Selective changes in the levels of nicotinic acetylcholine receptor protein and of corresponding mRNA species in the brains of patients with Parkinson's disease

Reductions in the number of neuronal nicotinic acetylcholine receptors (nAChRs) have been shown to occur in connection with Parkinson's disease (PD), but it is still unclear which subtype of this receptor is affected. In the present study we examined various nAChR subtypes employing ligand binding, as well as levels of subunit protein and mRNA in the brains of PD patients and age-matched controls. Binding of [3H]epibatidine and levels of alpha3 mRNA in the caudate nucleus and temporal cortex, but not in the hippocampus were significantly decreased in the PD brain. The level of the alpha3 protein subunit was significantly reduced in all these brain regions but there was no change in the level of alpha4. The level of the beta2 protein subunit in the temporal cortex and hippocampus and the beta2 mRNA in the temporal cortex was lowered. Both the levels of the alpha7 subunit protein and [125I]alpha-bungarotoxin binding were significantly increased in the temporal cortex of PD patients whereas the alpha7 mRNA level was unchanged. These findings reveal selective losses of the alpha3- and beta2-containing nAChRs and an increase in the alpha7 nAChRs that might be related to the pathogenesis of PD.     

The amyloid-β₄₂ proxy, amyloid-β(25-35), induces normal human cerebral astrocytes to produce amyloid-β₄₂

Amyloid-β (Aβ) peptides in the brain of patients with Alzheimer's disease (AD) assemble into various aggregation forms that differ in size, structure, and functional properties. Previous studies have shown that Aβ binds to nicotinic acetylcholine receptors (nAChRs) and activates signaling cascades that result in the disruption of synaptic functions. These findings suggest a possible link between impaired cholinergic neurotransmitter function in AD and Aβ pathogenesis. However, it is not yet known how the different Aβ assemblies interact with specific nAChR subtypes. In the present study, we demonstrate that neurotoxicity in neuronal cells in culture induced by fibrillar Aβ(1-40) is prevented through an α7 nAChR-dependent mechanism. The α7 nAChR agonists varenicline and JN403 increased binding of the amyloid ligand [3H]PIB to fibrillar Aβ in AD frontal cortex autopsy tissue. This suggests that the presence of nAChR agonists may inhibit interaction of Aβ with α7 nAChRs and prevent the formation of Aβ/α7 nAChR complexes. This interaction was confirmed in binding assays with [12?I]Aβ(1-40) and α7 nAChRs in autopsy brain tissue homogenates from the frontal cortex. The functional effects of Aβ fibrils and oligomers on nAChRs were examined by measuring intracellular calcium ([Ca(2+)](i) levels. Oligomeric, but not fibrillar Aβ(1-40), increased [Ca(2+)](i) in neuronal cells, and this effect was attenuated by varenicline. Our findings demonstrate that fibrillar Aβ exerts neurotoxic effects mediated partly through a blockade of α7 nAChRs, whilst oligomeric Aβ may act as a ligand activating α7 nAChRs, thereby stimulating downstream signaling pathways.     

High selective expression of alpha7 nicotinic receptors on astrocytes in the brains of patients with sporadic Alzheimer's disease and patients carrying Swedish APP 670/671 mutation: a possible association with neuritic plaques

In the present study, we have investigated the expression of nicotinic acetylcholine receptors (nAChRs) on astrocytes and neurons in the hippocampus and temporal cortex of subjects carrying the Swedish amyloid precursor protein (APP) 670/671 mutation (APPswe), patients with sporadic Alzheimer's disease (AD), and age-matched control subjects. Significant increases in the total numbers of astrocytes and of astrocytes expressing the alpha7 nAChR subunit, along with significant decreases in the levels of alpha7 and alpha4 nAChR subunits on neurons, were observed in the hippocampus and temporal cortex of both APPswe and sporadic AD brains. Both of these phenomena were more pronounced in APPswe than sporadic AD cases. Furthermore, the number of [(125)I]alpha-BTX binding sites (alpha7 nAChR) in the temporal cortex of the APPswe brain was significant lower than in the younger control group, reflecting the lower neuronal level of alpha7 nAChR. The increase in the level of expression of alpha7 nAChR on astrocytes was positively correlated with the extent of neuropathological alternations, especially the number of neuritic plaques, in the AD brain. The elevated expression of alpha7 nAChR on astrocytes might participate in Abeta cascade and formation of neuritic plaques, thereby playing an important role in the pathogenesis of AD.     

Decreased nicotinic receptors and cognitive deficit in rats intracerebroventricularly injected with beta-amyloid peptide(1-42) and fed a high-cholesterol diet

To investigate whether the changes in nicotinic receptors (nAChRs) and in learning and memory associated with Alzheimer's disease (AD) are influenced by both beta-amyloid peptide (Abeta) and cholesterol in vivo, we examined the effects of intracerebroventricular injection of Abeta(1-42) and/or a high-cholesterol diet on brain levels of nAChRs and learning and memory in rats. The levels of nAChR subunit proteins and the corresponding mRNA were measured by Western blotting and RT-PCR, respectively; and learning and memory were evaluated with the Morris Water Maze examination. Injection of Abeta(1-42) resulted in deposition of this peptide, activation of astrocytes, decreased levels of the alpha7 and alpha4 protein subunits of the nAChR, and elevated expression of alpha7 mRNA, as well as impaired learning and spatial memory. A high-cholesterol diet activated astrocytes and, more importantly, potentiated the toxic effects of Abeta on nAChR subunit levels and on learning and memory. These findings may be highly relevant to the mechanisms underlying the cognitive deficits associated with AD.     

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.     

Alpha7 nicotinic receptor up-regulation in cholinergic basal forebrain neurons in Alzheimer disease

BACKGROUND: Dysfunction of basocortical cholinergic projection neurons of the nucleus basalis (NB) correlates with cognitive deficits in Alzheimer disease (AD). Nucleus basalis neurons receive cholinergic inputs and express nicotinic acetylcholine receptors (nAChRs) and muscarinic AChRs (mAChRs), which may regulate NB neuron activity in AD. Although alterations in these AChRs occur in the AD cortex, there is little information detailing whether defects in nAChR and mAChR gene expression occur in cholinergic NB neurons during disease progression. OBJECTIVE: To determine whether nAChR and mAChR gene expression is altered in cholinergic NB neurons during the progression of AD. DESIGN: Individual NB neurons from subjects diagnosed ante mortem as having no cognitive impairment (NCI), mild cognitive impairment (MCI), or mild to moderate AD were analyzed by single-cell AChR expression profiling via custom-designed microarrays. SETTING: Academic research. PARTICIPANTS: Participants were members of the Rush Religious Orders Study cohort. MAIN OUTCOME MEASURES: Real-time quantitative polymerase chain reaction was performed to validate microarray findings. RESULTS: Cholinergic NB neurons displayed a statistically significant up-regulation of alpha7 nAChR messenger RNA expression in subjects with mild to moderate AD compared with those with NCI and MCI (P<.001). No differences were found for other nAChR and mAChR subtypes across the cohort. Expression levels of alpha7 nAChRs were inversely associated with Global Cognitive Score and with Mini-Mental State Examination performance. CONCLUSIONS: Up-regulation of alpha7 nAChRs may signal a compensatory response to maintain basocortical cholinergic activity during AD progression. Alternatively, putative competitive interactions of this receptor with beta-amyloid may provide a pathogenic mechanism for NB dysfunction. Increasing NB alpha7 nAChR expression may serve as a marker for the progression of AD.     

Selective changes in expression of different nicotinic receptor subtypes in brain and adrenal glands of mice carrying human mutated gene for APP or over-expressing human acetylcholinestrase

In this study, we investigated regulatory mechanisms and plasticity of the nicotinic acetylcholine receptors (nAChRs) in the brain and adrenal glands of two transgenic mice models over-expressing human beta-amyloid precursor protein (APP(SWE)Tg) and human AChE enzyme (hAChE-Tg), respectively. All animals were studied at 3 months of age. Binding studies showed higher (125)I-alpha-bungarotoxin (alpha7 nAChRs) and (3)H-epibatidine (alpha3 and alpha4 nAChRs) binding in the brain cortex and adrenal glands of hAChE-Tg mice compared to control mice. The APP(SWE)Tg mice showed a significantly lower relative level for the alpha4 mRNA in the brain cortex as well as a lower level of alpha3 mRNA, and higher level of alpha7 mRNA in the adrenal glands compared to control mice. A higher relative mRNA level of alpha3 and alpha4 nAChRs was observed in the brain as well as of alpha3 and alpha7 nAChRs in the adrenal glands of hAChE-Tg mice compared to control mice. Different nicotinic receptor plasticity is revealed in the brain cortex and adrenal glands in two transgenic mice models with different underlying pathophysiological mechanisms. Deposition of beta-amyloid (Abeta) may impair neurotransmitter activity in brain as well as in the adrenal gland.     

Chronic neonatal nicotine upregulates heteromeric nicotinic acetylcholine receptor binding without change in subunit mRNA expression

Smoking during pregnancy chronically exposes the fetus to nicotine resulting in long-term behavioral and cognitive deficits. Nicotine binds to neuronal nicotinic acetylcholine receptors (nAChRs), pentameric ligand-gated ion channels widely expressed in the nervous system. Chronic nicotine upregulates high-affinity nAChRs in animals and smokers. Here we determined if chronic nicotine treatment during a developmental period corresponding to the human third trimester regulates nAChR expression. Rat pups were intubated orally three times per day with or without nicotine (6 mg/kg/day) from postnatal day 1 to 8. Subunit mRNA expression was assessed by in situ hybridization. Expression of heteromeric and homomeric nAChR receptor was evaluated by autoradiography using (125)I-epibatidine and (125)I-alphabungarotoxin, respectively. nAChR expression was analyzed in cortex, hippocampus, thalamus and medial habenula from autoradiograms using computer assisted image analysis. Nicotine induced significant upregulation of heteromeric but not homomeric nAChRs in hippocampus, cortex and thalamus without changes in subunit mRNA expression. No effect of chronic nicotine on receptor expression was detected in the medial habenula, suggesting that nicotine's effect was mainly on alpha4beta2-type heteromeric nAChRs. The nicotine-induced upregulation was reversed after nicotine withdrawal. Receptor blockade by DHbetaE, an antagonist for heteromeric alpha4/beta2 nAChRs, did not prevent upregulation but increased expression to a similar degree as nicotine. Combination of both drugs had a cumulative effect. Thus, although transient, intermittent nicotine exposure as seen in smoking mothers is sufficient to upregulate heteromeric nAChRs during a critical period of brain development and could contribute to the behavioral deficits found in children whose mother smoked.     

Expression of the α7, α4 and α3 nicotinic receptor subtype in the brain and adrenal medulla of transgenic mice carrying genes coding for human AChE and β-amyloid

Human AChE-enzyme (hAChE) enhances the over-expression of beta-amyloid (Abeta) containing plaques in the brain of transgenic mice (APP(SWE)/hAChE-Tg) carrying mutated genes for human amyloid precursor protein (APP(SWE)) and hAChE. In this study, we showed that interaction of hAChE with Abeta affects the plasticity of the alpha7 nicotinic acetylcholine receptors (nAChRs) both in the brain and adrenal medulla. An age-related increase in the (125)I-alphabungarotoxin ((125)I-alphaBTX) binding (specific to alpha7 nAChRs) was observed in the adrenal medulla of 3, 7 and 10 months old control mice. In contrast, a significant decrease in (125)I-alphaBTX binding was detected in the adrenal medulla of 10 months old APP(SWE)/hAChE-Tg. A significantly higher alpha7 nAChR mRNA level was observed in the brain of APP(SWE)/hAChE-Tg at 3 and 7 months of age and in the adrenal medulla at 3 and 10 months of age compared to those of the control mice. The alpha3 nAChR mRNA level was significantly higher in the brain of APP(SWE)/hAChE-Tg at 3 months of age and in the adrenal medulla at 10 months of age. The alpha4 nAChR mRNA level remained unchanged in the brain and adrenal medulla of APP(SWE)/hAChE-Tg for all age groups. Based on these observations, we conclude that a high load of Abeta and an over-expression of hAChE induce differences in the expression of the nAChR subtypes at various ages in the brain and in the adrenal medulla of hAChE/APP(SWE)Tg mice. The findings may have implications for a better understanding the underlying mechanism for AD-related pathogenesis.     

Neuronal nicotinic receptors in synaptic functions in humans and rats: physiological and clinical relevance

The present report describes the participation of nicotinic receptors (nAChRs) in controlling the excitability of local neuronal circuitries in the rat hippocampus and in the human cerebral cortex. The patch-clamp technique was used to record responses triggered by the non-selective agonist ACh and the alpha7-nAChR-selective agonist choline in interneurons of human cerebral cortical and rat hippocampal slices. Evidence is provided that functional alpha7- and alpha4beta2-like nAChRs are present on somatodendritic and/or preterminal/terminal regions of interneurons in the CA1 field of the rat hippocampus and in the human cerebral cortex and that activation of the different nAChR subtypes present in the preterminal/terminal areas of the interneurons triggers the tetrodotoxin-sensitive release of GABA. Modulation by nAChRs of GABAergic transmission, which can result either in inhibition or disinhibition of pyramidal neurons, depends both on the receptor subtype present in the interneurons and on the agonist acting upon these receptors. Not only do alpha7 nAChRs desensitize faster than alpha4beta2 nAChRs, but also alpha7 nAChR desensitization induced by ACh lasts longer than that induced by choline. These mechanisms, which appear to be retained across species, might explain the involvement of nAChRs in cognitive functions and in such neurological disorders as Alzheimer's disease and schizophrenia.     

Localization of nAChR subunit mRNAs in the brain of Macaca mulatta

We present here a systematic mapping of nAChR subunit mRNAs in Macaca mulatta brain. A fragment, from the transmembrane segments MIII to MIV of Macaca neuronal nAChR subunits was cloned, and shown to exhibit high identity (around 95%) to the corresponding human subunits. Then, specific oligodeoxynucleotides were synthesized for in situ hybridization experiments. Both alpha4 and beta2 mRNA signals were widely distributed in the brain, being stronger in the thalamus and in the dopaminergic cells of the mesencephalon. Most brain nuclei displayed both alpha4 and beta2 signals with the exception of some basal ganglia regions and the reticular thalamic nucleus which were devoid of alpha4 signal. alpha6 and beta3 mRNA signals were selectively concentrated in the substantia nigra and the medial habenula. The strongest signals for alpha3 or beta4 mRNAs were found in the epithalamus (medial habenula and pineal gland), whereas there were no specific alpha3 or beta4 signals in mesencephalic dopaminergic nuclei. alpha5 and alpha7 mRNA signals were found in several brain areas, including cerebral cortex, thalamus and substantia nigra, although at a lower level than alpha4 and beta2. The distribution of alpha3, alpha4, alpha5, alpha6, alpha7, beta2, beta3 and beta4 subunit mRNAs in the monkey is substantially similar to that observed in rodent brain. Surprisingly, alpha2 mRNA signal was largely distributed in the Macaca brain, at levels comparable with those of alpha4 and beta2. This observation represents the main difference between rodent and Macaca subunit mRNA distribution and suggests that, besides alpha4beta2*, alpha2beta2* nAChRs constitute a main nAChR isoform in primate brain.     

Association analysis of the partially duplicated alpha7 nicotinic acetylcholine receptor genetic variant and Alzheimer's disease.

Changes in the nicotinic acetylcholine receptors (nAChRs) have been demonstrated for Alzheimer's disease (AD). Of these receptors, the alpha7 nAChRs, which are abundant on hippocampal interneurons, have been implicated in the cytotoxic role of the beta-amyloid. Increased mRNA levels of alpha7 nAChR in the peripheral lymphocytes and hippocampus of AD patients have been reported. We tested the hypothesis that the allelic variant, 2bp deletion, of the partially duplicated alpha7 nAChR gene confers susceptibility to Alzheimer's disease. The -2bp polymorphism was examined in 120 patients with AD and 98 normal controls. The distribution of the partially duplicated alpha7 nAChR genotypes (p = 0.372) and alleles (p = 0.465) did not differ significantly for AD patients and controls. This negative finding suggests that the partially duplicated alpha7 nAChR genetic polymorphism contributes no major effect to the development of AD. However, we suggest that the other genetic variation of the alpha7 nAChR gene, related to AD or the associated symptomatology, merits further investigation.     

Airway-related vagal preganglionic neurons express multiple nicotinic acetylcholine receptor subunits

Nicotine acting centrally increases bronchomotor tone and airway secretion, suggesting that airway-related vagal preganglionic neurons (AVPNs) within the rostral nucleus ambiguus (rNA) express nicotinic acetylcholine receptors (nAChRs). In the present study, we examined the three main functionally characterized subtypes of nAChRs in the CNS, the alpha7 homomeric and alpha4beta2 heteromeric receptors. First, we characterized the expression of these subunits at the message (mRNA) and protein levels in brain tissues taken from the rNA region, the site where AVPNs are located. In addition, double labeling fluorescent immunohistochemistry and confocal laser microscopy were used to define the presence of alpha7, alpha4, and beta2 nAChRs on AVPNs that were retrogradely labeled with cholera toxin beta subunit (CTb), injected into the upper lung lobe (n=4) or extrathoracic trachea (n=4). Our results revealed expression of all three studied subunits at mRNA and protein levels within the rNA region. Furthermore, virtually all identified AVPNs innervating intrapulmonary airways express alpha7 and alpha4 nAChR subunits. Similarly, a majority of labeled AVPNs projecting to extrathoracic trachea contain alpha7 and beta2 subunits, but less than half of them show detectable alpha4 nAChR traits. These results suggest that AVPNs express three major nAChR subunits (alpha7, alpha4, and beta2) that could assemble into functional homologous or heterologous pentameric receptors, mediating fast and sustained nicotinic effects on cholinergic outflow to the airways.     

Neuronal nicotinic acetylcholine receptor subunits in autism: an immunohistochemical investigation in the thalamus

The cholinergic system has been implicated in the development of autism on the basis of neuronal nicotinic acetylcholine receptor (nAChR) losses in cerebral and cerebellar cortex. In the present study, the first to explore nAChRs in the thalamus in autism, alpha4, alpha7 and beta2 nAChR subunit expression in thalamic nuclei of adult individuals with autism (n=3) and age-matched control cases (n=3) was investigated using immunochemical methods. Loss of alpha7- and beta2- (but not alpha4-) immunoreactive neurons occurred in the paraventricular nucleus (PV) and nucleus reuniens in autism. Preliminary results indicated glutamic acid decarboxylase immunoreactivity occurred at a low level in PV, co-expressed with alpha7 in normal and autistic cases and was not reduced in autism. This suggested loss of neuronal alpha7 in autism is not caused by loss of GABAergic neurons. These findings indicate nicotinic abnormalities that occur in the thalamus in autism which may contribute to sensory or attentional deficits.     

Nicotinic acetylcholine receptor immunohistochemistry in Alzheimer's disease and dementia with Lewy bodies: differential neuronal and astroglial pathology

Alzheimer's disease (AD) and dementia with Lewy bodies (DLB) are common forms of dementia in the elderly. The neuropathology of AD and DLB is related to cholinergic dysfunctions, and both alpha4 and alpha7 nicotinic acetylcholine receptor (nAChR) subunits are decreased in several brain areas in both diseases. In this immunohistochemical study, we compared neuronal and astroglial alpha4 and alpha7 subunits in AD, DLB and age-matched controls in the hippocampal formation. The numbers of alpha4 reactive neurons were decreased in layer 3 of the entorhinal cortex of AD and DLB, whereas those of alpha7 reactive neurons were decreased in layer 2 of the subiculum of AD and DLB and in layer 3 of the entorhinal cortex of DLB. In contrast, the intensity of alpha7 reactive neuropil was significantly higher in AD than in controls or DLB in a number of areas of the hippocampus (CA3/4 and stratum granulosum), subiculum and entorhinal cortex. An increase in alpha7 immunoreactivity in AD was also associated with astrocytes. The number of astrocytes double-labelled with alpha7 and glial fibrillary acidic protein (GFAP) antibodies was increased in most areas of the hippocampus and entorhinal cortex in AD compared with controls and DLB. Increased astrocyte alpha7 nAChRs in AD may be associated with inflammatory mechanisms related to degenerative processes specific to this disease.