstrated pH gradient (DeltapH)-dependent Ca2+ uptake by mammalian brain synaptic vesicles (Goncalves et al., 1998, 2000). Moreover, this antiport activity is effective at [Ca2+] ranging from approximately 100 to 800 microM (max. at approximately 500 microM) (Goncalves et al., 1998, 2000). We now show that the time course of acetylcholine (ACh) secretion in Torpedo neuroelectrocytic synapse is modified by bafilomycin A1 (baf.), which compromises antiport activity. Along with this mechanism, synaptic vesicles also have a P-type Ca2+ ATPase, exhibiting half-maximal activation for 0.6 microM Ca2+ (Goncalves et al., 2000). Here, we demonstrate the role of P-type Ca2+ ATPase in preventing desensitization of the release mechanism by inhibiting it with orthovanadate.
        
Title: Buprofezin inhibits acetylcholinesterase activity in B-biotype Bemisia tabaci Cottage EL, Gunning RV Ref: Journal of Molecular Neuroscience, 30:39, 2006 : PubMed
B-biotype Bemisia tabaci is a severe insect pest worldwide in many ornamental, agricultural, and horticultural industries. Control of this insect is hampered by resistance to many acetylcholinesterase (AChE)-inhibiting insecticides, such as organophosphates and carbamates. Consequently, insect growth regulators such as buprofezin, which act by inhibiting chitin synthesis, are being investigated for use against B-biotype B. tabaci in Australia. This study discusses the effects of buprofezin on B. tabaciAChE.
        
Title: Role of CaCMKII in the cross talk between ionotropic nucleotide and nicotinic receptors in individual cholinergic terminals Diaz-Hernandez M, Sanchez-Nogueiro J, Miras-Portugal MT Ref: Journal of Molecular Neuroscience, 30:177, 2006 : PubMed
Ionotropic P2X receptors for ATP are formed, to date, by seven different subunits named P2X (Torres et al., 1999; Cunha and Ribeiro, 2000; North and Surprenant, 2000; Pintor et al., 2000; Hervas et al., 2003; Miras-Portugal et al., 2003; Illes and Ribeiro, 2004), which are cloned from various mammalian species (Illes and Ribeiro, 2004). These subunits can occur as homo- or hetero-oligomeric assemblies of more than one subunit (North and Surprenant, 2000), except P2X (Miras-Portugal et al., 2003) receptor, which has been described not to coassemble with other subunits (Torres et al., 1999). They are abundantly expressed in the peripheral and central nervous systems and exhibit high permeability to Ca2+ ions (Cunha and Ribeiro, 2000). The existence of presynaptic ionotropic receptors for nucleotides, either for ATP or dinucleotides, has been reported in isolated synaptic terminals from mammalian brain, and both exhibit good permeability to Ca2+ ions (Pintor et al., 2000; Hervas et al., 2003; Miras-Portugal et al., 2003). Studies on isolated single terminals have confirmed the existence of independent and specific responses to ATP and dinucleotides on the same or different terminals (Miras-Portugal et al., 1999; Diaz-Hernandez et al., 2002; Hervas et al., 2005; Sanchez-Nogueiro et al., 2005). The activation of presynaptic ionotropic nucleotide receptors can induce the release of other neurotransmitters such as acetylcholine, glutamate, or GABA. In these specific terminals, ionotropic nucleotide receptors can be modulated by interaction with metabotropic receptors, such as GABAB and adenosine receptors (Khakh and Henderson, 1998; Gomez-Villafuertes et al., 2001), and ionotropic, such as nicotinic cholinergic receptors (Diaz-Hernandez et al., 2004; Sanchez-Nogueiro et al., 2005). Here, we discuss a relevant finding on the interaction between ionotropic nucleotide and nicotinic receptors in cholinergic synaptic terminals and the role of CaCMKII in this interaction.
        
Title: Desensitization characteristics of the human alpha7nAChR/5HT3A chimera receptor Dinklo T, Lesage AS, Grantham CG Ref: Journal of Molecular Neuroscience, 30:109, 2006 : PubMed
The alpha7-nicotinic acetylcholine receptor (alpha7) is an important ionotropic receptor in the central nervous system, which becomes permeable to cations upon binding of its natural agonist acetylcholine (ACh). alpha7 kinetics are characterized by rapid activation, followed by fast desensitization of the current. As the wild-type (WT) alpha7 is difficult to express heterologously in mammalian cellular systems, frequently a more easily expressible chimera consisting of the extracellular domain of the alpha7 and the transmembrane domain of the 5HT3A receptor is used to study alpha7 pharmacology (chick alpha7/mouse 5HT3A [Eisele et al., 1993]; human alpha7/mouse 5HT3A [Graig et al., 2004]). Desensitization characteristics of these chimera receptors have been described as intermediate compared with the fast desensitizing alpha7 and the more slowly desensitizing 5HT3A receptors. Here, we describe a fully human chimera receptor (h-alpha7/5HT3A), which is characterized by desensitization, and recovery kinetics that deviate from the human WT alpha7.
        
Title: The role of the cytoskeleton in neuromuscular junction formation Dobbins GC, Zhang B, Xiong WC, Mei L Ref: Journal of Molecular Neuroscience, 30:115, 2006 : PubMed
The cytoskeleton plays a vital role in neuromuscular junction (NMJ) formation. It is responsible for shaping synaptic membrane into folds opposed to presynaptic active zones and anchoring acetylcholine receptors (AChRs) to the crest of the junctional folds. Acetylcholine receptors (AChRs) associate with the actin cytoskeleton, the disruption of which affects spontaneous and agrin-induced AChR clusters (Prives et al., 1982; Connolly, 1984; Peng and Phelan, 1984; Bloch, 1986; Dai et al., 2000). How AChRs are tethered to the actin cytoskeleton remains unclear.
        
Title: Acetylcholine release in rapid synapses: two fast partners--mediatophore and vesicular Ca2+/H+ antiport Dunant Y Ref: Journal of Molecular Neuroscience, 30:209, 2006 : PubMed
Rapid neurotransmission is like lightning: a spark of calcium in the nerve terminal, a spark of transmitter in the cleft, and the signal is over. But "time is gained at the expense of sensitivity" (Katz, 1988); transmission relies on low-affinity, high-speed reactions. These fast processes are modulated by regulating reactions that do not need to be so rapid.
        
Title: Cognitive enhancement in man with ispronicline, a nicotinic partial agonist Dunbar GC, Kuchibhatla R Ref: Journal of Molecular Neuroscience, 30:169, 2006 : PubMed
Cholinergic mechanisms are clearly involved in memory deficits associated with Alzheimer's disease (AD) (Perry et al., 1977). Recently, there has been growing interest in the nicotinic approach to the treatment of AD; however, compounds have failed in the clinic because of a lack of separation between central and peripheral nicotinic effects (Potter et al., 1999). Ispronicline (TC-1734) is an orally active, selective, partial agonist of the central alpha4beta2 neuronal nicotinic acetylcholine receptor (nAChR), with high binding affinity to membrane preparations from rat brain or mammalian cells expressing recombinant human alpha4beta2 receptor (Gatto et al., 2004). Ispronicline has no detectable effects on muscle or ganglionic nAChRs, indicating a marked CNS over PNS selectivity. In animal models ispronicline potently improved cognitive function. A long duration of memory enhancement was displayed in object recognition and radial arm maze tests. Ispronicline pharmacokinetics (half-life of 2 h in rats) contrasts with the long-lasting improvement of working memory (18 h to 2 d) (Gatto et al., 2004).
        
Title: Phenotype comparison of three acetylcholinesterase knockout strains Duysen EG, Lockridge O Ref: Journal of Molecular Neuroscience, 30:91, 2006 : PubMed
The phenotypes of three mouse strains that carry the acetylcholinesterase knockout (AChE KO) mutation have been compared. The AChE KO mouse was developed from embryonic stem (ES) cells, originating from a 129/Sv blastocyst. Animals generated from strain 129/Sv suffer from dysgenesis of the corpus callosum and possibly a number of other neuroanatomical deficiencies. To determine the contribution of background genes to phenotype, 129/Sv AChE heterozygote (AChE+/-) mice were backcrossed 10 generations with wild-type inbred C57/BL6 (C57) mice and with wild-type outbred CD1(R) mice. AChE-/- mice in strains C57 and CD1died during seizures before postnatal day (P) 21, whereas mice in strain 129/Sv lived to adulthood.
Neuronal nicotinic receptors for acetylcholine (nAChRs) are among the ionotropic receptors that suffer the most desensitization upon prolonged exposure to their agonists. This is particularly true for the alpha7 subtype of nAChRs, although alpha3beta4 receptors also suffer quick desensitization. This study was planned to test the hypothesis that even after suffering desensitization, a given nAChR might still afford cell protection against a noxious stimulus. Of the many agonists developed for nAChRs, we selected the poorly desensitizing ligand dimethylphenylpiperazinium (DMPP) (Britt and Brenner, 1997) and the highly desensitizing agent epibatidine (EPB) (Marks et al., 1996). We have measured nAChR currents, catecholamine secretory responses, and changes of [Ca2+]c elicited by stimulation of nAChRs with DMPP or EPB. We have also investigated cytoprotection elicited by DMPP and EPB against the cytotoxic effects of veratridine in bovine chromaffin cells.
Ligand-gated ion channels (LGICs) constitute an important family of complex membrane proteins acting as receptors for neurotransmitters (Barnard, 1992; Ortells and Lunt, 1995). The nicotinic acetylcholine receptor (nAChR) from Torpedo is the most extensively studied member of the LGIC family and consists of a pentameric transmembrane glycoprotein composed of four different polypeptide subunits (alpha, beta, gamma, and delta) in a 2:1:1:1 stoichiometry (Galzi and Changeux, 1995; Hucho et al., 1996) that are arranged pseudosymmetrically around a central cation-selective ion channel. Conformational transitions, from the closed (nonconducting), to agonist-induced open (ion-conducting), to desensitized (nonconducting) states, are critical for functioning of the nAChR (Karlin, 2002). The ability of the nAChR to undergo these transitions is profoundly influenced by the lipid composition of the bilayer (Barrantes, 2004). Despite existing information on lipid dependence of AChR function, no satisfactory explanation has been given on the molecular events by which specific lipids exert such effects on the activity of an integral membrane protein. To date, several hypotheses have been entertained, including (1) indirect effects of lipids through the alteration of properties of the bilayer, such as fluidity (an optimal fluidity hypothesis [Fong and McNamee, 1986]) or membrane curvature and lateral pressure (Cantor, 1997; de Kruijff, 1997), or (2) direct effects through binding of lipids to defined sites on the transmembrane portion of the protein (Jones and McNamee, 1988; Blanton and Wang, 1990; Fernandez et al., 1993; Fernandez-Ballester et al., 1994), which has led to the postulation of a possible role of certain lipids as peculiar allosteric ligands of the protein. In this paper we have reconstituted purified AChRs from Torpedo into complex multicomponent lipid vesicles in which the phospholipid composition has been systematically altered. Stopped-flow rapid kinetics of cation translocation and Fourier transform-infrared (FT-IR) spectroscopy studies have been used to illustrate the lipid dependence of both AChR function and AChR secondary structure, respectively.
Three-finger-fold toxins, isolated from various snake venoms, are recognized by high affinity and various specificities by different nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs, respectively) present in peripheral, as well as central, nervous systems (Karlsson et al., 2000; Servent and Menez, 2001; Nirthanan and Gwee, 2004). The goal of our studies is (1) to identify, at the molecular level, the functional determinants involved in the various interaction profiles of nicotinic or muscarinic toxins on their respective receptors subtypes, (2) to model some of these toxin-receptor complexes using distance constraints obtained from cycle-mutant experiments, and (3) to understand how a unique scaffold (the three-finger fold) is able to support these different functional profiles and how molecular determinants have been selected during the evolution process to create these different specific properties. Finally, these structure/function analyses should be exploited to engineer non-natural peptides with new binding and functional properties useful as pharmacological tools or therapeutic agents.
        
Title: Expression of MuSK in in vitro-innervated human muscle Gajsek N, Jevsek M, Grubic Z Ref: Journal of Molecular Neuroscience, 30:27, 2006 : PubMed
Unlike rodent or avian muscle, which forms clusters of acetylcholine receptors (AChRs) on its surface, exhibits cross striations, and contracts spontaneously even if cultured in the absence of the nerve, human muscle must be innervated to reach such differentiation level under in vitro conditions (Kobayashi and Askanas, 1985; Mars et al., 2001). Because it is known that AChR clustering and other aspects of neuromuscular junction (NMJ) formation necessitate the activation of muscle-specific kinase (MuSK), one explanation of this inability of human muscle is that it has no MuSK or that it cannot be activated in the absence of the nerve. To test this hypothesis we analyzed cultured human muscle for the expression of MuSK at two stages of differentiation: postfusion myotube and innervated, contracting myotube. Analyses were carried out at the mRNA level, as no reliable anti-MuSK antibodies are available for the immunocytochemical demonstration of MuSK in cultured human muscle. The presence of MuSK, however, can be tested indirectly, as it can be activated in the absence of the nerve simply by growing muscle culture on laminin coating (Kummer et al., 2004). In the second part of our study, we therefore tested human myotubes for the presence and activation of MuSK by exposing them to laminin coating and by analyzing them afterwards for the areas of postsynaptic differentiation typical for NMJ formation.
        
Title: Expression of nicotinic acetylcholine receptors on murine alveolar macrophages Galvis G, Lips KS, Kummer W Ref: Journal of Molecular Neuroscience, 30:107, 2006 : PubMed
Neuronal nicotinic acetylcholine receptors (nAChRs) play an essential role in immunomodulation of macrophages. In particular, the alpha7 subunit confers inhibition of the systemic inflammatory response to bacterial lipopolysaccharide, thereby being the crucial element of the cholinergic anti-inflammatory pathway (Borovikova et al., 2000; Pavlov et al., 2003; Wang et al., 2003). In the murine lung, nicotine also exerts anti-inflammatory effects (Blanchet et al., 2004), but at least in murine alveolar macrophage cell lines the alpha7 subunit has not been detected (Matsunaga et al., 2001). On this background we investigated the expression of the nAChR subunits (alpha2-alpha7, alpha9, alpha10, beta2-beta4) on freshly isolated murine alveolar macrophages by immunohistochemistry and RT-PCR.
        
Title: A comparison between acetylcholine-like action potentials and square depolarizing pulses in triggering calcium entry and exocytosis in bovine chromaffin cells Garcia de Diego AM, Arnaiz JJ, Gandia L, Hernandez-Guijo JM, Garcia AG Ref: Journal of Molecular Neuroscience, 30:57, 2006 : PubMed
Depending on experimental conditions, cell model, and pattern and type of depolarizing stimuli, the relationship between calcium entry ([Ca2+]c) and the release of neurotransmitters and hormones varies from exponential (power of 3-4) to near linear (power of 1.5) or linear function. Here, we present a study using the more physiological stimulation pattern based on acetylcholine (ACh)-like action potentials, in voltage-clamped bovine chromaffin cells, with the perforated-patch configuration of the patch-clamp technique and 2 mM extracellular calcium. Trains of ACh-like action potentials or square depolarizing pulses of increasing length were applied, and calcium currents (ICa), total calcium entry (QCa), and exocytosis (DeltaCm) measured.
The five muscarinic acetylcholine receptors (M1-M5 mAChRs) mediate a very large number of important physiological functions (Caulfield, 1993; Caulfield and Birdsall, 1998; Wess, 2004). Because of the lack of small molecule ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues or cell types express two or more mAChR subtypes, identification of the physiological and pathophysiological roles of the individual mAChR subtypes has proved to be a challenging task. To overcome these difficulties, we recently generated mutant mouse lines deficient in each of the five mAChR genes (M1R-/- mice, M2R-/- mice, M3R-/- mice, etc. [Wess, 2004]). Phenotyping studies showed that each of the five mutant mouse lines displayed characteristic physiological, pharmacological, behavioral, biochemical, or neurochemical deficits (Wess, 2004). This chapter summarizes recent findings dealing with the importance of the M2mAChR for cognitive processes and the roles of the M1 and M3 mAChRs in mediating stimulation of glandular secretion.
        
Title: Remodeling of the neuromuscular junction in mice with deleted exons 5 and 6 of acetylcholinesterase Girard E, Bernard V, Camp S, Taylor P, Krejci E, Molgo J Ref: Journal of Molecular Neuroscience, 30:99, 2006 : PubMed
At the vertebrate skeletal neuromuscular junction (NMJ), two closely related enzymes can hydrolyze acetylcholine (ACh): acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Advances in mouse genomics offer new approaches to assess the role of specific cholinesterases involved in neuromuscular transmission (Minic et al., 2003). AChE knockout mice provide a valuable tool for examining the effects of long-term complete and selective abolition of AChE activity (Xie et al., 2000). AChE and BChE genes encode two functional domains--the catalytic domain (exons 2, 3, and 4 of AChE, or exon 2 of BChE) and a C-terminal domain (exon 5 or 6 of AChE, or exon 3 of BChE)--that dictate the targeting of the enzymes (Massoulie, 2002). In mammals, the AChE gene produces three types of coding regions by deleting 5'- splice acceptor sites, which generate proteins; these proteins possess the same catalytic domain associated with distinct C-terminal peptides. AChE subunits of type R (readthrough) produce soluble monomers; they are expressed during development and are thought to be induced in the mouse brain by stress (Kaufer et al., 1998). AChE subunits of type H (hydrophobic) produce GPI-anchored dimers, mainly in blood cells. Subunits of type T (tailed) exist for both AChE and BChE. They represent the predominant AChE variant expressed in cholinergically innervated tissues (muscle and nerve). These subunits generate a variety of quaternary structures, including homomeric oligomers (monomers, dimers, tetramers), as well as hetero-oligomeric assemblies with anchoring proteins ColQ (Krejci et al., 1997) and PRiMA (Perrier et al., 2002). At the NMJ, AChE is clustered by the interaction of the coding sequence of exon 6 with ColQ (Feng et al., 1999). The deletion of exons 5 and 6 in the AChE gene transforms anchored AChE into a soluble enzyme (Camp et al., 2004). The present study was designed to evaluate neuromuscular transmission and nicotinic ACh receptor (nAChR) distribution in muscles from mutant mice with deletions of these two spliced exons (AChE-del-exons-5+6-/-).
        
Title: Identification of two critical residues within the Cys-loop sequence that determine fast-gating kinetics in a pentameric ligand-gated ion channel Grutter T, de Carvalho LP, Dufresne V, Taly A, Changeux JP Ref: Journal of Molecular Neuroscience, 30:63, 2006 : PubMed
Pentameric ligand-gated ion channels (LGICs) are fast-gating receptors, represented by cationic nicotinic acetylcholine (nAChR) and serotonin (5HT3R) receptors, and by anionic GABA and glycine (GlyR) receptors. Because of a highly conserved sequence of 13 amino acids flanked by two canonical cysteine residues shared by all members of the family, these receptors are also known as the Cys-loop family. These receptors are allosteric transmembrane proteins made of five identical (or not) subunits arranged (pseudo) symmetrically around a central ion pore in the membrane. In nAChR, upon ACh binding, the receptor interconverts into discrete allosteric states, with each state corresponding to a different physiological state: resting (closed), active (open), and desensitized (closed).
        
Title: Inhibition of carbamate-insensitive acetylcholinesterase by piperonyl butoxide in Helicoverpa armigera Gunning RV Ref: Journal of Molecular Neuroscience, 30:21, 2006 : PubMed
The cotton bollworm, Helicoverpa armigera, is a cosmopolitan, insecticide-resistant insect pest of food and fiber (Gunning et al., 1992). Acetylcholinesterase (AChE) is the insect target site for carbamate insecticides, and H. armigera has developed an insensitive form of AChE as a resistance mechanism (Gunning et al., 1996). Insensitive AChE is normally considered an intractable resistance mechanism in pests. The methenedioxphenyl compound, piperonyl butoxide (PBO), has a long history as an insecticide synergist in the control of resistant arthropod pests; it is known to inhibit mono-oxygenases and nonspecific esterases (Gunning et al., 1998). This work discusses PBO inhibition of AChE in H. armigera and explores synergism.
Nicotinic acetylcholine receptors (nAChRs) are well-characterized allosteric transmembrane proteins involved in the rapid gating of ions elicited by ACh. These receptors belong to the Cys-loop superfamily of ligand-gated ion channels, which also includes GABAA and GABAC, 5-HT3, and glycine receptors. The nAChRs are homo- or heteromeric pentamers of structurally related subunits that encompass an extracellular N-terminal ligand-binding domain, four transmembrane-spanning regions that form the ion channel, and an extended intracellular region between spans 3 and 4. Ligand binding triggers conformational changes that are transmitted to the transmembrane-spanning region, leading to gating and changes in membrane potential. The four transmembrane spans on each of the five subunits create a substantial region of hydrophobicity that precludes facile crystallization of this protein. However the freshwater snail, Lymnaea stagnalis, produces a soluble homopentameric protein, termed the ACh-binding protein (AChBP), which binds ACh (Smit et al., 2001). Its structure was determined recently (Brejc et al., 2001) at high resolution, revealing the structural scaffold for nAChR, and has become a functional and structural surrogate of the nAChR ligand-binding domain. We have characterized an AChBP from Aplysia californica and determined distinct ligand-binding properties when compared to those of L. stagnalis, including ligand specificity for the nAChR alpha7 subtype-specific alpha-conotoxin ImI (Hansen et al., 2004).
        
Title: Cholinergic modulation of cortical function Hasselmo ME, Giocomo LM Ref: Journal of Molecular Neuroscience, 30:133, 2006 : PubMed
Extensive physiological research has demonstrated a number of common effects of acetylcholine within cortical structures, including the hippocampus, piriform cortex, and neocortex (Hasselmo, 1995, 1999). This article will provide a description of how the different physiological effects of acetylcholine could interact to alter specific functional properties of the cortex. The physiological effects of acetylcholine serve to enhance the influence of feed- forward afferent input to the cortex while decreasing background activity by suppressing excitatory feedback connections within cortical circuits. By enhancing the response to sensory input, high levels of acetylcholine enhance attention to sensory stimuli in the environment and enhance encoding of memory for specific stimuli. Interference from prior memory is reduced by suppressing synapses modified by prior learning (Sevilla et al., 2002; Linster et al., 2003).
        
Title: Peripheral choline acetyltransferase is expressed by monocytes and upregulated during renal allograft rejection in rats Hecker A, Lips KS, Pfeil U, Kummer W, Padberg W, Grau V Ref: Journal of Molecular Neuroscience, 30:23, 2006 : PubMed
Acetylcholine (ACh) has been shown to modulate the function of mononuclear leukocytes, both by muscarinic and nicotinic ACh receptors. Acute stimulation of lymphocytes with ACh or muscarinic agonists enhances proinflammatory functions, whereas chronic application of the ACh agonist nicotine has an anti-inflammatory effect (Geng et al., 1996; Kawashima and Fujii, 2003). In macrophages, acute treatment with nicotine down-modulates effector functions (Wang et al., 2003, 2004). ACh regulating leukocytes might originate from the nervous system. However, once released, ACh is quickly degraded. Relevant concentrations occur only in the direct vicinity of nerve endings. Non-neuronal ACh acting in a paracrine or autocrine fashion is more likely to influence immune functions. Lymphocytes express all enzymes needed for ACh synthesis, including choline acetyltransferase (ChAT). In the rat, alternative splicing generates common ChAT and peripheral ChAT (pChAT). Up to now, ChAT expression by monocytes has not been demonstrated. We investigate pChAT in monocytes in an experimental model of acute renal allograft rejection. Inside the blood vessels of the transplant, huge numbers of activated, cytotoxic monocytes accumulate and probably contribute to graft destruction (Grau et al., 2001).
        
Title: Structural dynamics of the acetylcholine binding protein: hydrodynamic and fluorescence anisotropy decay analyses Hibbs RE, Johnson DA, Shi J, Taylor P Ref: Journal of Molecular Neuroscience, 30:73, 2006 : PubMed
Recently, several crystal structures have become available for the acetylcholine binding protein (AChBP), a soluble nicotinic receptor extracellular domain (ECD) surrogate in its unliganded state, as well as in complex with agonists and antagonists. In these studies we sought to better understand how the dynamic receptor ECD surrogate from Lymnaea stagnalis behaves in solution, with and without ligand present. To accomplish this, we studied first the overall size and shape of the macromolecule, using hydrodynamic (sedimentation) techniques, and how these parameters were perturbed by the binding of various ligands. Analysis of sedimentation and frictional coefficients indicated that bound alpha-bungarotoxin (a three-fingered peptide toxin) is not oriented as a rigid body extending radially from the cylinder of the protein but, rather, that the toxin has inherent segmental flexibility such that it has a limited effect on the frictional coefficient of the pentamer. These results were supported by anisotropy decay studies of segmental motion of the toxin when free in solution and bound to AChBP. We selected the C-loop of AChBP, a region where ligand-elicited changes in conformation are substantial, and studied neighboring regions at higher resolution in terms of alpha-carbon backbone flexibility by decay of fluorescence anisotropy. Several single cysteine substitutions were labeled selectively with the fluorescent probe MTS-4-fluorescein. The covalently conjugated mutants, at two sites on the C-loop (S182C, D194C), and one on the opposing side of the subunit interface (Y164C), revealed similar alpha-carbon backbone flexibility with no ligand present but underwent distinctive changes in backbone mobility after ligand binding. At the sites we studied on the C-loop, agonists always segregated together in terms of their effects on backbone mobility; however antagonists did not reveal a similarly conserved pattern. At Y164C, however, we did observe segregating effects on backbone flexibility between agonists and antagonists. As a structural and functional surrogate of the nicotinic acetylcholine receptor, the AChBP reveals ligand-mediated changes in conformation, mimicking that of the receptor.
        
Title: Allosteric modulators and selective agonists of muscarinic receptors Holzgrabe U, De Amici M, Mohr K Ref: Journal of Molecular Neuroscience, 30:165, 2006 : PubMed
Allosteric modulators of ligand-receptor interactions are found for a variety of receptors (Christopoulos, 2002). Allosteric agents attach to a binding site being topographically distinct from the site for conventional (orthosteric) agonists or antagonists. In the case of the muscarinic receptor, a huge selection of structurally divergent modulators has been described for different receptor subtypes (Mohr et al., 2003). Alkane-bisammonio-type compounds carrying lateral phthalimido substituents are known to have a high affinity for the common allosteric binding site of the muscarinic acetylcholine M2 receptor (mAChR M2), which is already occupied by the orthosteric antagonist N-methylscopolamine (NMS). The resulting allosteric inhibition of the dissociation of [3H]NMS from the M2 receptors in porcine cardiac homogenates served to indicate binding of the test compounds to the allosteric site. Additionally, allosteric modulators can strongly influence equilibrium binding of the orthosteric ligand: Its binding can be reduced, left unaltered or elevated, and encoded as negative, neutral, and positive cooperativity, respectively (Christopoulos and Kenakin, 2002). The cooperativity is strongly dependent on the pair of allosteric/orthosteric ligands and on the receptor subtype.
        
Title: Determinants of positive cooperativity between strychnine-like allosteric modulators and N-methylscopolamine at muscarinic receptors Jakubik J, Dolezal V Ref: Journal of Molecular Neuroscience, 30:111, 2006 : PubMed
It has been shown previously that the third extracellular loop (o3) and its vicinity play a critical role in allosteric modulation at muscarinic acetylcholine receptors (mAChRs) (Ellis et al., 1993; Krejci and Tucek, 2001; Buller et al., 2002). In this study interaction of four chemically related substances (strychnine, its dimethoxy derivate brucine, precursor for synthesis of strychnine Wieland-Gumlich aldehyde (WGA), and precursor for synthesis of alcuronium propargyl-WGA) with orthosteric antagonist N-methylscopolamine (NMS) was investigated on the M3 subtype of mAChRs mutated at the o3 loop.
        
Title: Microarray screen for synaptic genes in the neuromuscular junction Jevsek M, Burden SJ Ref: Journal of Molecular Neuroscience, 30:29, 2006 : PubMed
The formation of neuromuscular synapses requires a complex exchange of signals between motor neurons and skeletal muscle fibers. Essential for the formation of neuromuscular junction (NMJ) is the activation of MuSK, a muscle-specific receptor tyrosine kinase (DeChiara et al., 1996). In mice lacking MuSK, motor axons fail to stop and differentiate, acetylcholine receptors (AChRs) fail to cluster, and AChR genes are expressed uniformly in muscle (DeChiara et al., 1996; Gautam et al., 1996). The retrograde signals for presynaptic differentiation are not known. Because synapse-specific transcription, like presynaptic differentiation, is MuSK-dependent, it is possible that retrograde signals for presynaptic differentiation might be encoded by genes that are expressed preferentially by synaptic nuclei. To identify such synapse-specific genes we screened Affymetrix microarrays with RNA from the dissected, synapse-enriched, and extrasynaptic regions of skeletal muscle and further studied those genes that encode for the secreted or cell-surface proteins.
        
Title: Alpha7 nicotinic acetylcholine receptor expression in Alzheimer's disease: receptor densities in brain regions of the APP(SWE) mouse model and in human peripheral blood lymphocytes Jones IW, Westmacott A, Chan E, Jones RW, Dineley K, O'Neill MJ, Wonnacott S Ref: Journal of Molecular Neuroscience, 30:83, 2006 : PubMed
The brains of people with Alzheimer's disease (AD) display several characteristic pathological features, including deposits (plaques) of beta-amyloid 1-42 (Abeta1-42), intraneuronal accumulations (tangles) of hyperphosphorylated tau, degeneration of the basal forebrain cholinergic pathway, and gliosis. Abeta1-42 plaques develop in specific brain regions, including hippocampus and cortex, as well as in the vasculature. Abeta1-42 might promote neurodegeneration through the induction of free radicals and disruption of Ca2+ homeostasis, giving rise to the symptoms of AD. Abeta1-42 interacts with the alpha7 subtype of the nicotinic acetylcholine receptor (alpha7 nAChR), which is widely expressed throughout the central and peripheral nervous systems, as well as in several nonneuronal loci, such as epithelial cells, lymphoid tissues, and peripheral blood lymphocytes. Western blot and autoradiographic analyses indicate that the alpha7 nAChR subunit protein is up-regulated in human brain samples from Alzheimer patients, as well as in animal models of AD (Dineley et al., 2001; Bednar et al., 2002), and might be involved in nicotine-mediated reduction of Abeta1-42 deposition (Hellstrom et al., 2004), although the nature of this relationship remains ill-defined. We have undertaken a semiquantitative histological evaluation of alpha7 nAChR expression in a mouse model of AD pathology, as well as a comparison of alpha7 nAChR levels in lymphocytes from AD patients and control subjects.
        
Title: Conclusions and comments for the XII ISCM Karczmar AG Ref: Journal of Molecular Neuroscience, 30:237, 2006 : PubMed
The first International Symposium on Cholinergic Mechanism (ISCM), organized by the late Edith Heilbronn, was held in Skokloster in 1970; Alicante's XII ISCM shows the exponential progress made in the cholinergic field in barely 30 years! Thus, Alzheimer's disease was not a topic at the first ISCM. The concept of homeostatic mechanisms regulating choline levels in the brain was not conceived of as yet. Three-dimensional pictures and the the protein structure of cholinergic receptors were not even thought of, as in 1970, we had only an "abstract" knowledge of receptors, based on SAR notions of Everhardus Ariens, Robert Furchgott, and Peter Pauling; in fact the Nobel Prize winner Furchgott stated in 1964 that "... with rare exceptions, we cannot ... identify the receptor as an individual chemical entity." Similarly, three-dimensional images of cholinesterases (ChEs) and the ChE "gorges" were unknown (Furchgott, 1964). The Whittakerian notion of synaptic vesicular release of acetylcholine (ACh) was the only version of the mode of ACh release, and the unorthodox opinions of Yves Dunant, Maurice Israel, Bruno Ceccarelli and Jacopo Meldolesi were still to be promulgated. Little was known about cholinergic correlates of behaviors such as learning and aggression, and there was no notion of cholinergic aspects of self-awareness (consciousness), free will, and the active subconscious. And modern methodologies were unknown, including the measurements of ACh, such as the gas chromatography-mass spectrometry (GCMS) method, discovered by Israel Hanin, Don Jenden, and Bo Holmstedt in the 1950s, the chemiluminescence developed by Maurice Israel, Yves Dunant, and their associates (Israel et al., 1983), and crystallography and molecular biology techniques, such as the "knockout" (KO) mouse models.
        
Title: Interaction of pyridinium oximes with acetylcholinesterase and their effect on organophosphate-poisoned mice Kovarik Z, Calic M, Vrdoljak AL, Radic B Ref: Journal of Molecular Neuroscience, 30:113, 2006 : PubMed
The progressive inhibition of acetylcholinesterase (AChE [EC 3.1.1.7]) by organophosphates (OPs), such as the nerve agents tabun and soman, is due to phosphorylation of the active center serine characterized by the formation of conjugates and inactivation of this essential enzyme involved in neurotransmission. Presently, a combination of an antimuscarinic agent, e.g., atropine, and an AChE reactivator, oxime, is used for the treatment of organophosphorus compound poisoning. The increased concern about terrorist use of nerve agents prompted us to search for new, more effective oximes against tabun and soman poisoning. We investigated the interactions of five bispyridinium oximes with human erythrocyte AChE and their effects on tabun- and soman-poisoned mice.
For the murine trachea, it has been reported that constriction evoked by serotonin (5-HT) is largely dependent on acetylcholine (ACh) released from the epithelium, owing to the sensitivity of the 5-HT response to epithelium removal, sensitivity to atropine, and insensitivity to tetrodotoxin (Moffatt et al., 2003). Consistent with this assumption, the respiratory epithelium contains ACh, its synthesizing enzyme, and the high-affinity choline transporter CHT1 (Reinheimer et al., 1996; Pfeil et al., 2003; Proskocil et al., 2004). Recently, we demonstrated that ACh can be released from non-neuronal cells by corticosteroid-sensitive polyspecific organic cation transporters (OCTs), which are also expressed by airway epithelial cells (Lips et al., 2005). Hence, we proposed that 5-HT evokes release of ACh from epithelial cells via OCTs and that this epithelial-derived ACh induces bronchoconstriction. We tested this hypothesis in a well-established model of videomorphometric analysis of bronchial diameter in precision-cut murine lung slices utilizing epithelium removal to assess the role of the epithelium, OCT mouse knockout (KO) strains to assess the role of OCT isoforms, and muscarinic receptor M2/M3 double-KO mice to assess the cholinergic component of 5-HT induced bronchoconstriction, as bronchi of this strain are entirely unresponsive to cholinergic stimulation(Struckmann et al., 2003).
        
Title: Gene transfer of acetylcholinesterase protects the knockout mouse from the toxicity of DFP Li B, Duysen EG, Lockridge O Ref: Journal of Molecular Neuroscience, 30:79, 2006 : PubMed
Acetylcholinesterase (AChE) has a clear role in nerve impulse transmission. Organophosphorus esters are highly toxic chemicals used as pesticides, fire retardants, plasticizers, and chemical warfare agents. The acute toxicity of organophosphorus poisons is attributed to inhibition of AChE in nerve synapses. This leads to seizures, respiratory arrest, and death. Our goal was to find a new therapeutic for protection against the toxicity of organophosphates (OPs). We investigated the feasibility of using a gene therapy vector to deliver AChE over long time periods and in quantities sufficiently high to provide protection against diisopropylfluorophosphate (DFP) toxicity. We used the AChE-/- mouse for these studies because this mouse has no endogenous AChE activity (Xie et al., 2000). Any AChE activity found in tissues could only come from the viral vector.
        
Title: Production of the butyrylcholinesterase knockout mouse Li B, Duysen EG, Saunders TL, Lockridge O Ref: Journal of Molecular Neuroscience, 30:193, 2006 : PubMed
The butyrylcholinesterase (BChE [EC 3.1.1.8]) knockout mouse is a model for BChE deficiency in humans. The existence of genetic variants of human BChE was discovered after a new muscle relaxant, succinylcholine, was introduced into the practice of medicine in the late 1950s. People with the atypical variant were unable to breathe for 2 h after receiving a dose intended to paralyze for 3-5 min (Kalow and Gunn, 1957, 1959). The atypical variant was later found to have a single-amino-acid mutation at Asp-70 (McGuire et al., 1989), which decreased the affinity of BChE for all positively charged compounds. Though the atypical BChE mutant is the one most commonly encountered in cases of succinylcholine apnea, an additional 58 mutations in the BChE coding sequence have been reported. The frequency of BChE mutations in the American population is known (Lockridge, 1990). One person out of 25 carries one atypical allele (D70G), whereas 1 out of 2500 is homozygous for D70G. The most frequent mutation, A539T, is carried by 1 person out of every 4 and is found in homozygous form in 1 person out of 69 (Bartels et al., 1992). The homozygous A539T form is associated with a 33% decrease in plasma BChE activity. Some people have no detectable BChE activity in plasma, owing to a mutation that truncates the protein, or inactivates it. The frequency of silent BChE is 1 out of 160 for carriers, and 1 out of 110,000 for homozygotes. The BChE knockout mice are models for silent BChE in humans. The literature contains no documentation of the health of people with silent BChE, other than to say they are healthy. We know nothing about their life expectancy, fertility, risk of cognitive impairment, risk of heart disease, or susceptibility to toxins. The BChE knockout mouse will allow us to test the hypothesis that the function of BChE is to detoxify poisons and will allow us to test the role of BChE in other physiological functions.
        
Title: Ispronicline: a novel alpha4beta2 nicotinic acetylcholine receptor-selective agonist with cognition-enhancing and neuroprotective properties Lippiello P, Letchworth SR, Gatto GJ, Traina VM, Bencherif M Ref: Journal of Molecular Neuroscience, 30:19, 2006 : PubMed
To date, the primary treatments for Alzheimer's disease with proven efficacy have been acetylcholinesterase inhibitors that prevent the hydrolysis of acetylcholine (ACh) in the synaptic cleft, thereby prolonging its activity. Although these agents have some benefit in alleviating cognitive impairment, they have limited clinical utility because of insufficient efficacy and marginal tolerability. Within the last decade, there has been much experimental support for the use of therapeutics that directly target nicotinic ACh receptors (nAChRs) to improve cognitive function and slow neurodegenerative disease progression. These findings have spurred considerable research efforts to develop ligands selective for nAChRs, such as ABT-418 (Arneric et al., 1995), SIB-1553 (Bontempi et al., 2001), TC-2403 (Lippiello et al., 1996), and TC-2559 (Bencherif et al., 2000). There is abundant evidence that nAChR modulators have the potential to alleviate cognitive impairment in demented states. In addition to improving cognitive function, a large body of research implicates a role for nAChRs in neuroprotection, suggesting potential for disease modification. An impact of nAChR agonists on disease progression would provide an advantage over currently available treatments for memory loss. The profile of previous nAChR-targeted clinical candidates has not been adequate to warrant further development owing to poor oral bioavailability, side effects, and/or lack of efficacy. Thus, a challenge in nAChR drug design and development has been the reduction of undesirable effects that result from activity at specific nAChRs in the CNS and PNS, including cardiovascular toxicity, emesis, seizures, and hypothermia.
Fast excitatory synaptic transmission in sympathetic ganglia is mediated by nicotinic acetylcholine receptors (nAChRs). Although it is known that the nAChR alpha7-subunit occurs in sympathetic ganglia, the expression of the recently cloned subunit alpha10 (Elgoyhen et al., 2001; Lustig et al., 2001; Sgard et al., 2002) has not been analyzed. Until now, functional receptors containing alpha10-subunits have been found only in combination with alpha9-subunits (Elgoyhen et al., 2001; Lustig et al., 2001; Sgard et al., 2002). The alpha9-subunit exhibits a restricted expression pattern, whereas the alpha10-subunit is expressed more widely. This broad distribution resembles more closely that known for subunit alpha7 than for subunit alpha9. On this background, we investigated the distribution of nAChR subunits alpha7, alpha9, and alpha10 in rat sympathetic ganglia and studied a possible interaction between subunit alpha7 and potential partners by double-labeling immunofluorescence and fluorescence resonance energy transfer (FRET) (Kam et al., 1995; Jares-Erijman and Jovin, 2003).
        
Title: Can cholinesterase inhibitors provide additional effects to cholinergic neurotransmission enhancement? Lopez MG, Arias E, Sobrado M, Lorrio S, Roda JM, Garcia AG Ref: Journal of Molecular Neuroscience, 30:141, 2006 : PubMed
The most frequent of the primary degenerative dementias is Alzheimer's disease (AD). The gradual loss of memory and attention in patients suffering from this illness are accompanied by aphasia, apraxia, agnosia, and alterations in visual-spatial perception. This group of symptoms is completed by emotional alterations, psychic instability, and changes in personality that appear in advanced phases of the illness. Different histopathological alterations have been described, like marked atrophy of the cerebral cortex with loss of cortical and subcortical neurons. Other histopathological hallmarks are the formation of senile plaques composed of beta-amyloid (Abeta) and neuro fibrillary tangles composed of hyperphosphorylation of tau protein.
        
Title: Docosahexaenoic acid supports cell growth and expression of choline acetyltransferase and muscarinic receptors in NG108-15 cell line Machova E, Novakova J, Lisa V, Dolezal V Ref: Journal of Molecular Neuroscience, 30:25, 2006 : PubMed
A large body of evidence indicates that adequate intake of polyunsaturated fatty acids is essential for brain development in early ontogenesis and positively impacts various pathological states connected with aging, as well as other neurodegenerative diseases (Jump, 2002; Bazan, 2003; Ruxton et al., 2004). In the present experiments, we investigated the possible effects of polyunsaturated docosahexanoic acid (DHA [22:6, n = 3]) on the expression of cholinergic phenotype-represented by choline acetyltransferase (ChAT) activity and a number of surface muscarinic receptors-as well as on cell growth in the cholinergic cell line NG108-15(Hamprecht, 1977; Hamprecht et al., 1985). However, chemical composition of different batches of sera is neither stable nor defined, and this fact complicates investigations on in vitro effects of substances that are natural constituents of serum. To avoid this restraint we employed defined medium in which fatty acid-free bovine albumin as a carrier of DHA replaced serum. Growth of most cell lines, as well as cells in primary cultures, depends strictly on the presence of serum in growth medium. As expected, withdrawal of serum resulted in growth arrest exemplified by a decrease in protein content compared with control cells grown in the presence of serum and also caused a decrease in ChAT activity (Fig. 1, lower left). DHA, at a concentration of 10 mumol/L, largely prevented both growth arrest in defined medium with fatty acid-free bovine albumin as a carrier of DHA and the attenuation of ChAT activity. DHA at concentrations 10 times higher had no further effect. At a concentration of 100 mumol/L, DHA also significantly increased the number of surface muscarinic receptors compared with cells grown in serum-containing as well as serum-free medium (Fig. 1, upper right). These data demonstrate the ability of DHA at low micromolar concentrations to support cell growth and expression of ChAT activity. Although it is not possible to stipulate a mechanism of action on the expression of ChAT and muscarinic receptors, a plausible explanation could be prevention of apoptosis, evidenced by a sharp decrease in executive caspase-3 activity (Fig. 1, lower right). Apoptosis is a process with a high requirement for energy. An improved metabolic state of cells consequent to suppression of apoptosis might thus better fulfill requirements for protein synthesis and targeting.
        
Title: The C-terminal T peptide of cholinesterases: structure, interactions, and influence on protein folding and secretion Massoulie J, Bon S Ref: Journal of Molecular Neuroscience, 30:233, 2006 : PubMed
Mammalian cholinergic tissues mostly express the T splice variant of acetylcholinesterase, in which the catalytic domain is associated with a C-terminal peptide of 40 residues, called the t peptide (Massoulie, 2002). Homologous t peptides exist in all vertebrate cholinesterases, acetylcholinesterases (AChEs), and butyrylcholinesterases (BChEs): they contain a series of seven conserved aromatic residues, including three tryptophans, and a cysteine at position-4 of their C-terminus. The major AChE isozyme of the nematode Caenorhabditis elegans also contains a similar peptide. Although the C-terminal t peptides do not seem to affect the catalytic activity of cholinesterases, they determine their physiological function, because they allow cholinesterase subunits of type T to form oligomers and to associate with structural anchoring proteins. When reduced to their catalytic domain, AChE subunits without a t peptide are active but remain monomeric and soluble.
Although the in vitro effect of organophosphorus (OP) compounds on acetylcholine-esterase (AChE) has been studied extensively, the hypothesis that OP inhibition of AChE is the primary mechanism of acute in vivo OP toxicity has been controversial. For example, a recent review (Pope and Liu, 2004) suggested that OP compounds have direct toxic effects on other enzymes, ACh receptors, and receptor/ channel complexes that are independent of AChE inhibition. The purpose of this report is to examine the hypothesis that AChE inhibition is the mechanism of acute toxicity of OP compounds by mathematically modeling the in vivo lethal effects of highly toxic OP compounds and determining the amount of variation in OP toxicity that is explained by AChE inhibition.
        
Title: Effects of acetylcholinesterase gene silencing on its activity in cultured human skeletal muscle Mis K, Mars T, Golicnik M, Jevsek M, Grubic Z Ref: Journal of Molecular Neuroscience, 30:31, 2006 : PubMed
In spite of several reports demonstrating that acetylcholinesterase (AChE [EC 3.1.1.7]) expression is importantly regulated at the level of its mRNA, we still know little about the relationship between AChE mRNA level and the level of mature, catalytically active enzyme in the cell. Better insight into this relationship is, however, essential for our understanding of the molecular pathways underlying AChE synthesis in living cells. We have approached this problem previously (Grubic et al., 1995; Brank et al., 1998; Mis et al., 2003; Jevsek et al., 2004); however, recently introduced small interfering RNA (siRNA) methodology, which allows blockade of gene expression at the mRNA level, opens new possibilities in approaching the AChE mRNA-AChE activity relationship. With this technique one can eliminate AChE mRNA in the cell, specifically and at selected times, and follow the effects of such treatment at the mature enzyme level. In this study we followed AChE activity in siRNA-treated cultured human myoblasts. Our aim was to find out how the temporal profile of the AChE mRNA decrease is reflected at the level of AChE activity under normal conditions and after inhibition of preexisting AChE by diisopropyl phosphorofluoridate (DFP).AChE activity was determined at selected time intervals after siRNA treatment in both myoblast homogenates and in culture medium to follow the effects of siRNA treatment at the level of intracellular AChE synthesis and at the level of AChE secreted from the cell.
Nicotinic acetylcholine receptors (nAChRs) in the brain exhibit diverse functional properties and ubiquitous distribution. Yet, except for providing a receptor for the exogenously applied nicotine of tobacco products, their role in the normal functioning of the brain has remained elusive. We have used a lentiviral expression vector to re-express the beta2 subunit specifically in the ventral tegmental area (VTA) of beta2-/- mice. The viral vector efficiently expresses beta2- subunit protein leading to new nAChR-binding sites. VTA neurons transduced by the lentiviral vector are responsive to intravenous nicotine when analyzed using in vivo electrophysiology. Nicotine-induced dopamine release from the nucleus accumbens (NuAcc) was also restored in re-expressing beta2-/- mice. Intra-VTA injection of nicotine was found to be reinforcing in both wild-type and beta2-subunit re-expressing beta2-/- mice, but not in beta2-/- mice. Furthermore, in the absence of applied nicotine, the spontaneous slow exploratory behavior of the mice was restored, whereas fast navigation did not change. This latter behavioral analysis suggests a role for beta2* nAChR, specifically expressed in the VTA, in mammalian cognitive function.
Apart from the hydrolysis of acetylcholine (ACh), acetyl- (AChE) and butyrylcholinesterase (BChE), through noncatalytic mechanisms, intervene in hematopoiesis, morphogenesis, and neurogenesis (Layer and Willbold, 1995; Soreq and Seidman, 2001). Cholinesterase (ChE) molecules occur as globular (G1, G2, and G4) and asymmetric (A4, A8, and A12) forms (Legay, 2000; Massoulie, 2002). The G species might display amphiphilic (GA) or hydrophilic (GH) properties (Perrier et al., 2002). The involvement of ChEs in tumorigenesis is supported by the measurement of ChE activity in tumors (Garcia-Ayllon et al., 2001; Ruiz-Espejo et al., 2003), the amplification of ChE genes in leukemias and ovarian tumors, and the relationship between the expression of AChE and the aggressiveness of astrocytomas(Perry et al., 2002). This research was undertaken to determine whether ChE activity is altered in gut carcinomas.
In vitro studies carried out on liposomes of defined composition showed that nicotinic acetylcholine receptors (nAChRs) are fully functional when they are reconstituted in a heterogeneous lipid matrix, such as that provided by crude soybean (asolectin [R-Aso]) lipids. However, when they are reconstituted in plain phosphatidylcholine (R-PC) lipids, their functional activity is completely lost (Fong and McNamee, 1986). This kind of study also pointed out that phosphatidic acid (PA) and cholesterol (Chol) play an important role in preserving the ability of this protein to exhibit an optimal channel activity (Fong and McNamee, 1986). Furthermore, it has been shown recently that nAChR, itself, induces the formation of specific PA-rich lipid domains (Poveda et al., 2002). Because Xenopus oocytes incorporate functionally into their plasma membrane nAChRs after intracellular injection of liposomes bearing this protein (Morales et al., 1995), the aim of this work was to determine the effect of the reconstitution lipid matrix on the functional properties of the transplanted nAChRs.
Short- and long-chain alpha-neurotoxins from snake venoms are potent blockers of nicotinic acetylcholine receptors (nAChRs). Short alpha-neurotoxins consist of 60-62 amino acid residues and include 4 disulfide bridges, whereas long alpha-neurotoxins have 66-75 residues and 5 disulfides. The spatial structure of these toxins is built by three loops, I-III "fingers," confined by four disulfide bridges; the fifth disulfide of long-chain alpha-neurotoxins is situated close to the tip of central loop II. An accurate knowledge of the mode of alpha-neurotoxin-nAChR interaction is important for rational design of new nAChR agonists and antagonists for medical purposes. Ideas on the topography of toxin-nAChR complexes were based until recently on nAChR interactions with selectively labeled alpha-neurotoxins, mutations in toxins, nAChR, or both. Recently, crystal structures have been solved for the Torpedo marmorata nAChR (4A[Unwin, 2005]) and for the acetylcholine-binding protein (AChBP) complexed with mollusk alpha-conotoxin (2.4 A[Celie et al., 2005]) or alpha-cobratoxin, long-chain alpha-neurotoxin (4 A [Bourne et al., 2005]). However, there were no angstrom-resolution models for complexes of short-chain alpha-neurotoxins. Here, we report the model of the Torpedo californica nAChR extracellular domain complexed to a short-chain alpha-neurotoxin II (NTII) from Naja oxiana cobra venom.
        
Title: Stoichiometry and pharmacology of two human alpha4beta2 nicotinic receptor types Moroni M, Bermudez I Ref: Journal of Molecular Neuroscience, 30:95, 2006 : PubMed
The alpha4beta2 nicotinic acetylcholine receptor (nAChR) is the most abundant nAChR subtype in the brain, where it forms the high-affinity binding site for nicotine. The alpha4beta2 nAChR belongs to a gene family of ligand-gated ion channels that also includes muscle nAChRs, GABAA receptors, and glycine receptors and that assembles into pentameric structures. alpha4 and beta2 nAChR subunits expressed heterologously in Xenopus laevis oocytes assemble into a mixture of high- and low-affinity functional receptors, giving rise to biphasic ACh concentration-response curves (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001). High- and low-affinity alpha4beta2 nAChRs differ significantly in their functional and pharmacological properties (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001; Nelson et al., 2003) and result from the assembly of alpha4 and beta2 subunits into two distinct stoichiometric arrangements: (alpha4)2(beta2)3(high-affinity subtype) and (alpha4)3(beta2)2 (low-affinity subtype) (Nelson et al., 2003). In this study we have examined the functional and pharmacological properties of high- and low-affinity alpha4beta2 receptors using two-electrode voltage clamp procedures on Xenopus oocytes transfected with high (1:10) or low (10:1) ratios of alpha4/beta2 cDNAs, which yield high (1:10)- or low (10:1)- affinity receptors with monophasic ACh concentration- response curves. Furthermore, to determine the stoichiometry of high- and low-affinity receptors expressed heterologously by Xenopus oocytes, we have determined the stoichiometry of high- and low-affinity alpha4beta2 receptors by mutating a highly conserved hydrophobic residue in the middle (position 9') of the pore-lining domain, which increases agonist potency in a manner that allows predictions on subunit composition (Cooper et al., 1991; Revah et al., 1991; Labarca et al., 1995; Boorman et al., 2000).
        
Title: Photo-induced covalent attachment of agonists as a tool to study allosteric mechanisms of nicotinic acetylcholine receptors Mourot A, Kotzyba-Hibert F, Goeldner M, Bamberg E Ref: Journal of Molecular Neuroscience, 30:3, 2006 : PubMed
Muscular and neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels and contain either two or five binding sites for acetylcholine (ACh). Binding of ACh molecules on the nAChR will trigger the fast opening of the channel and subsequent slow desensitization process. Neuronal alpha7 nicotinic receptors are made up of five identical subunits and possess five binding sites for ACh; this raises the question of how many sites must be occupied before channel opening. However, the effect of each ligand binding on gating is difficult to assess because of the reversible aspect of ligand binding at each site. One solution is to photochemically tether agonists to their binding sites. Such methodology has been applied elegantly and successfully on the homotetrameric cyclic-nucleotide-gated channels to evaluate the functional effects of each ligand binding on gating (Ruiz and Karpen, 1997). We therefore decided to develop a similar approach on Torpedo and neuronal alpha7 nAChRs with the photoactivatable agonist AC5 to investigate the effect of binding site occupancy on allosteric transitions of the receptor. In the dark, AC5 (see structure below) evokes robust currents on oocytes expressing Torpedo nAChR, displaying maximal amplitude comparable to ACh, with EC50 = 1.2 microM (Mourot et al., 2005). When the voltage-clamp oocyte was exposed to UV light in the presence of 30 microM AC5 for 50 s, there was a prolonged activation of the Torpedo nAChR, not reversible by washing, but inhibited by the noncompetitive blockers tetracaine and proadifen (see structure below). Both UV light and AC5 are required for this effect. However, further studies are required to determine whether the gradual decrease of the inward current reflects a slow desensitization process. AC5 is thus a potent photoactivatable agonist of the nAChR, which is able, upon UV irradiation, to incorporate covalently into the ACh-binding sites and to prolong activation of the nAChR. By extending this methodology to patch-clamp experiments, we will be able to incorporate one or several AC5s covalently into the muscular and neuronal nAChR at the single-channel level. Such study will help us understand the observed cooperative effect of gating and will contribute decisively to the controversial concerted vs sequential models for nAChR allosteric transitions.
The nicotinic acetylcholine receptor (nAChR) from fish electric organs and vertebrate neuromuscular junctions is a well-characterized transmembrane allosteric protein, composed of four polypeptide chains assembled into a heterologous pentamer alpha2betagammadelta, which carries ACh-binding sites and contains cation-selective channel-forming elements. Topographical mapping of residues contributing to the ligand-binding domain (LBD) of Torpedo nAChR was achieved with different site-directed antagonist or agonist probes. Over two decades of biochemical investigation led to the identification of three discontinuous domains on alpha subunits, with additional residues on gamma and delta subunits (Kotzyba- Hibert et al., 2004). This six binding-segment-domain model fits quite nicely with the three-dimensional positioning of the homologous residues in AChbinding protein (Brejc et al., 2001). However, little is known about the structural dynamics of the functioning receptor.
        
Title: CNS localization of neuronal nicotinic receptors Nashmi R, Lester HA Ref: Journal of Molecular Neuroscience, 30:181, 2006 : PubMed
Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of pentameric ligand-gated ion channels, which include GABA (A and C), serotonin, and glycine receptors. Currently, 12 neuronal nAChR subunits have been identified (alpha2-10 and beta2-4) and are generally grouped into alpha subunits, which contain two adjacent cysteine residues essential for ACh binding, and beta subunits, which lack these residues. The majority of neuronal nAChRs fall into two categories: those that bind agonist with high affinity (nM concentrations); and those that bind with lower affinity (microM concentrations). The low-affinity receptors are presumably homomeric alpha7 receptors that are alpha-bungarotoxin sensitive, whereas alpha4beta2 nAChRs account for >90% of the high-affinity nicotinic receptors in the brain (Whiting and Lindstrom, 1986). Their physiological contributions to neurotransmission, signaling, and behavior are not completely understood. Precise mapping of subcellular and neuroanatomical localizations of neuronal nAChR subunits will help elucidate the physiological role of neuronal nAChRs and their role in nicotine addiction.
A basic question regarding the integrative properties of the central nervous system is how transient motor commands or brief sensory stimuli are able to evoke persistent neural changes, mainly in the form of a sustained tonic rate of action potentials. Examples of this persistent neural activity have been reported in prefrontal (Fuster, 1997) and entorhinal (Egorov et al., 2002) cortices, as part of the neural mechanisms involved in short-term working memory (Goldman-Rakic, 1995). Interestingly enough, the general organization of motor systems assumes the presence of bursts of short-lasting motor commands encoding movement characteristics such as velocity, duration, and amplitude, followed by a maintained tonic firing encoding the position at which the moving appendage should be maintained (Robinson, 1981; Moschovakis, 1997). Thus, persistent neural activity seems to be necessary for both behavioral (positions of fixation) and cognitive (working memory) processes.
Lama2dy mice constitute an animal model for congenital muscular dystrophy (CMD) by merosin (laminin alpha2-chain) deficiency. This pathology affects the properties of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) of mouse skeletal muscle and nerves (Moral-Naranjo et al., 1999, 2002). AChE and BChE are involved through catalytic and noncatalytic actions in multiple processes, such as hydrolysis of acetylcholine (ACh), morphogenesis, hematopoiesis, and tumorigenesis (Soreq and Seidman, 2001). AChE and BChE molecules can be globular (G1, G2, and G4) or asymmetric forms (A4, A8, and A12) (Massoulie, 2002), and G molecules can show amphiphilic (detergent-interacting, GA) or hydrophilic (GH) behavior. AChE catalytic subunits are encoded by three mRNAs (T, H, or R) generated by alternative splicing. The presence of AChE in lymphoid tissues (Rossi et al., 1991; Nieto-Ceron et al., 2004), the role of immune responses in muscular dystrophy (Spencer and Tidball, 2001), the abnormalities of Lama2dy thymus (Magner et al., 2000), and the role of ACh in thymocyte function (Kawashima and Fujii, 2000) prompted us to investigate thymus AChE and the possible effect of merosin deficiency on it.
        
Title: Muscarinic receptors are affected by corticotropin-releasing hormone and c-fos gene disruptions: is there a mutual connection to adrenoceptors? Novakova M, Benes J, Kubovcakova L, Kvetnansky R, Myslivecek J Ref: Journal of Molecular Neuroscience, 30:69, 2006 : PubMed
In the last decade, progress in gene disruption technology has allowed the study of the effects of the single-gene knockout (KO) on different molecules involved in the signaling cascade activated via muscarinic receptors. Many KO mice targeting muscarinic receptors have been developed, that is, all (M1-M5) muscarinic receptor KO mice (Wess, 2003) and acetylcholinesterase (AChE) KO mice(Xie et al., 2000). Recently, we have shown that these (AChE-/-) mice not only reveal changes in the number of muscarinic receptors in the heart, lung, cortex, and cerebellum but also in the number of adrenoceptors (Teplicky et al., 2004). Next, we studied whether the disruption of corticotropin-releasing hormone (CRH) or c-Fos could affect the properties of muscarinic receptors and adrenoceptors in the lungs and hearts of mice. The effects of immobilization stress in CRH KO animals were also studied.
        
Title: Quaternary ammonium anticholinesterases have different effects on nicotinic receptors: is there a single binding site? Olivera-Bravo S, Ivorra I, Morales A Ref: Journal of Molecular Neuroscience, 30:205, 2006 : PubMed
Copious current research is devoted to finding new modulators of nicotine acetylcholine receptors (nAChRs), in view of their high relevance for nervous system physiology and because alterations in their number and/or function are related to several neurological disorders and neurodegenerative diseases (Pereira et al., 2002). Recently, we have shown that 1,5-bis(4-allyldimethyl ammonium phenyl) pentan-3-one dibromide (BW284c51), a quaternary ammonium cholinesterase inhibitor (QChEI), selectively inhibits nicotinic currents (IACh) by blocking the nAChR channel (Olivera et al., 2005). Interestingly, we found different effects of BW284c51 on nAChRs from those described previously for other QChEIs (Bertrand et al., 1992; Yost and Maestrone, 1994), such as decamethonium and edrophonium. Therefore, this work was aimed at characterizing, comparing, and elucidating the mechanisms underlying the action of different QChEIs on nAChRs.
Expression of cholinesterase (ChE) activity during phases of embryonic development is a general phenomenon in embryonic tissues. To elucidate the role(s) of ChEs during embryonic development, one line of research followed the assumption of a primitive muscarinic system involved in morphogenesis (Hohmann et al., 1995). This means that ChE functioning during development fits into the classical cholinergic neurotransmitter system: acetylcholine (ACh), as a signal, binds to ACh receptors and then is degraded by acetylcholinesterase (AChE) as the terminating enzyme. However, this is just one of the possible mechanisms. The other line of research was driven by evidence for noncholinergic functions of ChE proteins (AChE and butyrylcholinesterase [BChE]). There is accumulating data that other sites on AChE could exert nonclassical roles related to cell differentiation, neurite outgrowth, and adhesion.
The gene of mammalian acetylcholinesterase (AChE) generates multiple molecular forms, by alternative splicing of its transcripts and association of the tailed variant (AChET) with structural proteins. In the mammalian brain, the major AChE species consists of AChET tetramers anchored to the cell membrane of neurons by the PRiMA protein (Perrier et al., 2002). Stress and anticholinesterase inhibitors have been reported to induce rapid and long-lasting expression of the readthrough variant (AChER) in the mouse brain (Kaufer et al., 1998). In the readthrough transcript, there is no splicing after the last exon encoding the catalytic domain, so that the entire alternatively spliced 3' region is maintained. It encodes a C-terminal peptide with no specific interaction properties: COS cells transfected with AChER produce a soluble, nonamphiphilic monomeric form. We quantified AChER and total AChE expression in the mouse brain after an immobilization stress and after heat shock in neuroblastoma cells, and compared the observed effects with those induced by irreversible AChE inhibition (Perrier et al., 2005).
        
Title: The N-butylcarbamate derivative of galantamine acts as an allosteric potentiating ligand on alpha7 nicotinic receptors in hippocampal neurons: clinical implications for treatment of Alzheimer's disease Popa RV, Pereira EF, Lopes C, Maelicke A, Albuquerque EX Ref: Journal of Molecular Neuroscience, 30:227, 2006 : PubMed
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive impairments that become severe enough to interfere with the daily activities of patients and eventually lead to death (Chung and Cummings, 2000). Arecent study reports that approx 24 million people suffer from dementia worldwide. If the mortality rate does not change and no curative or preventive treatment is developed, this number is expected to double every 20 yr worldwide (Ferri et al., 2005). Although the causes of AD remain obscure, it has been reported that incremental loss of cholinergic neurons and of nicotinic receptor (nAChR) function/expression in specific brain regions correlates well with the severity of the symptoms at early stages of the disease (Hellstrom-Lindahl et al., 1999; Nordberg, 2001; Perry et al., 2001; Wevers et al., 1999). In patients with more advanced stages of AD, such a correlation between the magnitude of nAChR loss and of cognitive decline does not appear to exist (Sabbagh et al., 2001). The nicotinic cholinergic system plays a central role in modulating different forms of associative learning known to be impaired in AD patients, including the eyeblink classical conditioning (Woodruff-Pak, 2001), and in maintaining neuronal viability. Neuroprotection and cognitive improvement result from increasing the activity of different nAChR subtypes, including those bearing the alpha7 subunit (Carlson et al., 1998; Hejmadi et al., 2003; Kihara et al., 1997; Levin et al., 2006). Thus, increasing nAChR activity in the brain was proposed as a mechanism to slow down the progression of the disease (Maelicke and Albuquerque, 1996).
        
Title: Ethanol consumption produces changes in behavior and on hippocampal alpha7 and alpha4beta2 nicotinic receptors Robles N, Sabria J Ref: Journal of Molecular Neuroscience, 30:119, 2006 : PubMed
Ethanol consumption produces a wide range of effects on the central nervous system, most of them related to changes in neural receptors. In vitro studies have demonstrated that ethanol increases neural nicotinic acetylcholine receptor (nnAChR) affinity for ACh (Narahashi et al., 1991) and have also reported differences in sensitivity of nnAChRs for ethanol, depending on the subunit composition of the receptor (Cardoso et al., 1999). There is evidence that ethanol induces changes in density of nnAChRs in cellular cultures (Gorbounova et al., 1998). However, there are no clear results concerning the effects of chronic ethanol on nicotinic receptors and on behavior in rats nonselected by their preference to ethanol. A number of studies demonstrate that nnAChRs participate in a variety of functions, including memory and learning processes, neurodegeneration, and neuroprotection (Picciotto et al., 2000). In the present work, we found significant alterations in the Fixed-Interval Behavioral Test, as well as in density and affinity parameters of hippocampal main subtypes of nnAChRs: alpha7 homopentamers and alpha4beta2 heteropentamers of ethanol-drinking rats nonselected by their preference to ethanol.
        
Title: Interaction between P2X and nicotinic acetylcholine receptors in glutamate nerve terminals of the rat hippocampus Rodrigues RJ, Almeida T, de Mendonca A, Cunha RA Ref: Journal of Molecular Neuroscience, 30:173, 2006 : PubMed
Nicotinic acetylcholine receptors (nAChRs [constituted by pentameric association of alpha2-10 and beta2-4 subunits]) and P2X receptors (P2XRs [activated by ATP and constituted by multimeric association of P2X1-7 subunits]) are both ionotropic receptors permeable to cations, which have in common the disparity between the wealth of data showing their presence in the brain and little evidence of their participation in mediating synaptic transmission. This has led to the proposal that both nAChRs and P2XRs might primarily modulate rather than directly mediate synaptic transmission, which is in accordance with the predominant presynaptic localization of both receptor subtypes (Role and Berg, 1996; Cunha and Ribeiro, 2000). Interestingly, both functional neurochemical (Allgaier et al., 1995; Salgado et al., 2000; Diaz-Hernandez et al., 2002) and electrophysiological studies (Barajas-Lopez et al., 1998; Searl et al., 1998; Zhou and Calligan, 1998; Khakh et al., 2000) indicated a close interaction between nAChRs and P2XRs, which is paralleled by a co-release of ATPand ACh from central terminals (e.g., Richardson and Brown, 1987). Because glutamate release in the hippocampus is controlled by both nAChRs (e.g., McGehee et al., 1995) and P2XRs (Khakh et al., 2003; Rodrigues et al., 2005), we investigated if there was a functional interaction between these two presynaptic ionotropic receptors in the control of glutamate release in the rat hippocampus.
        
Title: Neuroprotection by nicotine in hippocampal slices subjected to oxygen-glucose deprivation: involvement of the alpha7 nAChR subtype Rosa AO, Egea J, Gandia L, Lopez MG, Garcia AG Ref: Journal of Molecular Neuroscience, 30:61, 2006 : PubMed
Nicotine (NIC) is neuroprotective against glutamate and hypoxia-induced neurotoxicity, preventing neuronal death and apoptosis in primary neuronal cultures. This effect is mediated by activation of both alpha7 and alpha4beta2 subtypes of nicotinic receptors for acetylcholine (nAChR) (Kaneko et al., 1997; Hejmadi et al., 2003). Furthermore, it seems that activation of alpha7 nAChR is the mechanism by which galantamine protects against thapsigargin and beta-amyloid-induced cell death (Arias et al., 2004), as well as in neuroprotection exerted by NIC against tumor necrosis factor alpha (Gahring et al., 2003). In this context we studied possible protection produced by NIC in an oxygen-glucose deprivation (OGD) model of rat and mouse hippocampal slices. The involvement of alpha7 nAChR in neuroprotection was proved by using wild-type and alpha7 knockout (KO) mice.
        
Title: Effect of acetylcholine on tau phosphorylation in human neuroblastoma cells Rubio A, Avila J, Perez M Ref: Journal of Molecular Neuroscience, 30:185, 2006 : PubMed
Alzheimer's disease (AD) is a senile dementia characterized by a progressive loss of memory, together with cognitive and behavioral impairments. In the past it was indicated that the disease was associated with a loss of acetylcholine (ACh) in the cerebral cortex (Bowen et al., 1976; Davies and Maloney, 1976); afterward, it was indicated that the severity of dementia was correlated with the extent of cholinergic loss (Perry et al., 1981). Because one of the biochemical features of AD is modification by phosphorylation of the microtubule-associated protein tau (for review, see Avila et al., 2004), in this work we indicate the effect of ACh on tau phosphorylation at specific sites recognized by 12E8 and PThr50 antibodies in human neuroblastoma SH-SY5Y cells. Two sites in which modification might regulate the binding of tau to microtubules (Novak et al., 1991; Feijoo et al., 2004).
        
Title: Engineered site-directed labeling of nicotinic acetylcholine receptors using reactive epibatidine derivatives: appraisal of epibatidine-docking models in neuronal and muscular receptors Sakr E, Kotzyba-Hibert F, Grande J, Hovius R, Vogel H, Bertrand S, Bertrand D, Goeldner M Ref: Journal of Molecular Neuroscience, 30:35, 2006 : PubMed
We developed an engineered site-directed labeling method (Foucaud et al., 2001) to investigate ligand receptor interactions on the acetylcholine (ACh)- binding site of nicotinic acetylcholine receptors (nAChRs). The method uses cysteine receptor mutants, together with cysteine-reactive ligand analogs, to generate a site-directed covalent reaction within the binding site. We selected epibatidine (EPB) as a prototypical ligand, acting at all types of nAChRs with sufficient affinity to allow this study. Accordingly, we synthesized three cysteine-reactive derivatives, all modified at the C-3 of the pyridine ring of the alkaloid with NCS; -NHCOCH2Cl, and -CH2Cl groups, respectively (Fig. 1). The binding properties have been established on rat brain, alpha7-5HT3 chimera, and Torpedo membranes, respectively, whereas the functional properties were tested on alpha4beta2 and alpha7 receptor expressed in oocytes and Cys-less muscular receptor expressed in HEK cells (Sakr et al., 2005).
Current antidotal regimens for organophosphorus compound (OP) poisoning consist of a combination of pretreatment with a spontaneously reactivating AChE inhibitor such as pyridostigmine bromide, and postexposure therapy with anticholinergic drugs such as atropine sulfate and oximes such as 2-PAM chloride (Gray, 1984). Although these antidotal regimens are effective in preventing lethality of animals from OP poisoning, they do not prevent postexposure incapacitation, convulsions, performance deficits, or, in many cases, permanent brain damage (Dunn and Sidell, 1989). These problems stimulated the development of enzyme bioscavengers as a pretreatment to sequester highly toxic OPs before they reach their physiological targets. Several studies over the last two decades have demonstrated that exogenously administered human serum butyrylcholinesterase (Hu BChE) can be used successfully as a safe, efficacious, and single prophylactic treatment to counteract the toxicity of OPs. It also has potential use for first responders (civilians) reacting to terrorist nerve gas release, pesticide overexposure, or succinylcholine-induced apnea. A dose of 200 mg of Hu BChE in humans is envisioned as a prophylactic treatment that can protect from exposure of 2-5 x LD50 of nerve agents (Ashani, 2000).
        
Title: Modulation of calcium-dependent and -independent acetylcholine release from motor nerve endings Searl TJ, Silinsky EM Ref: Journal of Molecular Neuroscience, 30:215, 2006 : PubMed
Inhibition of acetylcholine (ACh) release by adenosine is an important mechanism by which the secretory apparatus is regulated at both mammalian (Ginsborg and Hirst, 1972; Hirsh et al., 2002; Silinsky, 2004) and amphibian (Silinsky, 1980; Silinsky and Solsona, 1992; Redman and Silinsky, 1993, 1994; Robitaille et al., 1999) neuromuscular junctions (NMJs). ACh is known to be costored with ATP in cholinergic vesicles (Zimmermann, 1994), and it has been demonstrated that at amphibian NMJs, adenosine derived from neurally released ATPis the mediator of neuromuscular depression exhibited at low frequencies of nerve stimulation (Redman and Silinsky, 1994) (Fig. 1). At the mouse motor nerve ending the inhibitory actions of adenosine on transmitter release are linked to a reduction in the nerve-terminal calcium current associated with neurotransmitter release (Silinsky, 2004). In contrast, at the frog motor nerve, inhibition of ACh release by adenosine occurs in the absence of any effect on nerve-terminal calcium currents (Silinsky and Solsona, 1992; Redman and Silinsky, 1994; Robitaille et al., 1999). That is, at the frog NMJ adenosine inhibits ACh release through an effect on a process that takes place downstream from calcium entry. Although the exact site at which adenosine inhibits transmitter release is unknown, both the speed (50-100 ms; E. M. Silinsky, unpublished observations) and the stimulation-independent nature of inhibition suggest that this process must occur through an action on vesicles that are already primed and ready for release. Thus, the likely sites for mediating the action of adenosine are those core components of the neurotransmitter release process, the three SNARES (SNAP-25, syntaxin, and synaptobrevin), and synaptotagmin. However, there are difficulties in addressing which of these individual elements of the secretory apparatus might be involved in the actions of adenosine. We could use fractions of botulinum toxin to eliminate individual components of the secretory apparatus. However, each of these core components of the release machinery is individually essential for the neurotransmitter release process. Therefore, we decided to approach this problem by alternative means.
Several neurodegenerative disorders present deficiencies in the cholinergic system. Scarce research on prion encephalopathies has examined the levels of cholinergic pathway-related enzymes. Acetylcholinesterase (AChE) is expressed as several molecular forms. The potential importance of these variants is increased by the possibility that AChE has roles other than acetylcholine hydrolysis. We investigated the levels of AChE, its molecular forms, and glycosylation in the cerebrospinal fluid (CSF) from Creutzfeldt-Jakob disease (CJD) patients.
Acetylcholine-binding protein (AChBP) is a water-soluble protein released from molluscan glial cells and modulates ACh-mediated synaptic transmission. Acetylcholine-binding protein (AChBP) is a water-soluble homolog of the ligand-binding domain of nicotinic receptors and other members of the pharmaceutically important family of pentameric ligand-gated ion channels (LGICs), GABAA, GABAC, 5-HT3 serotonin, and glycine receptors. The crystal structure of AChBP from Lymnaea stagnalis has become an established model for the extracellular domain of the pentameric LGICs, and homology models have been generated to analyze receptor-ligand interactions. AChBP has pharmacological properties similar to the homomeric alpha7 subtype of nicotinic ACh receptors (nAChRs), with relatively weak affinity for ACh and a 10-fold higher affinity for nicotine.
        
Title: Nicotinic acetylcholine receptors containing subunits alpha3 and alpha5 in rat nociceptive dorsal root ganglion neurons Spies M, Lips KS, Kurzen H, Kummer W, Haberberger RV Ref: Journal of Molecular Neuroscience, 30:55, 2006 : PubMed
Nociceptive primary afferent neurons carry nicotinic acetylcholine receptors (nAChRs). Using RTPCR, mRNAs for all alpha-subunits have been identified in rat dorsal root ganglia (DRG) (Genzen et al., 2001; Lips et al., 2002), but the responses of nociceptive neurons to nicotine are not uniform and the cellular distribution of nAChRs within DRG, in general, and among functionally different subtypes of primary afferent neurons, in particular, are only partially resolved (Rau et al., 2005). These diverse actions might suggest the presence of various nAChR isoforms that are operative under different conditions. The present study was aimed to extend previous studies on nAChRs that contain subunits alpha4, alpha7, and alpha10 in providing data for alpha3- and alpha5-subunit-containing nAChRs (Haberberger et al., 2004; Papadopolou et al., 2004). To this end, calcium-imaging and double-labeling immunofluorescence with nAChR alpha-subunit-specific antibodies, in combination with markers for nociceptive neurons (TRPV1, I-B4), were applied.
Cholesterol-sphingolipid microdomains, or lipid rafts, are major regulators of molecular interactions in membrane organization. Because lipid rafts can move laterally and cluster into larger patches, they have been proposed to play a role in the redistribution of specific molecules to specialized cellular structures. Rafts have been shown to favor formation and maintenance of synaptic receptor clusters in neurons of the central nervous system. However, little is known about their role in formation of the neuromuscular junction (NMJ). To determine whether lipid rafts are involved in acetylcholine receptor (AChR) cluster formation and stabilization in myogenic cells, two standard tools were employed: (1) Perturbation of lipid rafts by drugs that deplete membrane cholesterol was carried out to verify that cholesterol is required for AChR clustering in agrin-treated C2C12 myotubes; and (2) detergent resistance of lipid-ordered domains was also used to demonstrate that AChRs, as well as key components of the postsynaptic membrane of the NMJ, are associated with rafts.
        
Title: Enhancement of secretion by threshold nicotinic stimulation in bovine chromaffin cells Tapia L, Eguiagaray JG, Egea J, Garcia AG, Gandia L Ref: Journal of Molecular Neuroscience, 30:81, 2006 : PubMed
Synaptic release of neurotransmitters displays activity-dependent changes such as enhancement (facilitation, augmentation, or potentiation) or diminution (depression), which have been studied widely because of their implication in synaptic efficacy, neuronal plasticity, and formation and consolidation of learning and memory. Some of these types of modulation of secretion displayed by neurons are also present in neuroendocrine chromaffin cells, for instance, facilitation or augmentation, which seem to be related to mild changes in the transients of cytosolic concentration of calcium ([Ca2+]i) and the degree of refilling of the primed vesicle pool (Zucker, 1996; Neher, 1998). Desensitized nicotinic acetylcholine receptors (nAChRs) and their possible role in this short-term synaptic plasticity was investigated in populations of bovine chromaffin cells.
Alpha-conotoxins, neurotoxic peptides from poisonous Conus marine snails, can be subdivided into several groups targeting distinct subtypes of nicotinic acetylcholine receptors (nAChRs). Such alpha-conotoxins as, for example, GI, MI, or SIA potently block muscle-type nAChRs from muscles and from the electric organ of Torpedo ray, whereas others target distinct neuronal nAChRs: alpha-conotoxins ImI and PnIB block pentaoligomeric alpha7 nAChRs, and alpha-conotoxins MII or PnIA inhibit heteromeric nAChRs made of combinations of alpha3 or alpha6 subunits with beta2 subunit. alpha-Conotoxins interact with N-terminal extracellular ligand-binding domains of nAChRs and are indispensable tools for distinguishing various subtypes of AChRs at normal and pathological states. Although many alpha-conotoxins have been isolated from Conus venoms, there is still a great need in more potent and selective tools, which in principle can be obtained by design and synthesis of novel alpha-conotoxin analogs.
        
Title: Transcriptional control of different acetylcholinesterase subunits in formation and maintenance of vertebrate neuromuscular junctions Tsim KWK, Xie HQ, Ting AK, Siow NL, Ling KK, Kong LW Ref: Journal of Molecular Neuroscience, 30:189, 2006 : PubMed
Acetylcholinesterase (AChE; EC 3.1.1.7) is a highly polymorphic enzyme (Massoulie, 2002). Asingle ACHE gene produces several types of catalytic subunits by alternative splicing, but a single splice variant, called type T (AChET), is expressed in adult mammalian muscle and brain. Catalytic subunits of AChET produce amphiphilic monomers and dimers, nonamphiphilic homotetramers, as well as heteromeric associations with anchoring proteins, ColQ (collagenous subunit) and PRiMA (proline-rich membrane anchor), which allow their functional localization in cholinergic synapses (Massoulie, 2002). ColQ characterizes the collagen-tailed forms (Aforms) of AChE and butyrylcholinesterase (BChE), which are localized in the basal lamina at neuromuscular junctions (NMJs) of vertebrates (Krejci et al., 1999); in these molecules (A4, A8, A12), one, two, or three tetramers of catalytic subunits are disulfide-linked to the strands of a triple helix of ColQ collagen. The cDNAs encoding ColQ, which have two transcripts, have been cloned: ColQ-1a predominantly in fast-twitch muscle, and ColQ-1 predominantly in slow-twitch muscle. The tetrameric globular (G4) form of AChE is characterized by linkage to PRiMA. PRiMAcDNA encodes a single-pass approximately 20-kDa type-I transmembrane protein and, similar to that of ColQ, contains a short PRAD (proline-rich attachment domain) that is able to organize AChE catalytic subunits into tetramers and anchor the enzyme at the surface of neuron and muscle (Massoulie, 2002).
In vertebrate neuromuscular junction, acetylcholinesterase (AChE) is colocalized with acetylcholine receptor (AChR). This synaptic expression of AChE requires precise regulation of the AChE gene. However, the gene regulation pattern has species variation. Previous studies (Massoulie, 2002) indicated that AChE activities in muscles decreased in rat but increased in chicken after denervation. The spatial arrangement of regulatory elements in promoters among animals therefore might be varied. The genomic structures of AChE have been analyzed in Torpedo, mouse, rat, and human but not in chick, and the molecular mechanism(s) responsible for contrary regulation of AChE between chick and mammal has been proposed (Choi et al., 2001) but not fully understood. Here, we report the cloning of the chick AChE promoter, the regulation of which is being characterized.
        
Title: MuSK signaling at the neuromuscular junction Wang Q, Zhang B, Xiong WC, Mei L Ref: Journal of Molecular Neuroscience, 30:223, 2006 : PubMed
The neuromuscular junction (NMJ) is a peripheral cholinergic synapse that conveys signals from motor neurons to muscle cells (Sanes and Lichtman, 1999; Sanes and Lichtman, 2001). The formation of the NMJ requires communication between motoneurons and muscle fibers. Three molecules are essential for NMJ formation: agrin, MuSK, and rapsyn. MuSK appears to be involved in every aspect of NMJ development and maintenance. The paper reviews agrin-MuSK cascades and its potential cross talk with Wnt signaling pathways.
        
Title: Markers of organophosphate exposure in human serum Wieseler S, Schopfer LM, Lockridge O Ref: Journal of Molecular Neuroscience, 30:93, 2006 : PubMed
The goal of this work is to identify novel serum proteins that are labeled with organophosphates (OPs) and to create a protocol for identification using mass spectroscopy. The use of OP-labeled proteins for identification of exposure is useful because such proteins will remain in circulation for weeks (Van Der Schans et al., 2004). Currently, both butyrylcholinesterase (BChE) and albumin have been shown to bind OPs in blood. Peeples et al. (2005) showed that albumin is labeled by OPs, specifically 6-Nbiotinylaminohexyl isopropyl phosphorofluoridate hemihydrate, in living mice. Albumin is the major protein in human serum, and its reaction with OPs tends to overwhelm the identification of other proteins. In vitro studies of human serum require removal of the serum albumin without depleting the less abundant proteins. Following this step, identifying the remaining proteins is simply a matter of labeling the proteins with an OP, separating the labeled from nonlabeled proteins, and using Q-trap mass spectrometry for identification.
        
Title: Nicotinic receptors modulate transmitter cross talk in the CNS: nicotinic modulation of transmitters Wonnacott S, Barik J, Dickinson J, Jones IW Ref: Journal of Molecular Neuroscience, 30:137, 2006 : PubMed
Neuronal nicotinic acetylcholine receptors (nAChRs) in the CNS appear to exert a predominantly modulatory influence on brain mechanisms, despite being fast-acting ligand-gated ion channels. Many nAChRs have an extrasynaptic location on somatodendritic regions or presynaptic terminals. They influence local excitability by depolarization and can initiate short- and long-term changes by interfacing with Ca2+ signaling pathways (Dajas- Bailador and Wonnacott, 2004). The modulation of neurotransmitter release by presynaptic nAChRs is well-documented (Wonnacott, 1997): Both Na+ and Ca2+ fluxes associated with nAChR activation can influence transmitter release. It is also emerging that nAChRs, especially the alpha7 subtype, can exert an indirect effect on transmitter release, through modulation of amino acid transmitters. This complex scenario facilitates transmitter cross talk, which is the subject of this short review.
        
Title: Acetylcholine, cognition, and consciousness Woolf NJ Ref: Journal of Molecular Neuroscience, 30:219, 2006 : PubMed
Acetylcholine (ACh) is a neuromodulator inextricably involved with higher mental functions. The organization of central pathways enables this role, as do the complex responses to ACh. This chapter focuses on intradendritic responses to ACh.
        
Title: Readthrough acetylcholinesterase: a multifaceted inducer of stress reactions Zimmerman G, Soreq H Ref: Journal of Molecular Neuroscience, 30:197, 2006 : PubMed
Stress insults induce hyperexcitation of cholinergic circuits, both peripherally in the sympathetic pathway (Tracey, 2002) and at the central nervous system (CNS) (Sapolsky, 1996). This reaction can serve to ensure survival but might also entail a risk to the hyperactivated neurons. Consequent changes in the expression of a series of proteins related to acetylcholine (ACh) metabolism might protect the organism from the potentially detrimental effects of this increase in ACh. Of particular interest among these effects is the induction by alternative splicing of the alternative, usually rare, readthrough variant of acetylcholinesterase (AChE), AChE-R. AChE-R is one of the first proteins conveying the signal that the organism has entered a state of alert. Viewing AChER as a stress signal can therefore serve to answer the question "How do we expect a stress signal to operate"? This facilitates the generation of hypotheses regarding the triggering of such signals and the effects it exerts at the molecular, cellular, and physiological levels.
The therapeutic potential of acetylcholinesterase (AChE) inhibitors has been strengthened recently by evidence showing that besides their role in cognitive function, they might contribute to slow down the neurodegeneration in Alzheimer's disease (AD) patients. It is known that AChE exerts secondary noncholinergic functions, related to its peripheral anionic site, in cell adhesion and differentiation, and recent findings also support its role in mediating the processing and deposition of beta-amyloid (Abeta) peptide. AChE is one of the proteins that colocalizes with Abeta peptide deposits in the brain of AD patients and promotes Abeta fibrillogenesis by forming stable AChEA beta complexes. Additionally, it has also been postulated that AChE binds through its peripheral site to the Abeta nonamyloidogenic form and acts as a pathological chaperone inducing a conformational transition to the amyloidogenic form (Inestrosa et al., 1996; Bartolini et al., 2003). Anew series of dual binding site AChE inhibitors has been designed and synthesized as new potent AChE inhibitors, which might simultaneously alleviate cognitive deficits and behave as disease-modifying agents by inhibiting Abeta peptide aggregation through binding to both catalytic and peripheral sites of the enzyme.