Expression of nicotinic receptors on primary cultures of rat astrocytes and up-regulation of the alpha7, alpha4 and beta2 subunits in response to nanomolar concentrations of the beta-amyloid peptide(1-42)

Neuronal nicotinic acetylcholine receptors (nAChRs) are thought to be involved in the pathogenesis of Alzheimer's disease (AD). Interestingly, in the brains of patients with this disease, losses of several subtypes of nAChRs on neurons have been reported, while an increase in alpha7 nAChRs was recently detected in the astrocytes. However, little is presently known about the expressions of individual subunits of nAChR on rat astrocytes in primary culture or the possible influence of exposure to beta-amyloid peptide (Abeta), a neuropathological hallmark of AD, on this expression. Thus, in the present investigation the levels of individual nAChR subunits on primary rat astrocytes and the possible direct influence of Abetas on the receptors were examined by RT-PCR, Western blotting, monitoring intracellular free calcium and immunohistochemistry. The alpha4, alpha7, beta2 and beta3 subunits and related calcium channel responses were found in these cells, whereas neither alpha2 nor alpha3 could be detected. Elevation in the levels of alpha7, alpha4 and beta2 mRNAs and proteins were observed in astrocytes exposed to 0.1-100nM Abeta(1-42). In contrast, incubation with 1muM Abeta(1-42) or Abeta(35-25) did not affect these levels. We propose that the enhanced expression of alpha7, alpha4 and beta2 nAChRs by astrocytes stimulated directly by nanomolar concentrations of Abeta(1-42) might be related to ongoing defensive or compensative mechanisms.     

Hierarchical control of dopamine neuron-firing patterns by nicotinic receptors

Nicotine elicits dopamine release by stimulating nicotinic acetylcholine receptors (nAChRs) on dopaminergic neurons. However, a modulation of these neurons by endogenous acetylcholine has not been described. We recorded, in vivo, the spontaneous activity of dopaminergic neurons in the VTA of anaesthetized wt and nAChR knockout mice and their response to nicotine injections. Deleting alpha7 or beta2 subunits modified the spontaneous firing patterns, demonstrating their direct stimulation by endogenous acetylcholine. Quantitative analysis further revealed four principal modes of firing, each depending on the expression of particular nAChR subunits and presenting unique responses to nicotine. The prominent role of the beta2 subunit was further confirmed by its selective lentiviral reexpression in the VTA. These data suggest a hierarchical control of dopaminergic neuron firing patterns by nAChRs: activation of beta2*-nAChR switches cells from a resting to an excited state, whereas activation of alpha7*-nAChRs finely tunes the latter state but only once beta2*-nAChRs have been activated.     

Amyloid peptide Abeta(1-42) binds selectively and with picomolar affinity to alpha7 nicotinic acetylcholine receptors

We have recently reported evidence that a very high affinity interaction between the beta-amyloid peptide Abeta(1-42) and the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) may be a precipitating event in the formation of amyloid plaques in Alzheimer's disease. In the present study, the kinetics for the binding of Abeta(1-42) to alpha7nAChR and alpha4beta2nAChR were determined using the subtype-selective nicotinic receptor ligands [(3)H]methyllycaconitine and [(3)H]cytisine. Synaptic membranes prepared from rat and guinea pig cerebral cortex and hippocampus were used as the source of receptors. Abeta(1-42) bound to the alpha7nAChR with exceptionally high affinity, as indicated by K(i) values of 4.1 and 5.0 pM for rat and guinea pig receptors, respectively. When compared with the alpha7nAChR, the affinity of Abeta(1-42) for the alpha4beta2nAChR was approximately 5,000-fold lower, as indicated by corresponding K(i) values of 30 and 23nM. The results of this study support the concept that an exceptionally high affinity interaction between Abeta(1-42) and alpha7nAChR could serve as a precipitating factor in the formation of amyloid plaques and thereby contribute to the selective degeneration of cholinergic neurons that originate in the basal forebrain and project to the cortex and hippocampus.     

Differential physiologic responses of alpha7 nicotinic acetylcholine receptors to beta-amyloid1-40 and beta-amyloid1-42

The beta-amyloid peptides (Abeta), Abeta(1-40) and Abeta(1-42), have been implicated in Alzheimer's disease (AD) pathology. Although Abeta(1-42) is generally considered to be the pathological peptide in AD, both Abeta(1-40) and Abeta(1-42) have been used in a variety of experimental models without discrimination. Here we show that monomeric or oligomeric forms of the two Abeta peptides, when interact with the neuronal cation channel, alpha7 nicotinic acetylcholine receptors (alpha7nAChR), would result in distinct physiologic responses as measured by acetylcholine release and calcium influx experiments. While Abeta(1-42) effectively attenuated these alpha7nAChR-dependent physiology to an extent that was apparently irreversible, Abeta(1-40) showed a lower inhibitory activity that could be restored upon washings with physiologic buffers or treatment with alpha7nAChR antagonists. Our data suggest a clear pharmacological distinction between Abeta(1-40) and Abeta(1-42).     

beta-Amyloid(1-42) binds to alpha7 nicotinic acetylcholine receptor with high affinity. Implications for Alzheimer's disease pathology

Alzheimer's disease pathology is characterized by the presence of neuritic plaques and the loss of cholinergic neurons in the brain. The underlying mechanisms leading to these events are unclear, but the 42-amino acid beta-amyloid peptide (Abeta(1-42)) is involved. Immunohistochemical studies on human sporadic Alzheimer's disease brains demonstrate that Abeta(1-42) and a neuronal pentameric cation channel, the alpha7 nicotinic acetylcholine receptor (alpha7nAChR), are both present in neuritic plaques and co-localize in individual cortical neurons. Using human brain tissues and cells that overexpress either alpha7nAChR or amyloid precursor protein as the starting material, Abeta(1-42) and alpha7nAChR can be co-immunoprecipitated by the respective specific antibodies, suggesting that they are tightly associated. The formation of the alpha7nAChR.Abeta(1-42) complex can be efficiently suppressed by Abeta(12-28), implying that this Abeta sequence region contains the binding epitope. Receptor binding experiments show that Abeta(1-42) and alpha7nAChR bind with high affinity, and this interaction can be inhibited by alpha7nAChR ligands. Human neuroblastoma cells overexpressing alpha7nAChR are readily killed by Abeta(1-42), whereas alpha7nAChR agonists such as nicotine and epibatidine offered protection. Because Abeta(1-42) inhibits alpha7nAChR-dependent calcium activation and acetylcholine release, two processes critically involved in memory and cognitive functions, and the distribution of alpha7nAChR correlates with neuritic plaques in Alzheimer's disease brains, we propose that interaction of the alpha7nAChR and Abeta(1-42) is a pivotal mechanism involved in the pathophysiology of Alzheimer's disease.     

Activity of cytisine and its brominated isosteres on recombinant human alpha7, alpha4beta2 and alpha4beta4 nicotinic acetylcholine receptors

Effects of cytisine (cy), 3-bromocytisine (3-Br-cy), 5-bromocytisine (5-Br-cy) and 3,5-dibromocytisine (3,5-diBr-cy) on human (h) alpha7-, alpha4beta2- and alpha4beta4 nicotinic acetylcholine (nACh) receptors, expressed in Xenopus oocytes and cell lines, have been investigated. Cy and its bromo-isosteres fully inhibited binding of both [alpha-(125)I]bungarotoxin ([alpha-(125)I]BgTx) to halpha7- and [(3)H]cy to halpha4beta2- or halpha4beta4-nACh receptors. 3-Br-cy was the most potent inhibitor of both [alpha-(125)I]BgTx and [(3)H]cy binding. Cy was less potent than 3-Br-cy, but 5-Br-cy and 3,5-diBr-cy were the least potent inhibitors. Cy and 3-Br-cy were potent full agonists at halpha7-nACh receptors but behaved as partial agonists at halpha4beta2- and halpha4beta4-nACh receptors. 5-Br-cy and 3,5-diBr-cy had low potency and were partial agonists at halpha7- and halpha4beta4-nACh receptors, but they elicited no responses on halpha4beta2-nACh receptors. Cy and 3-Br-cy produced dual dose-response curves (DRC) at both halpha4beta2- and halpha4beta4-nACh receptors, but ACh produced dual DRC only at halpha4beta2-nACh receptors. Low concentrations of cy, 3-Br-cy and 5-Br-cy enhanced ACh responses of oocytes expressing halpha4beta2-nACh receptors, but at high concentrations they inhibited the responses. In contrast, 3,5-diBr-cy only inhibited, in a competitive manner, ACh responses of halpha4beta2-nACh receptors. It is concluded that bromination of the pyridone ring of cy produces marked changes in effects of cy that are manifest as nACh receptor subtype-specific differences in binding affinities and in functional potencies and efficacies.     

Inhibiting gene expression of alpha3 nicotinic receptor in SH-SY5Y cells with the effects on APP metabolism and antioxidation in Alzheimer's disease

In order to examine the effects of alpha3 nicotinic acetylcholine receptor (nAChR) in connection with the pathogenesis of Alzheimer's disease (AD), neuroblastoma (SH-SY5Y) cells were transfected with small interference RNAs (siRNAs) that target specifically towards alpha3 nAChR. The expressions of alpha3 nAChR mRNA and protein were measured by real-time PCR and Western blotting, respectively. The levels of the alpha-form of secreted amyloid precursor protein (alphaAPPs) and total-APP were determined by Western blotting. SH-SY5Y cells transfected with siRNA were then treated with 1muM beta-amyloid peptide (Abeta)(1-42), following which the levels of lipid peroxidation, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the reduction rate of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were characterized by utilizing spectrophotometric procedures. As compared to controls, SH-SY5Y cells transfected with siRNA expressed the decreases in the levels of alpha3 nAChR mRNA and protein by 98% and 66% lower levels, respectively; exhibited reduced level of the alphaAPPs; and demonstrated enhanced lipid peroxidation, decreased rate of MTT reduction, and declined activities of SOD and GSH-Px. Inhibited gene expression of the alpha3 nAChR enhanced the toxicity exerted by Abeta. These results indicate that alpha3 nAChR may improve cleavage of APP by alpha-secretase, enhance antioxidation and inhibit the toxicity of Abeta, suggesting that the receptor might play an important role in AD.     

Statins prevent cholinesterase inhibitor blockade of sympathetic alpha7-nAChR-mediated currents in rat superior cervical ganglion neurons

Statins are reported to be beneficial in treating a multitude of disorders including dementia due to Alzheimer disease (AD) and vascular dementia (VaD) with varying, yet-to-be determined mechanisms of actions. Although cholinesterase inhibitors (ChEIs) are still recommended as the primary drug of choice for AD and related diseases, their efficacy is frequently questioned. We recently reported that alpha7-neuronal acetylcholine nicotinic receptor (alpha7-nAChR)-mediated neurogenic vasodilation of porcine cerebral arteries was blocked by ChEIs, and this blockade was prevented by statin pretreatment. The exact mechanism of interaction between ChEIs and statins remains unclear. Activation of alpha7-nAChRs located on perivascular postganglionic sympathetic nerve terminals releases norepinephrine, which then acts on presynaptic beta(2)-adrenoceptors located on neighboring nitrergic nerve terminals, resulting in nitric oxide release and vasodilation. The present study, therefore, was designed to determine whether interaction of ChEIs and statins occurs at the alpha7-nAChR level. We examined effects of concurrent application of ChEIs and statins on alpha7-nAChR-mediated inward currents in primary neuronal cultures of rat superior cervical ganglion cells, the origin of the perivascular sympathetic innervation to the cerebral arteries. The results indicated that physostigmine, neostigmine, and galantamine inhibited choline- and nicotine-induced whole cell currents in a concentration-dependent manner. This inhibition, which was noncompetitive in nature, was prevented by concurrent application of mevastatin and lovastatin in a concentration-dependent manner. These results suggest that statins protect alpha7-nAChR function directly at the receptor level. Since alpha7-nAChR is neuroprotective, having beneficial effects on memory and cerebral vascular function, its functional inhibition by ChEIs may explain in part the limitation of its effectiveness in AD and VaD therapy. Protection of alpha7-nAChR function from ChEI inhibition by concurrent administration of statins may provide an alternative strategy in improving the efficacy of AD and VaD therapy.     

Synthesis, nicotinic acetylcholine receptor binding, antinociceptive and seizure properties of methyllycaconitine analogs

A series of methyllycaconitine (1a, MLA) analogs was synthesized where the (S)-2-methylsuccinimidobenzoyl group in MLA was replaced with a (R)-2-methyl, 2,2-dimethyl-, 2,3-dimethyl, 2-phenyl-, and 2-cyclohexylsuccinimidobenzoyl (1b-f) group. The analogs 1b-f were evaluated for their inhibition of [(125)I]iodo-MLA binding at rat brain alpha7 nicotinic acetylcholine receptors (nAChR). In order to determine selectivity, MLA and the analogs 1b-f were evaluated for inhibition of binding to rat brain alpha,beta nAChR using [(3)H]epibatidine. At the alpha7 nAChR, MLA showed a K(i) value of 0.87 nM, analogs 1b-e possessed K(i) values of 1.67-2.16 nM, and 1f showed a K(i) value of 26.8 nM. Surprisingly, the analog 1e containing the large phenyl substituent (K(i)=1.67 nM) possessed the highest affinity. None of the compounds possessed appreciable affinity for alpha,beta nAChRs. MLA antagonized nicotine-induced seizures with an AD(50)=2 mg/kg. None of the MLA analogs were as potent as MLA in this assay. MLA and all of the MLA analogs, with the exception of 1b, antagonized nicotine's antinociceptive effects in the tail-flick assay. Compound 1c (K(i)=1.78 nM at alpha7 nAChR) with an AD(50) value of 1.8 mg/kg was 6.7 times more potent than MLA (AD(50)=12 mg/kg) in antagonizing nicotine's antinociceptive effects but was 5-fold less potent than MLA in blocking nicotine-induced seizures. Since MLA has been reported to show neuroprotection against beta-amyloid(1-42), these new analogs which have high alpha7 nAChR affinity and good selectivity relative to alpha,beta nAChRs will be useful biological tools for studying the effects of alpha7 nAChR antagonist and neuroprotection.     

Memantine inhibits α3β2-nAChRs-mediated nitrergic neurogenic vasodilation in porcine basilar arteries

Memantine, an NMDA receptor antagonist used for treatment of Alzheimer's disease (AD), is known to block the nicotinic acetylcholine receptors (nAChRs) in the central nervous system (CNS). In the present study, we examined by wire myography if memantine inhibited α3β2-nAChRs located on cerebral perivascular sympathetic nerve terminals originating in the superior cervical ganglion (SCG), thus, leading to inhibition of nicotine-induced nitrergic neurogenic dilation of isolated porcine basilar arteries. Memantine concentration-dependently blocked nicotine-induced neurogenic dilation of endothelium-denuded basilar arteries without affecting that induced by transmural nerve stimulation, sodium nitroprusside, or isoproterenol. Furthermore, memantine significantly inhibited nicotine-elicited inward currents in Xenopous oocytes expressing α3β2-, α7- or α4β2-nAChR, and nicotine-induced calcium influx in cultured rat SCG neurons. These results suggest that memantine is a non-specific antagonist for nAChR. By directly inhibiting α3β2-nAChRs located on the sympathetic nerve terminals, memantine blocks nicotine-induced neurogenic vasodilation of the porcine basilar arteries. This effect of memantine is expected to reduce the blood supply to the brain stem and possibly other brain regions, thus, decreasing its clinical efficacy in the treatment of Alzheimer's disease.     

Decreased protein levels of nicotinic receptor subunits in the hippocampus and temporal cortex of patients with Alzheimer's disease

Deficits of cortical nicotinic acetylcholine receptors (nAChRs) have been observed in Alzheimer's disease (AD) by receptor binding assays. Little is known about the receptor subunit specificity influenced by AD, and it might be of importance for therapeutic strategies. In the present study, the protein levels of nAChR alpha3, alpha4, alpha7, and beta2 subunits were investigated using western blot analysis on postmortem brains of patients with AD and age-matched controls. The results showed that in human postmortem brain samples, bands with molecular masses of 52, 42, and 50 kDa were detected by anti-alpha4, anti-alpha7, and anti-beta2 antibodies, respectively. When anti-alpha3 antibody was used, one major band of 49 kDa and two minor bands of 70 and 38 kDa were detected. In AD patients, as compared with age-matched controls, the alpha4 subunit was reduced significantly by approximately 35 and 47% in the hippocampus and temporal cortex, respectively. A significant reduction of 25% in the alpha3 subunit was also observed in the hippocampus and a 29% reduction in the temporal cortex. For the alpha7 subunit, the protein level was reduced significantly by 36% in the hippocampus of AD patients, but no significant change was detected in the temporal cortex. In neither the hippocampus nor the temporal cortex was a significant difference observed in the beta2 subunit between AD patients and controls. These results reveal brain region-specific changes in the protein levels of the nAChR alpha3, alpha4, and alpha7 subunits in AD.     

Effect of 1,1-dimethylphenyl 1,4-piperazinium on mouse tracheal smooth muscle responsiveness

Bronchial hyperresponsiveness is one of the main features of asthma. A nicotinic receptor agonist, 1,1-dimethylphenyl 1,4-piperazinium (DMPP), has been shown to have an inhibitory effect on airway response to methacholine in an in vivo model of asthma. The aims of this study were to 1) verify whether nicotinic acetylcholine receptors (nAChR) were present on mouse tracheal smooth muscle, 2) verify whether bronchoprotection observed in mice was due to a direct effect on airway smooth muscle, and 3) compare the effects of nicotinic agonists to that of salbutamol. Alpha3-, alpha4-, and alpha7-nAChR subunits were detected by immunofluorescence on tracheal tissues from normal BALB/c mice. The effect of DMPP on tracheal responsiveness was verified by an isometric method. Tracheas were isolated from normal mice, placed in organ baths, and contracted with a single dose of methacholine. Cumulative doses of DMPP or salbutamol were added to the baths. Results show that mouse tracheal smooth muscle is positive for alpha4- and alpha7-nAChR subunits and that the epithelium is positive for alpha3-, alpha4-, and alpha7-subunits. DMPP induced a greater dose-dependent relaxation of tracheal smooth muscles precontracted with methacholine than with salbutamol. These results suggest that the smooth muscle-relaxing effect of DMPP could have some interest in the treatment of obstructive pulmonary diseases.     

Up-regulation of cell-surface alpha4beta2 neuronal nicotinic receptors by lower temperature and expression of chimeric subunits.

The predominant nicotinic acetylcholine receptor (nAChR) expressed in vertebrate brain is a pentamer containing alpha4 and beta2 subunits. In this study we have examined how temperature and the expression of subunit chimeras can influence the efficiency of cell-surface expression of the rat alpha4beta2 nAChR. Functional recombinant alpha4beta2 nAChRs, showing high affinity binding of nicotinic radioligands (K(d) = 41 +/- 22 pM for [(3)H]epibatidine), are expressed in both stably and transiently transfected mammalian cell lines. Despite this, only very low levels of alpha4beta2 nAChRs can be detected on the cell surface of transfected mammalian cells maintained at 37 degrees C. At 30 degrees C, however, cells expressing alpha4beta2 nAChRs show a 12-fold increase in radioligand binding (with no change in affinity), and a 5-fold up-regulation in cell-surface receptors with no increase in total subunit protein. In contrast to "wild-type" alpha4 and beta2 subunits, chimeric nicotinic/serotonergic subunits ("alpha4chi" and "beta2chi") are expressed very efficiently on the cell surface (at 30 degrees C or 37 degrees C), either as hetero-oligomeric complexes (e.g. alpha4chi+beta2 or alpha4chi+beta2chi) or when expressed alone. Compared with alpha4beta2 nAChRs, expression of complexes containing chimeric subunits typically results in up to 20-fold increase in nicotinic radioligand binding sites (with no change in affinity) and a similar increase in cell-surface receptor, despite a similar level of total chimeric and wild-type protein.     

The beta-amyloid protein of Alzheimer's disease binds to membrane lipids but does not bind to the alpha7 nicotinic acetylcholine receptor

Accumulation of the amyloid protein (Abeta) in the brain is an important step in the pathogenesis of Alzheimer's disease. However, the mechanism by which Abeta exerts its neurotoxic effect is largely unknown. It has been suggested that the peptide can bind to the alpha7 nicotinic acetylcholine receptor (alpha7nAChR). In this study, we examined the binding of Abeta1-42 to endogenous and recombinantly expressed alpha7nAChRs. Abeta1-42 did neither inhibit the specific binding of alpha7nAChR ligands to rat brain homogenate or slice preparations, nor did it influence the activity of alpha7nAChRs expressed in Xenopus oocytes. Similarly, Abeta1-42 did not compete for alpha-bungarotoxin-binding sites on SH-SY5Y cells stably expressing alpha7nAChRs. The effect of the Abeta1-42 on tau phosphorylation was also examined. Although Abeta1-42 altered tau phosphorylation in alpha7nAChR-transfected SH-SY5Y cells, the effect of the peptide was unrelated to alpha7nAChR expression or activity. Binding studies using surface plasmon resonance indicated that the majority of the Abeta bound to membrane lipid, rather than to a protein component. Fluorescence anisotropy experiments indicated that Abeta may disrupt membrane lipid structure or fluidity. We conclude that the effects of Abeta are unlikely to be mediated by direct binding to the alpha7nAChR. Instead, we speculate that Abeta may exert its effects by altering the packing of lipids within the plasma membrane, which could, in turn, influence the function of a variety of receptors and channels on the cell surface.     

Antidepressants noncompetitively inhibit nicotinic acetylcholine receptor function

Nicotinic acetylcholine receptors (nAChRs) are diverse members of the neurotransmitter-gated ion channel superfamily and play critical roles in chemical signaling throughout the nervous system. The present study establishes for the first time the acute functional effects of sertraline (Zoloft), paroxetine (Paxil), nefazodone (Serzone), and venlafaxine (Effexor) on two human and one chick nAChR subtype. This study also confirms previous findings of nAChR functional block by fluoxetine (Prozac). Function of human muscle-type nAChR (alpha1/beta gammadelta) in TE671/RD cells, human autonomic nAChR (alpha3/beta4alpha5 +/- beta2) in SH-SY5Y neuroblastoma cells, or chick V274T mutant alpha7-nAChR heterologously expressed in native nAChR-null SH-EP1 epithelial cells was measured using 86Rb+ efflux assays. Functional blockade of human muscle-type and autonomic nAChRs is produced by each of the drugs in the low to intermediate micromolar range, and functional blockade of chick V274T-alpha7-nAChR is produced in the intermediate to high micromolar range. Functional blockade is insurmountable by increasing agonist concentrations at each nAChR subtype tested for each of these drugs, suggesting noncompetitive inhibition of nAChR function. These studies open the possibilities that nAChR subtypes in the brain could be targets for therapeutic antidepressants and could play roles in clinical depression.     

Apolipoprotein E and beta-amyloid (1-42) regulation of glycogen synthase kinase-3beta

Glycogen synthase kinase-3beta (GSK-3beta) is implicated in regulating apoptosis and tau protein hyperphosphorylation in Alzheimer's disease (AD). We investigated the effects of two key AD molecules, namely apoE (E3 and E4 isoforms) and beta-amyloid (Abeta) 1-42 on GSK-3beta and its major upstream regulators, intracellular calcium and protein kinases C and B (PKC and PKB) in human SH-SY5Y neuroblastoma cells. ApoE3 induced a mild, transient, Ca2+-independent and early activation of GSK-3beta. ApoE4 effects were biphasic, with an early strong GSK-3beta activation that was partially dependent on extracellular Ca2+, followed by a GSK-3beta inactivation. ApoE4 also activated PKC-alpha and PKB possibly giving the subsequent GSK-3beta inhibition. Abeta(1-42) effects were also biphasic with a strong activation dependent partially on extracellular Ca2+ followed by an inactivation. Abeta(1-42) induced an early and potent activation of PKC-alpha and a late decrease of PKB activity. ApoE4 and Abeta(1-42) were more toxic than apoE3 as shown by MTT reduction assays and generation of activated caspase-3. ApoE4 and Abeta(1-42)-induced early activation of GSK-3beta could lead to apoptosis and tau hyperphosphorylation. A late inhibition of GSK-3beta through activation of upstream kinases likely compensates the effects of apoE4 and Abeta(1-42) on GSK-3beta, the unbalanced regulation of which may contribute to AD pathology.     

Prostate apoptosis response-4 enhances secretion of amyloid beta peptide 1-42 in human neuroblastoma IMR-32 cells by a caspase-dependent pathway

Prostate apoptosis response-4 (Par-4) is a leucine zipper protein that promotes neuronal cell death in Alzheimer's disease (AD). Neuronal degeneration in AD may result from extracellular accumulation of amyloid beta peptide (Abeta) 1-42. To examine the effect of Par-4 on Abeta secretion and to reconcile amyloid/apoptosis hypotheses of AD, we generated IMR-32 cell lines that overexpress Par-4 and/or its leucine zipper domain. Overexpression of Par-4 did not significantly affect levels of the endogenously expressed beta amyloid precursor protein but drastically increased the Abeta(1-42)/Abeta(total) ratio in the conditioned media about 6-8 h after trophic factor withdrawal. Time course analysis of caspase activation reveals that Par-4 overexpression exacerbated caspase activation, which is detectable within 2 h after trophic factor withdrawal. Furthermore, inhibition of caspase activity by the broad spectrum caspase inhibitor BD-fmk significantly attenuated the Par-4-induced increase in Abeta 1-42 production. In addition, the effects of Par-4 on secretion of Abeta 1-42 were consistently blocked by co-expression of the leucine zipper domain, indicating that the effect of Par-4 on Abeta secretion may require its interaction with other protein(s). These results suggest that Par-4 increases secretion of Abeta 1-42 largely through a caspase-dependent pathway after apoptotic cascades are initiated.     

Peptide blockers of the inhibition of neuronal nicotinic acetylcholine receptors by amyloid beta

Alzheimer disease (AD) is characterized by accumulation of the neurotoxic amyloid beta peptide (Abeta) and by the loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs) throughout the brain. Direct inhibition of nAChRs by Abeta has also been suggested to contribute to cholinergic dysfunction in AD. In an effort to find ligands capable of blocking Abeta-induced inhibition of nAChRs, we have screened a phage display library to identify peptides that bind to Abeta. Using this approach, we identified a heptapeptide denoted IQ, which binds with nanomolar affinity to Abeta and is homologous to the acetylcholine-binding protein and to most subtypes of nAChRs. Rapid kinetic whole-cell current-recording measurements showed that Abeta inhibits nAChR function in a dose-dependent manner in neuronal differentiated PC12 cells and that nanomolar concentrations of IQ completely block the inhibition by Abeta. These results indicate that the Abeta binding site in nAChRs is homologous to the IQ peptide and that this is a relevant target for Abeta neurotoxicity in AD and, more generally, for the regulation of nAChR function by soluble Abeta in a physiological context. Furthermore, the results suggest that the IQ peptide may be a lead for the development of novel drugs to block the inhibition of nAChRs in AD.     

Beta 3: a new member of nicotinic acetylcholine receptor gene family is expressed in brain

Screening of a rat brain cDNA library with a radiolabeled probe made from an alpha 3 cDNA (Boulter, J., Evans, K., Goldman, D., Martin, G., Treco, D., Heinemanns, S., and Patrick, J. (1986) Nature 319, 368-374) resulted in the isolation of a clone whose sequence encodes a protein, beta 3, which is homologous (40-55% amino acid sequence identity) to previously described neuronal nicotinic acetylcholine receptor subunits. The encoded protein has structural features found in other nicotinic acetylcholine receptor (nAChR) subunits. Two cysteine residues that correspond to cysteins 128 and 142 of the Torpedo nAChR alpha subunit are present in beta 3. Absent from beta 3 are 2 adjacent cysteine residues that correspond to cysteines 192 and 193 of the Torpedo subunit. In situ hybridization histochemistry, performed using probes derived from beta 3 cDNAs, demonstrated that the beta 3 gene is expressed in the brain. Thus, beta 3 is the fifth member of the nAChR gene family that is expressed in the brain. The pattern of beta 3 gene expression partially overlaps with that of the neuronal nAChR subunit genes alpha 3, alpha 4, or beta 2. These results lead us to propose that the beta 3 gene encodes a neuronal nAChR subunit.     

Nicotinic acetylcholine receptor subunits and receptor activity in the epithelial cell line HT29

In the present study we have used RT-PCR to investigate nicotinic acetylcholine receptor (nAChR) subunit expression, and studied the effect of nicotine on TNFalpha-induced cytokine (IL-8) release in the epithelial cell line HT29. RNA was extracted using a commercial kit and amplified by RT-PCR. RT-PCR products were separated by electrophoresis and visualised using ethidium bromide. IL-8 release was measured by ELISA from cells activated for 6 h with TNFalpha (50 ng ml(-1)) in the absence and presence of nicotine (10(-11)-10(-6) M). HT29 cells contained mRNA for beta1, alpha4, alpha5, and alpha7 nAChR subunits. Activation of HT29 cells increased IL-8 release from undetectable amounts to 3.92 +/- 0.51 ng ml(-1) (n = 5). Nicotine significantly inhibited TNFalpha-induced IL-8 release in a concentration related manner with peak inhibition occurring at 10(-7) M (2.39 +/- 0.78 ng ml(-1), n = 5). Our data suggests that, while HT29 cells express mRNA for nAChR subunits, the only nAChR subunits that could form functional receptors and inhibit IL-8 release are alpha7.