α7 nicotinic acetylcholine receptor expression in Alzheimer's disease

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.     

Cellular protein and mRNA expression of β1 nicotinic acetylcholine receptor (nAChR) subunit in brain,skeletal muscle and placenta

The β1 nicotinic acetylcholine receptor (nAChR) subunit is a muscle type subunit of this family and as such, is found predominantly in muscle. Recent reports document its expression in other tissues and cell lines including adrenal glands, carcinomas, lung and brain. However, the majority of studies were of tissue lysates, thus the cellular distribution was not determined. This study aimed to determine the cellular distribution of the β1 nAChR subunit in the brain, at both the mRNA and protein levels, using non-radioactive in situ hybridization (ISH) and immunohistochemistry (IHC), respectively, and to compare it to two muscle tissue types, skeletal and placenta. Tissue was formalin fixed and paraffin embedded (all tissue types) and frozen (placenta) from humans. Additional control tissue from the piglet and mouse brain were also studied, as was mRNA for the α3 nAChR and N-methyl-d-aspartate receptor 1 (NR1) subunit. We found no β1 nAChR subunit mRNA expression in the human and piglet brain despite strong protein expression. Some signal was seen in the mouse brain but considered inconclusive given the probes designed were not of 100% homology to the mouse. In the skeletal muscle and placenta tissues, β1 nAChR subunit mRNA expression was prominent and mirrored protein expression. No α3 nAChR or NR1 mRNA was seen in the skeletal muscle, as expected, although both subunit mRNAs were present in the placenta. This study concludes that further experiments are required to conclusively state that the β1 nAChR subunit is expressed in the human, piglet and mouse brain.     

Suppressed expression of nicotinic acetylcholine receptors by nanomolar β-amyloid peptides in PC12 cells

Summary. A line of evidence has shown that a link between the common pathological features of β-amyloid peptide (Aβ) deposition and cholinergic degeneration observed in Alzheimer's disease (AD) may exist, however, no experimental evidence has shown that exposure to Aβ can decrease expression of nicotinic acetylcholine receptors (nAChRs), which have been shown to play roles in brain cognitive functions. Here, we report that treatment with Aβ_1–40 and Aβ_25–35 at nanomolar concentrations significantly decreased the [³H]epibatidine and [¹²⁵I]α-bungarotoxin binding sites, the protein and mRNA levels of nAChR α3, α7 and β2 subunits in PC12 cells. Aβ_1–40 and Aβ_25–35 at the concentrations used in the treatment study neither bound to nAChRs nor induced apoptosis, but significantly inhibited the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl tetrazolium bromide) reduction. These data suggest that the decreased biosynthesis of nAChRs induced by Aβ may be attributable partially to perturbances of some intracellular signal transduction pathways. The results presented in this study lead to a hypothesis that Aβ can degenerate nAChRs early in the course of AD before the formation of abundant Aβ fibrils. Received May 3, 2001; accepted August 23, 2001     

Inhibition of neuronal nicotinic acetylcholine receptor channels expressed in Xenopus oocytes by β-amyloid1–42 peptide

Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in a variety of physiological processes, including cognition and development. Dysfunctions in nAChRs have been linked to Alzheimer's disease (AD), a human neurological disorder that is the leading cause of dementia. AD is characterized by an increasing loss of cognitive function, nAChRs, cholinergic neurons, and choline acetyltransferase activity. A major hallmark of AD is the presence of extracellular neuritic plaques composed of the beta-amyloid (Abeta1-42) peptide; however, the link between Abeta1-42 and the loss of cognitive function has not been established. Many groups have shown direct interactions between Abeta1-42 and nAChR function, however, with differing results. For example, in rat hippocampal CA1 interneurons in slices, we found that Abeta1-42 inhibits nAChR channels directly, and non-alpha7 receptors were more sensitive to block than alpha7 receptors. However, some groups have found that alpha7 subtypes were potently blocked by Abeta1-42, whereas other groups reported that Abeta1-42 can activate nAChRs (i.e., both alpha7 and non-alpha7 subtypes). To further investigate the link between nAChR function and Abeta1-42, we expressed various subtypes of nAChRs in Xenopus oocytes (e.g., alpha4beta2, alpha2beta2, alpha4alpha5beta2, and alpha7) and found that Abeta1-42 blocked these various non-alpha7 nAChRs, without any effect on alpha7 nAChRs. Furthermore, none of these channels was activated by Abeta1-42. The relative block by Abeta1-42 was dependent on the subunit makeup and apparent stoichiometry of these receptors. These data further support our previous findings that Abeta1-42 directly and preferentially inhibits non-alpha7 nAChRs.     

BDNF up-regulates α7 nicotinic acetylcholine receptor levels on subpopulations of hippocampal interneurons

In the hippocampus, brain-derived neurotrophic factor (BDNF) regulates a number of synaptic components. Among these are nicotinic acetylcholine receptors containing α7 subunits (α7-nAChRs), which are interesting because of their relative abundance in the hippocampus and their high relative calcium permeability. We show here that BDNF elevates surface and intracellular pools of α7-nAChRs on cultured hippocampal neurons and that glutamatergic activity is both necessary and sufficient for the effect. Blocking transmission through NMDA receptors with APV blocked the BDNF effect; increasing spontaneous excitatory activity with the GABA_A receptor antagonist bicuculline replicated the BDNF effect. BDNF antibodies blocked the BDNF-mediated increase but not the bicuculline one, consistent with enhanced glutamatergic activity acting downstream from BDNF. Increased α7-nAChR clusters were most prominent on interneuron subtypes known to directly innervate excitatory neurons. The results suggest that BDNF, acting through glutamatergic transmission, can modulate hippocampal output in part by controlling α7-nAChR levels.     

Role of mouse cerebellar nicotinic acetylcholine receptor (nAChR) α(4)β(2)- and α(7) subtypes in the behavioral cross-tolerance between nicotine and ethanol-induced ataxia

We have demonstrated that nicotine attenuated ethanol-induced ataxia via nicotinic-acetylcholine-receptor (nAChR) subtypes α(4)β(2) and α(7). In the present study, ethanol (2g/kg; i.p.)-induced ataxia was assessed by Rotorod performance following repeated intracerebellar infusion of α(4)β(2)- and α(7)-selective agonists. Localization of α(4)β(2) and α(7) nAChRs was confirmed immunohistochemically. Cerebellar NO(x) (nitrite+nitrate) was determined flurometrically. Repeated intracerebellar microinfusion of the α(4)β(2)-selective agonist, RJR-2403 (for 1, 2, 3, 5 or 7 days) or the α(7)-selective agonist, PNU-282987 (1, 2, 3 or 5 days), dose-dependently attenuated ethanol-induced ataxia. These results suggest the development of cross-tolerance between ethanol-induced ataxia and α(4)β(2) and α(7) nAChR agonists. With RJR-2403, the cross-tolerance was maximal after a 5-day treatment and lasted 48h. Cross-tolerance was maximal after a 1-day treatment with PNU-282987 and lasted 72h. Pretreatment with α(4)β(2)- and α(7)-selective antagonists, dihydro-β-erythroidine and methyllycaconitine, respectively, prevented the development of cross-tolerance confirming α(4)β(2) and α(7) involvement. Repeated agonist infusions elevated cerebellar NO(x) 16h after the last treatment while acute ethanol exposure decreased it. Pretreatment with repeated RJR-2403 or PNU-282987 reversed ethanol-induced decrease in NOx. The NO(x) data suggests the involvement of the nitric oxide (NO)-cGMP signaling pathway in the cross-tolerance that develops between α(4)β(2)- and α(7)-selective agonists and ethanol ataxia. Both α(4)β(2) and α(7) subtypes exhibited high immunoreactivity in Purkinje but sparse expression in molecular and granular cell layers. Our results support a role for α(4)β(2) and α(7) nAChR subtypes in the development of cross-tolerance between nicotine and ethanol with the NO signaling pathway as a potential mechanism.     

Distribution of the α7 nicotinic acetylcholine receptor subunit in the developing chick cerebellum

Previous studies of the nicotinic acetylcholine receptor (nAChR) subunits in adult mammalian and avian brains have demonstrated a spatially restricted distribution of these subunits; little, however, is known about the nAChR subunit developmental distribution. The present study demonstrated a transient pattern of distribution of the neuronal nAChR subunit, α7, in the developing chick cerebellum by using immunohistochemical techniques. This transient distribution may suggest a critical period for the development of the cholinergic system in the cerebellum.     

Identification of α7 nicotinic acetylcholine receptor on hippocampal astrocytes cultured in vitro and its role on inflammatory mediator secretion

The present study found expressions of α7 nicotinic acetylcholine receptor on hippocampal slices and hippocampal astrocytes using double immunofluorescence stainings. Expression of glial fibrillary acidic protein in the cultured hippocampal slices and hippocampal astrocytes significantly increased, and levels of macrophage inflammatory protein 1α, RANTES, interleukin-1β, interleukin-6, and tumor necrosis factor-α increased in the supernatant of cultured astrocytes following exposure to 200 nM amyloid β protein 1-42. Preconditioning of 10 μM nicotine, a nicotinic acetylcholine receptor agonist, could attenuate the influence of amyloid β protein 1-42 in inflammatory mediator secretion of cultured astrocytes. Experimental findings indicated that α7 nicotinic acetylcholine receptor was expressed on the surface of hippocampal astrocytes, and activated α7 nicotinic acetylcholine receptor was shown to inhibit inflammation induced by amyloid β protein 1-42.     

Melatonin and the time window for the expression of the α8 nicotinic acetylcholine receptor in the membrane of chick retinal cells in culture

We have previously shown that melatonin influences the development of α8 nicotinic acetylcholine receptor (nAChR) by measurement of the acetylcholine-induced increase in the extracellular acidification rate (ECAR) in chick retinal cell cultures. Cellular differentiation that takes place between DIV (days in vitro) 4 and DIV 5 yields cells expressing α8 nAChR and results in a significant increase in the ECAR acetylcholine-induced. Blocking melatonin receptors with luzindole for 48 h suppresses the development of functional α8 nAChR. Here we investigated the time window for the effect of melatonin on retinal cell development in culture, and whether this effect was dependent on an increase in the expression of α8 nAChR. First, we confirmed that luzindole was inhibiting the effects of endogenous melatonin, since it increases 2-[¹²⁵I] iodomelatonin (23 pM) binding sites density in a time-dependent manner. Then we observed that acute (15, 60 min, or 12 h) luzindole treatment did not impair acetylcholine-induced increase in the ECAR mediated by activation of α8 nAChR at DIV 5, while chronic treatment (from DIV 3 or DIV 4 till DIV 5, or DIV 3.5 till DIV 4.5) led to a time-dependent reduction of the increase in the acetylcholine-induced ECAR. The binding parameters for [¹²⁵I]-α-bungarotoxin (10 nM) sites in membrane were unaffected by melatonin suppression that started at DIV 3. Thus, melatonin surges in the time window that occurs at the final stages of chick retinal cell differentiation in culture is essential for development of the cells expressing α8 nAChR subtype in full functional form.     

Interaction of α-2-macroglobulin and HSV-1 during infection of neuronal cells

We describe the effect of pretreatment with alpha-2-macroglobulin (A2M) on the susceptibility of the human neuroblastoma SKNMC cell line to infection by herpes virus type 1 (HSV-1). ELISA and co-immunoprecipitation experiments confirmed the A2M-HSV-1 interaction in vitro. Indirect immunofluorescence shows that A2M exacerbated the cytopathic effect induced after HSV-1 infection. However, A2M-pretreated SKNMC cells notably produced fewer HSV-1 particles than did the untreated cells, suggesting that A2M could induce a restrictive infection. Furthermore, high levels of HSV-1 and A2M induced the production of nitric oxide (NO) in SKNMC. Preliminary results suggest that A2M might induce apoptosis in HSV-1-infected cells. These findings affirm the conclusion that A2M may interact directly with HSV-1 and modulate the course of the infection in SKNMC human neuroblastoma cells.     

α7 neuronal nicotinic receptor agonist (TC-7020) reverses increased striatal dopamine release during acoustic PPI testing in a transgenic mouse model of schizophrenia

Genetic and post mortem evidence has implicated the α7 neuronal nicotinic receptor (NNR) in the etiology of schizophrenia and related disorders. In schizophrenia, enhanced subcortical dopamine (DA) correlates with positive and cognitive of the disease, including impairments in sensorimotor gating. We measured the levels of extracellular DA and DA metabolites during an acoustic test session of prepulse inhibition (PPI) of the startle response, a measure of sensorimotor gating, by microdialysis and HPLC-EC in a transgenic mouse model of schizophrenia. In th-fgfr1(tk-) mice, blockade of fibroblast growth factor receptor 1 (FGFR1) signaling during development in catecholaminergic neurons results in reduced size and density of midbrain DA neurons of the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). These mice displayed reduced PPI and enhanced startle response relative to control mice as well as a potentiation of DA release in the dorsal striatum during a 30 minute PPI test session. Acute administration of a partial α7 NNR agonist TC-7020 (1.0 mg/kg) normalized PPI and startle deficits and attenuated increases of DA release during acoustic PPI testing. These results provide direct evidence of elevated striatal dopaminergic transmission with impaired sensorimotor gating that may underlie cognitive and positive symptoms and motor deficits in schizophrenia and related disorders. Also, systemic targeting of alpha7 NNRs may ameliorate these deficits by functionally suppressing striatal DA activity.     

Selective down-regulation of α4β2 neuronal nicotinic acetylcholine receptors in the brain of uremic rats with cognitive impairment

Cognitive impairment is common in patients with chronic kidney disease. Brain nicotinic acetylcholine receptors modulate cognitive functions, such as learning and memory. Pharmacological cholinergic enhancement is useful in patients with cognitive dysfunction. The major nicotinic acetylcholine receptor subtypes in the brain are heteromeric α4β2 and homomeric α7 receptors. To study the involvement of neuronal acetylcholine receptors in cognitive impairment in uremic rats, bilateral nephrectomy was performed. 24 weeks after nephrectomy, memory was assessed using the one trial step-down inhibitory avoidance test. Neuronal nicotinic acetylcholine receptors in the brain were studied by radioligand binding, immunoprecipitation, Western blot and sucrose gradient experiments. We demonstrated that rats with severe renal failure show disorders of short term memory. Long term memory was not altered in these rats. The number of functional α4β2 heteromeric neuronal nicotinic receptors was decreased in the brains of rats with severe renal failure. There was a significant correlation between the degree of renal impairment and the number of heteromeric nicotinic acetylcholine receptors in the brain. The down-regulation of functional α4β2 receptors in the brains of rats with severe renal failure was not due to a reduction of α4 or β2 subunit proteins. The number of α7 homomeric neuronal nicotinic acetylcholine receptors was not altered. These findings may have important clinical significance for the management of cognitive impairment in patients with chronic kidney disease.     

The role of receptor protein tyrosine phosphatase α in neuronal differentiation of embryonic stem cells

In the present study, we have investigated the function of the receptor protein tyrosine phosphatase α (RPTPα) in the neuronal differentiation of E14-embryonic stem (E14-ES) cells. RNAase protection and western blot analysis revealed that E14-ES cells up regulate RPTPα expression upon neuronal differentiation with retinoic acid. Overexpression of RPTPα, by stable DNA transfection, and subsequent differentiation with retinoic acid, resulted in a temporally enhanced expression of the neuronal markers GAP-43 and NF-164. Electrophysiological experiments demonstrated that RPTPα overexpression also enhanced the development of neurotransmitter responses during differentiation. These results indicate that RPTPα plays an important role in the cascade of molecular events that lead to the formation of neurons.     

A role for the calmodulin kinase II-related anchoring protein (αkap) in maintaining the stability of nicotinic acetylcholine receptors

αkap, a muscle specific anchoring protein encoded within the Camk2a gene, is thought to play a role in targeting multiple calcium/calmodulin kinase II isoforms to specific subcellular locations. Here we demonstrate a novel function of αkap in stabilizing nicotinic acetylcholine receptors (AChRs). Knockdown of αkap expression with shRNA significantly enhanced the degradation of AChR α-subunits (AChRα), leading to fewer and smaller AChR clusters on the surface of differentiated C2C12 myotubes. Mutagenesis and biochemical studies in HEK293T cells revealed that αkap promoted AChRα stability by a ubiquitin-dependent mechanism. In the absence of αkap, AChRα was heavily ubiquitinated, and the number of AChRα was increased by proteasome inhibitors. However, in the presence of αkap, AChRα was less ubiquitinated and proteasome inhibitors had almost no effect on AChRα accumulation. The major sites of AChRα ubiquitination reside within the large intracellular loop and mutations of critical lysine residues in this loop to arginine increased AChRα stability in the absence of αkap. These results provide an unexpected mechanism by which αkap controls receptor trafficking onto the surface of muscle cells and thus the maintenance of postsynaptic receptor density and synaptic function.