The nicotinic α7 acetylcholine receptor agonist ssr180711 is unable to activate limbic neurons in mice overexpressing human amyloid-β1–42

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

The nicotinic α7 acetylcholine receptor agonist ssr180711 is unable to activate limbic neurons in mice overexpressing human amyloid-β1–42 Andreas Søderman a,b , Morten S. Thomsen c , Henrik H. Hansen b , Elsebet Ø. Nielsen b , Morten S. Jensen a , Mark J. West a , Jens D. Mikkelsen b,d,⁎ a

Institute of Anatomy, University of Aarhus, Wilhelm Meyers Allé, 8000 Århus C, Denmark NeuroSearch A/S, Pederstrupvej 93, DK-2750 Ballerup, Denmark c Department of Neuroscience and Pharmacology, University of Copenhagen, Blegdamsvej 3, 2200 N, Denmark d Neurobiological Research Unit, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark b

A R T I C LE I N FO

AB S T R A C T

Article history:

Recent studies have demonstrated that amyloid-β1–42 (Aβ1–42) binds to the nicotinergic α7

Accepted 17 June 2008

acetylcholine receptor (α7 nAChR) and that the application of Aβ1–42 to cells inhibits the

Available online 25 June 2008

function of the α7 nAChR. The in vivo consequences of the pharmacological activation of the α7 nAChR have not been examined. The aim of this study has been to evaluate the efficacy of

Keywords:

α7 nAChR modulators in transgene mice that overexpress human amyloid precursor protein

Acetylcholine

and accumulate Aβ1–40 and Aβ1–42. In accordance with observations in human Alzheimer

Nicotine

tissues, we show here through the use of co-immunoprecipitation that human Aβ-

Amyloid

immunoreactive peptides bind to mice α7 nAChR in vivo. Agonists of the α7 nAChR improve

Alzheimer

memory and attentional properties and increase immediate early gene expression in the

Prefrontal cortex

prefrontal cortex and the nucleus accumbens. We show that acute systemic administration

Nucleus accumbens

of the α7 nAChR agonist SSR180711 (10 mg/kg) result in a significant increase in Fos protein levels in the shell of nucleus accumbens in wild-type mice, but has no effect in the transgene mice. There were fewer cell bodies expressing Fos in the prefrontal cortex of transgene mice, and in this region no induction was achieved after administration with SSR180711 in either of the two groups. These results suggest that overexpression of human Aβ peptides perhaps via direct interaction with α7 nAChR, inhibit α7 nAChR-dependent neurotransmission in vivo and emphasize that clinical trials testing α7 nAChR agonists should be related to the content of Aβ peptides in the patient's nervous system. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by progressive cognitive decline, and loss of neurons, especially cholinergic neurons and synapses in the basal forebrain, cerebral cortex and hippocampus (Kasa

et al., 1997). Based on the observation that the cholinergic neurons that originate in the basal forebrain are particularly vulnerable to degeneration in AD (Auld et al., 1998), one dominant strategy for treatment of AD has focused on enhancing acetylcholine (ACh)-dependent neurotransmission (O'Neill et al., 2002; Terry and Buccafusco, 2003).

⁎ Corresponding author. NeuroSearch A/S, Pederstrupvej 93, DK-2750 Ballerup, Denmark. Fax: +45 44608080. E-mail address: [email protected] (J.D. Mikkelsen). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.06.062

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The neuropathological features of AD include amyloid plaques, neurofibrillary tangles, and inflammation (Terry et al., 1991). Currently, the amyloid plaques are thought to arise from the gradual accumulation of amyloid-β(1–42) (Aβ1–42) extracellularly (Neve and Robakis, 1998). The mechanisms by which the Aβ1–42 is formed and its specific role in the disease are not yet clear. Evidence obtained from familial AD and transgene animals overexpressing human Aβ1–42 suggest that aberrant amyloid metabolism is involved in the disruption of normal neuronal function (Hsiao et al., 1996; Selkoe, 1998). It has been shown that Aβ1–42 binds to the nicotinergic α7 acetylcholine receptor (α7 nAChR) of transfected cells that express α7 nAChR (Palop et al., 2005; Spencer et al., 2006; Wang et al., 2000a; Wang et al., 2000b). A decline in density of α7 nAChR has been reported in postmortem brain tissue from patients with schizophrenia and AD (Banerjee et al., 2000; Freedman et al., 1995). These findings suggest that Aβ1–42 contributes to the pathology of AD via binding to and inhibition of α7 nAChR. The α7 nAChR is a homopentameric ligand-gated receptor that is widely distributed throughout the mammalian CNS (Seguela et al., 1993). Activation of this receptor in vitro leads to the influx of Ca2+ and the excitation of neurons (Papke et al., 2000). The systemic administration of α7 nAChR agonists has been demonstrated to improve performance in certain cognitive tasks (Jones et al., 1999; Newhouse et al., 2004; Pichat et al., 2007), and it has now become increasingly clear that α7 nAChR is involved in attention (Hashimoto et al., 2008; Pichat et al., 2007; Young et al., 2007). Further, increase of expression of immediate early genes in the frontal cortex and the nucleus accumbens has been demonstrated after administration with nicotine and the α7 nAChR agonist SSR180711 (Hansen et al., 2007; Kristensen et al., 2007; Schochet et al., 2005).

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The aim of this study has been to determine whether or not Aβ1–40/42 binds to α7 nAChR in situ and whether or not the increased expression of Aβ1–40/42 has any functional consequences for pharmacological activation of the α7 nAChR. We used detection of Fos immunoreactivity and stereological quantification of Fos positive nuclei as a marker of neuronal activity after acute administration of the selective α7 nAChR agonist SSR180711 in transgene and wild-type mice. Furthermore tissue from animals overexpressing Aβ1–42 that develop amyloid plaques similar to those seen in AD (Jankowsky et al., 2001) was used to examine whether Aβ1–40/42 and α7 nAChR coimmunoprecipitated.

2.

Results

2.1. Acute administration of SSR180711A induces Fos expression only in wild-type mice The number of c-Fos-immunoreactive cells was estimated using a stereological method in the subregions of the striatum and the prefrontal cortex of transgene and wild-type littermate mice treated with a single acute injection of the α7 nAChR agonist SSR180711 (10 mg/kg). This dose was selected after examining a dose–response relationship for doses of 1– 20 mg/ml in 9-month old female C57 mice. In these mice, a dose of 10 mg/kg was shown to induce a maximal effect on Fos in the nucleus accumbens shell region (not shown) in accordance with recent data from the rat (Hansen et al., 2007). In the brains of saline-treated wild-type mice, a moderate number of immunostained nuclei were seen in the nucleus accumbens shell region, whereas the basal level of Fos positive cells in the core region was low (Fig. 1). Virtually no

Fig. 1 – Photomicrographs of coronal sections through the nucleus accumbens of wild-type (A–C) and transgene mice (D–F) immunoreacted for Fos. The mice had been treated with saline (A, D), SSR180711 (B, E) or haloperidol (C, F). In the wild-type mice, a prominent increase was observed after both SSR180711 and haloperidol, whereas only haloperidol produced an increase in the transgene animals.

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Fig. 2 – Stereological quantification of the number of Fos positive nuclei in the nucleus accumbens shell subregion (A), core subregion (B) and the prefrontal cortex (C) wild-type (open bars) and transgene mice (shattered bars) acutely treated with SSR180711 (10 mg/kg). While Fos is strongly induced in the shell region after SSR180711 (A) in the wild-type animals, this effect is not seen in the transgene mice. No effect of SSR180711 was observed in the prefrontal cortex (C). However, there was a strong decline in the number of positive cells in the transgene animals independent of treatment. Two-way ANOVA ***P < .001.

Fos immunoreactivity was found in the dorsolateral striatum (not shown). The stereological approach estimated there to be 5,300 cells that contained Fos in the nucleus accumbens shell subregion of wild-type after treating with vehicle. This number increased to 11,300 after an acute injection with SSR180711 (Fig. 2A). Importantly, while SSR180711 induced a significant increase in the number of cells expressing Fos in the nucleus accumbens shell region in the wild-type mice, the same treatment had no effect on the transgene animals overexpressing Aβ (Figs. 1 and 2A). The compound had no significant effect on the number of neurons expressing Fos in either the core region (Fig. 2B) or in the dorsolateral striatum (not shown). In the prefrontal cortex the level of Fos after a single injection with saline was lower in the transgene compared to the littermate controls (Fig. 2C). Acute treatment with SSR180711 had no effect on the number of Fos positive cells in the prefrontal cortex of either the transgene animals or the littermate controls (Fig. 2C). To evaluate the ability of the striatal neurons to respond to other stimuli than the activation of α7 nAChR, the effects neurons have after haloperidol was determined. Fos was strongly induced in the nucleus accumbens shell and core regions as well as in the dorsolateral striatum after haloper-

idol treatment (Fig. 3). When comparing the effect of haloperidol there was no difference in the induction in transgene and control animals (Fig. 3). To investigate receptor specificity, nicotine was administered to transgene animals to determine whether or not Fos was induced in other areas activated by non-α7 nAChR. Nicotine elicited an increase in Fos neurons in the hypothalamic paraventricular nucleus that was much stronger than after SSR180711 (Fig. 4). A similar level of induction was observed in the lateral habenula and the medial terminal nucleus of the accessory optic tract (not shown).

2.2. Human Aβ1–42 interacts with endogenous α7 nAChR in vivo Western blot analysis demonstrated that human Aβ peptides only were expressed in the transgene mice and not in the wildtype. A 5 kDa band that most likely corresponds to Aβ1–40 or Aβ1–42 (Fig. 5A) and a 95 kDa band that likely corresponds to human amyloid precursor protein were identified (Figs. 5A, B). To study the possible interaction of endogenous α7 nAChR with Aβ1–42 in mouse cerebral cortex, we performed immunoprecipitation of Aβ1–40/42 and detection of α7 nAChR by Western

Fig. 3 – Quantification of the number of Fos cells in the striatum after acute administration of haloperidol in wild-type and transgene animals. Haloperidol produces a strong induction of Fos in all parts of the striatum, i.e. the nucleus accumbens shell (A), the nucleus accumbens core (B), and the dorsolateral part of the striatum (C) independent of phenotype. One-way ANOVA, *P < 0.05; **P < .01; ***P < .001.

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Fig. 4 – Nicotine but not SSR180711 produces a strong induction of Fos in the hypothalamic paraventricular nucleus of the hypothalamus. This effect is not dependent of the phenotype. One-way ANOVA, ***P < .001.

analysis. This revealed the presence of a band that was similar in size to that of α7 nAChR in extracts immunoprecipitated with an antiserum against Aβ1–40/42 from the cerebral cortex from transgene mice (Fig. 5C). By contrast, no bands were demonstrable in extracts from wild-type littermates (not shown). As a control, Western blotting analysis demonstrated the expression of α7 nAChR in cells stably expressing the receptor, and the specificity of the antiserum used (Fig. 5D).

3.

Discussion

We provide here evidence that endogenous human Aβ selectively inhibits α7 nAChR in vivo. The present results could be important in the development of novel α7 nAChR modulators for AD, because translation of the present results to the clinical situation would assume that activation of the α7 nAChR in the Alzheimer brain in the presence of high concentrations of Aβ would counteract the pharmacological effect. The data reported here show that acute treatment of the selective α7 nAChR agonist SSR180711 is unable to induce an immediate activation of the α7 nAChR in mice in which there is overexpression of mutant human presenilin-1 and amyloid precursor protein. The present data are not sufficient to conclude that the lack of Fos induction in the nucleus accumbens after acute administration is a direct result of the binding of Aβ1–40/42 and α7 nAChR. However, our results correlate well with a number of recent reports of a direct interaction between Aβ and α7 nAChR in vitro. Patch-clamp recordings from rat hippocampal neurons in culture or from transfected cells showed that Aβ peptides in nM concentrations blocked α7 nAChR in a specific and reversible manner (Liu et al., 2001; Pettit et al., 2001). In agreement with co-immunoprecipitation studies in Alzheimer brain tissues and in cells overexpressing α7 nAChR and Aβ1–42 (Wang et al., 2000a), we have been able to demonstrate that Aβ1–42 binds to α7 nAChR in the cerebral cortex of the mutant animals. The antiserum used in our immunoprecipitation studies recognized Aβ1–40 and Aβ1–42 but other studies have provided evidence that Aβ1–42 has the highest affinity for the receptor (Wang et al., 2000a).

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The concentration of Aβ1–42 needed to block α7 nAChR in vivo remains to be determined. Transgene animals expressing mutant forms of human amyloid precursor protein APP have Aβ1–42 concentrations that have been estimated to be in the low nM range (Mucke et al., 2000). However, as the concentration of Aβ peptides increases with age, it is likely that the binding of Aβ to the α7 nAChR would increase with age. Amyloid load in mice that express human Aβ has typically been characterized in terms of measurements of total Aβ or the volume of the brain occupied by amyloid plaques. However, it is more important to consider location and functional compartmentalization of various forms of Aβ molecules and aggregates. It is important to know whether or not Aβ can interact with α7 nAChR at both post- and presynaptic sites, and whether or not Aβ is located intra-or extracellularly and thereby can affect specific receptor domains. In addition, there are differences in localization and concentration of Aβ in different brain areas of both the transgenes and in AD patients. These considerations are important because a number of studies have shown that application of α7 nAChR modulators can counteract the cellular effect of Aβ peptides in vitro. For example α7 nAChR agonists are found to be neuroprotective in the presence of Aβ peptides (Kihara et al., 2001; Shimohama and Kihara, 2001) and both the α7 nAChR agonist PNU282987 and the α7 nAChR antagonist methyllycaconitine prevent Aβ1– 42 induced reduction in neurite outgrowth (Hu et al., 2007). These results do not directly conflict with our findings, because the α7 nAChR modulator and Aβ peptides are not necessarily interacting when added to the medium for a relatively short period of time compared to the long-term exposure in vivo as in transgene mice. The present data also correlates well with the observation that chronic administration of the partial α7 nAChR agonist 4OH-GTS-21 reduces the death and shrinkage of the cholinergic neurons in the basal forebrain of wild-type but not APP/PS1 transgene mice (Ren et al., 2007). By contrast, Spencer et al.

Fig. 5 – Solubilized protein samples from the cortex of 9-month old transgene mice and wild-type controls. Precipitation was carried out with a mouse monoclonal Aβ antibody and immunoreactivity was assessed on Western blots using another mouse monoclonal antibody specific for the Aβ molecule. Aβ-immunoreactivity was observed only in the transgene mice (A). In the transgene mouse there was an intense band at 98 kDa (B) which corresponds to the molecular weight of the human amyloid precursor protein (APP) (B). Figure C illustrates the stripped membrane, re-incubated with a goat anti-α7 nAChR antiserum that gives rise to a band at 55 kDa that corresponds to the molecular weight of α7 nAChR. Extract from HEK cells transfected with human α7 nAChR, incubated with the same goat anti-α7 nAChR antiserum resulted in one distinct band at 55 kDA (D).

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(2006) reported that the function of native α7 nAChR in hippocampal slices prepared from another mouse that overexpressed amyloid was normal. Systemic administration of α7 nAChR agonist results in an increase in the expression of various genes in the frontal cortex and the shell region of the accumbens, but not in the hippocampus (Kristensen et al., 2007; Schochet et al., 2005). It is possible that the mechanisms by which α7 nAChR activation stimulates neurons in the hippocampus, frontal cortex, septum, and the ventral striatum are different. As stated, the location and compartmentalization of Aβ molecules may be different between the hippocampus and in the other areas activated by SSR180711. The mechanism by which Aβ inhibits the function of α7 nAChR at the structural level is not fully elucidated. The interaction appears to occur at the N-terminal extracellular portion of the receptor and does not appear to interact with the bungarotoxin binding site (Liu et al., 2001). However, it has been reported that Aβ1–42 competes with α-bungarotoxin for binding to the α7 nAChR in neuroblastoma cell lines (Wang et al., 2000a,b). These discrepancies could in part be explained by Aβ1–42 binding to the receptor with the result that there are conformational changes in the receptor that change its functional properties. In contrast to what is known about the acute effect of Aβ, potential chronic interactions between Aβ1–42 and its impact on α7 nAChR function are more controversial. Long-term treatment with nicotine has been shown to reduce Aβ levels in the brain (Hellström-Lindahl et al., 2004; Zhang et al., 2006). Despite the fact that α7 nAChR is inhibited by amyloids, it remains to be determined whether or not the effect can be reduced if higher doses of nicotine are given, or if the drug is given during the progression of the disease rather when the plaque formation is well established. The basal expression of Fos in the prefrontal cortex correlates well with earlier observations that the expression of other genes decline as mice grew older and amyloid deposits accumulate (Dickey et al., 2004; Palop et al., 2005). These data suggests that Aβ peptides produce reductions in gene expression by interacting with cell signaling. The Fos induction was not observed in the prefrontal cortex in the wild-type mice. The induction of immediate early genes after nicotine and/or SSR180711 is more pronounced in younger than in older animals (Kristensen et al., 2007; Schochet et al., 2005), and it might be that the 9-month old animals as used in the present study are not receptive. This is in contrast to the activation by both haloperidol and nicotine via mechanisms that are independent of α7 nAChR. This further emphasizes the specificity of the inhibition of SSR180711 in the transgene mice. In summary, these data demonstrate that the build up of amyloid has a negative impact on the function of α7 nAChR and inhibition of amyloid–α7 nAChR binding may prove effective in reducing cognitive disturbances that accompany AD.

4.

Experimental procedures

4.1.

Compounds

SSR180711 (1,4-Diazabicyclo[3.2.2]nonane-4-carboxylic acid, 4bromophenyl ester) was synthesized in house. Haloperidol was

purchased from Janssen-Cilag, (Denmark), and nicotine from Sigma-Aldrich (St. Louis, USA). All compounds were dissolved in 0.9% sterile saline and administered subcutaneously.

4.2.

Animals and single administration of drugs

In a pilot study designed to determine the optimal dose of SSR180711 for the transgene animals, 9-month old female C57/ 6J mice from Harlan Inc. in Denmark were used. All animals were housed 6–8 animals per cage under standard housing and feeding conditions. The animals were organized in groups of 4 to 6 animals. The groups of animals were injected with either saline or various doses (1, 3, 10 and 20 mg/kg) of SSR180711. The animals were returned to their home cage and killed under the fixation procedure 60 min after the injection. In the experimental study, 9-month old female heterozygous APPswe/PS1ΔE9 transgene mice weighing approximately 35 g (Borchelt et al., 1996) were used in this study. These mice co-express the PS1ΔE9 mutation and a chimeric mouse–human APP695 (mutations K595N and M596L) driven by the mouse prion protein promoter (Borchelt et al., 1996). Mice were bred at the Anatomical Institute, Aarhus University, and genotyped using previously described methods (Gordon et al., 2002). Females with non-transgene genotype (wild-type) littermates were used as controls. These animals received a single acute injection of 10 mg/ kg SSR180711, and returned to their home cage for 60 min before perfusion as described below. All procedures were conducted according with the Danish National Guide for care and use of laboratory animals.

4.3.

Immunohistochemistry for Fos

All mice used for immunocytochemistry were deeply anesthetized with mebumal, perfused transcardially with ice-cold 0.9% saline for 1 min, fixed with 4% paraformaldehyde-PBS for 5 min, and finally immersed in the fixative overnight at 4 °C. The brains were then dehydrated in 30% sucrose-PBS for 24 h prior to sectioning. Sections of 40 μM thick were collected at 8 series intervals and stored in cryoprotectant. Before the immunocytochemical steps, the sections were rinsed for three times 10 min in 0.01 M PBS, for 10 min in 1% H2O2-PBS to block endogenous peroxidase activity, and for 30 min in 0.01 M PBS with 0.3% Triton X-100 (TX; Sigma-Aldrich, St. Louis, MO), 5% swine serum, and 1% bovine serum albumin (BSA) to block non-specific binding sites. The sections were then incubated at 4 °C for 24 h in a rabbit antisera against Fos (code #94012-5); diluted 1:4000 in 0.01 M PBS to which 0.3% Triton X-100 and 1% BSA had been added. After incubation in primary antiserum, immunoreactivity was detected by means of the avidin–biotin method using diaminobenzidine (DAB) as a chromogen. The sections were then washed in PBS with 0.1% TX and incubated for 60 min in biotinylated donkey anti-rabbit antiserum (The Jackson Laboratory, Bar Harbor, ME) diluted 1:800 in PBS with 1% BSA, washed again, and then transferred to the avidin–biotin complex reaction (Vector Laboratories, Burlingame, CA) diluted 1:250 in PBS-TX. After several washes, the sections were incubated in 0.05% diaminobenzidine (Sigma-Aldrich) with 0.05% H2O2 in PBS buffer for 10 min and then washed twice in PBS buffer. The sections were mounted

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on gelatinized glass slides, dried, and coverslipped with Pertex prior to light microscopy.

4.4.

Quantification of Fos cells

To quantify the effects of the drugs, an observer blind to the treatment of the animals counted total numbers of neurons expressing Fos using the optical fractionator stereological method (West et al., 1991). The brains were sectioned exhaustively in the frontal plane at a microtome setting of 40 μm. Every other section was stained for Fos and counterstained with toluidine blue. The borders of the regions were studied; the core (ACCcore) and the shell (ACCshell) of nucleus accumbens and the prefrontal cortex (PFC), were defined on a section immunoreacted for Fos and counterstained with toluidine. The rostral and caudal extent of ACCcore and ACCshell was defined by sections in the series that had the appearance of sections at inter-aural distances of −5.00 mm and −4.42 mm respectively. This resulted in about 8 sections. The rostral and caudal extent of PFC was defined by sections in the series that had the appearance of sections at inter-aural distances of −5.70 mm and −5.30 mm respectively. In the latter case, this resulted in about 5 sections per individual. The PVN was likewise defined according to the atlas resulting in analysis of approximately 4 sections per animal. Accordingly, the nuclei of Fos positive neurons were directly counted in a known fraction of these chosen brain regions, ΣQ− using optical dissectors and then multiplied by the reciprocal of the fraction of the volume of the respective brain regions to obtain an estimate of the total number, est N. The fraction of the volume of the tissue sampled by dissectors (i.e. in which neurons were counted) was calculated as the product of the fraction of the section thickness sampled, tsf, the fraction of the area of the sections sampled, asf, and the fraction of the sections sampled, ssf. est N ¼

X

Q  d1=ssfd1=asfd1=tsf

The sampling with optical dissectors was performed in a systematic random manner so that all parts of the respective brain regions had equal probabilities of being sampled. The amount of sampling, i.e. the number of nuclei counted to make an estimate of total stained cells, was a priori set to approximately 150 counts, on the basis of earlier studies that indicate that the variance of an estimate based on 150 counts in wild-type mice makes a smaller contribution to the observed group variance than does the true biological variance (inter-individual variance) (Slomianka and West, 2005). The sampling scheme for the shell subregion of the nucleus accumbens is summarized in Table 1. The data were analyzed by one- or two-way analysis of variance (ANOVA) followed by Tukey post hoc test. All data are presented as group means and standard error of mean (S.E.M.). Significance and levels of P b .05 were considered significant.

4.5.

Co-immunoprecipitation of Aβ and the α7 nAChR

Four 9-month old APPswe/PS1ΔE9 transgene mice and four wild-type controls were decapitated, and the brain placed on a cooled platform. The cortex and the hippocampus were

Table 1 – Sampling scheme for stereological analysis (nucleus accumbens shell)

a

n Ssf b Asf c Tsf d aframe e astep f hg Th i j

Wtalpha7

Wtvehicle

Tgalpha7

Tgvehicle

8 1/2 0.77 0.69 625 μm2 8100 5 7.2 193 11,570

8 1/2 0.77 0.69 625 mm2 8100 5 7.3 93 5370

8 1/2 0.77 0.70 625 mm2 8100 5 7.1 102 6479

8 1/2 0.77 0.69 625 mm2 8100 5 7.2 98 6457

a

Number of mice per group. Fraction of sections sampled. c Fraction of area of sections sampled, aframe/astep. d Thickness of sections sampled, h/t, μm. e Area of optical dissector counting frame, μm2. f Section area associated with stage movement to next sample, μm2. g Height of optical dissector, μm. h Section thickness, μm. i Number of neurons counted per individual (mean). j Mean coefficient of error of estimate in individuals. b

isolated and immediately transferred to tubes that contained 500 ml ice-cold Buffer A (50 mM sodium phosphate, 50 mM NaCl, 2 mM EDTA, 2 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4 and complete protease inhibitor cocktail (Roche Diagnostics)). The tissue was homogenized with a micropistile using a pellet pestle motor (Eppendorf, Hamburg, Germany). The homogenate was centrifugated (16,100 ×g 1 h, 4 °C) and the supernatant was discarded. The resulting pellets were then resuspended in Buffer A, homogenized, and recentrifugated. The resulting pellet was resuspended in icecold Buffer B (Buffer A containing 2% Triton X-100 and a cocktail of protease inhibitors (Roche Diagnostics), and incubated for 2 h on a rotor at 4 °C, centrifuged at 16,100 ×g rpm (1 h, 4 °C), and the supernatant was collected and assayed for Aβ1–42 and α7 nAChR immunoreactivity. Additionally, HEK293 cells that overexpress α7 nAChR were used to control the specific immunoreaction of the α7 nAChR antiserum. To construct the HEK293 cells that overexpressed α7 nAChR we used full-length human α7 nAChR cDNA, cloned between the BamHI and XhoI sites of pcDNA3 (Elliott et al., 1996). Empty HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml penicillin, streptomycin, and 4 mM glutamine. HEK-293 cells were sub-cultured the day before transfection and plated to achieve ∼ 60–70% confluency within 24 h. Cells were then transfected with wild-type human α7 nAChR in pcDNA3 using 3Al of FuGENE 6 transfection reagent (Roche, Lewes, UK). Transfected cells were incubated for 48 h at 37 °C before use. Cells were collected, washed, and centrifuged (3000 ×g) in ice-cold PBS (pH 7.4). The supernatant was removed and the cells were maintained at −80 °C until further processing. Aβ was precipitated from the solubilized protein fraction using a mouse monoclonal antibody directed against amino acids 17–24 of the human Aβ sequence (clone 4G8, Sigma, St.

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Louis, MI). Prior to immunoprecipitation, the antibody was pre-incubated with protein A/G agarose-coupled beads (Santa Cruz Biotechnology Inc., Santa Cruz, CA) in Buffer B (2 h, 4 °C). Thereafter, 1000 mg of protein was added and the immunoprecipitation reaction was performed on a rotor overnight at 4 °C. The beads were precipitated by centrifugation (6000 ×g, 5 min, 4 °C), and the pellet was rinsed twice with ice-cold Buffer B and Buffer A, respectively. The buffer used for rinsing was removed completely, 20 ml of Laemmli buffer was added to the beads and boiled for 5 min. The solution was then centrifuged at 16,100 ×g. The proteins in the resulting supernatant were separated on a 4–12% Tris– HCl gel (Bio-Rad Laboratories, Hercules, CA) and then electrotransferred in transfer buffer (80 mM Tris–HCl, 39 mM glycine, 20% methanol) to a nitrocellulose membrane (0.2 μm, Whatman Schleicher and Schuell, Dassel, Germany). The membrane was rinsed with Tris-buffered saline solution that contained Tween 20 (TBS-T; 10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.6) and treated with a blocking solution (5% non-fat dry milk in TBS-T) for 2 h at room temperature to prevent non-specific antibody binding. Comparable loading and transfer of proteins was initially confirmed by staining the membrane with Ponceau-S solution (Fluka, Buchs, Switzerland). The membrane was incubated overnight at 4 °C with a mouse monoclonal Aβ1–42 antibody (clone 6E10, directed against amino acids 1–16 of the human Aβ1–42 sequence, Signet, Inc.). Following incubation with goat antimouse horseradish peroxidase-labeled (HRP) secondary antibody (1:40,000; Pierce Chemical, Rockford, IL), at room temperature for 2 h, the immunoreactive proteins were visualized by enhanced chemiluminescence system (Pierce Chemical, Rockford, IL), and serial exposures were made on autoradiographic films (GE Healthcare). To determine whether or not the α7 nAChR co-immunoprecipitated with Aβ1–42, the membrane was subjected at a commercial stripping reagent (Pierce Chemical, Rockford, IL) for 5 min at 37 °C using and reprobed overnight (4 °C) with a goat anti-α7 nAChR antibody (sc-1447; directed against the Cterminus of the α7 nAChR, Santa Cruz, CA). Following TBS-T rinses, the membrane was incubated with a donkey anti-goat HRP labelled antibody (1:40,000, Vector Laboratories, Burlingame, CA) and developed as described above.

Acknowledgments The expert technical assistance of Ulla Borberg, Tine Engelbrecht, Katrine S. Hansen, Mia Billenberg Nielsen, and Pia Rovsing Sandholm is greatly appreciated. This work was supported by the Lundbeck and Alzheimer Foundations.

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