Design  Quantitative measures of 7 nAChR by [³H]methyllycaconitine binding and Aβ concentration by enzyme-linked immunosorbent assay in SFC were compared across subjects with antemortem clinical classification of no cognitive impairment, mild cognitive impairment, or mild to moderate AD, and with postmortem neuropathological diagnoses.

Setting  Academic medical center.

Subjects  Twenty-nine elderly retired clergy.

Main Outcome Measures  Quantitative measures of 7 nAChR binding and Aβ peptide concentration in SFC.

Results  Higher concentrations of total Aβ peptide in SFC were associated with clinical diagnosis of mild to moderate AD (P = .02), lower Mini-Mental State Examination scores (P = .003), presence of cortical Aβ plaques (P = .02), and likelihood of AD diagnosis by the National Institute on Aging–Reagan criteria (P = .002). Increased 7 nAChR binding was associated with National Institute on Aging–Reagan diagnosis (P = .02) and, albeit weakly, the presence of cortical Aβ plaques (P = .08). There was no correlation between the 2 biochemical measures.

Conclusions  These observations suggest that during the clinical progression from normal cognition to neurodegenerative disease state, total Aβ peptide concentration increases while 7 nAChRs remain relatively stable in SFC. Regardless of subjects' clinical status, however, elevated 7 nAChR binding is associated with increased Aβ plaque pathology, supporting the hypothesis that cellular expression of these receptors may be upregulated selectively in Aβ plaque–burdened brain areas.

METHODS

This study included 29 participants in the Religious Orders Study, a longitudinal clinical-pathological study of aging and AD in retired Catholic nuns, priests, and brothers.²⁰ Inclusion criteria and a description of the clinical evaluation have been published.^20-21 At the last clinical evaluation (<12 months prior to death), subjects were classified as having NCI, MCI, or mAD (Table 1). Diagnosis of AD dementia was made using standard criteria.²² Mild cognitive impairment was defined as impairment on neuropsychological testing but without a diagnosis of dementia by the examining neurologist,²⁰ criteria similar to those describing patients who were not cognitively intact but nonetheless did not meet the criteria for dementia.^23-26 A consensus conference of neurologists and neuropsychologists reviewed all the clinical and neuroimaging data, medical records, and interviews with family members and assigned a final diagnosis.

View this table: [in this window] [in a new window] [as a PowerPoint slide]   Table 1. Demographic, Clinical, and Neuropathological Characteristics by Diagnostic Group

NEUROPATHOLOGICAL EVALUATION

Neuropathological diagnosis of AD (possible, probable, or definite AD) or not AD (Table 1) was based on modified criteria by the Consortium to Establish a Registry for Alzheimer's Disease (CERAD),²⁷ which applied semiquantitative estimates of neuritic plaque density by a board-certified neuropathologist blinded to the clinical diagnosis.²⁸ Subjects were also assigned a National Institute on Aging (NIA)–Reagan neuropathological diagnosis²⁹ and a Braak score based on the presence of neurofibrillary tangles.³⁰ Subjects with pathology other than AD were excluded from the study.

[³H]METHYLLYCACONITINE BINDING ASSAY

Fresh frozen SFC (Brodmann area 9) gray matter was divided into aliquots for nAChR binding and Aβ peptide enzyme-linked immunosorbent assay (ELISA). For 7 nAChR binding, samples were homogenized in 10 volumes of 50mM Tris-hydrochloride (Tris-HCl) buffer (pH = 7.0), centrifuged twice at 40 000 x g for 10 minutes, resuspended in Tris buffer, and stored at –80°C. Samples were thawed and resuspended in an equal volume of Tris buffer containing 0.1% bovine serum albumin.^31-32 Samples, 0.5 mg of protein each, were combined with 9.5nM [³H]methyllycaconitine (MLA) (159.1x10¹⁰ Bq/mmol; Tocris Cookson Ltd, Bristol, England) in Tris-HCl buffer containing 0.1% bovine serum albumin. Nonspecific binding was measured in the presence of 1mM nicotine. After a 2-hour incubation on ice, bound ligand was separated from free ligand using Whatman GF/B filters (Whatman, Florham Park, New Jersey), presoaked in 0.3% polyethyleneimine. Filters were rinsed with Tris-HCl buffer, placed in scintillation vials, and shaken in scintillation fluid for 1 hour before radioactivity was determined. Specific binding was calculated as the difference between total and nonspecific binding. Results were expressed as femtomoles per milligram of protein.

Aβ ELISA ASSAY

The Aβ assay was performed using a previously reported protocol.³³ Frozen SFC samples were homogenized (150 mg of tissue wet weight/mL of phosphate-buffered saline, pH = 7.4) and 30 mg of homogenized tissue were sonicated in 70% formic acid and centrifuged at 109 000 x g at 4°C for 1 hour, resulting in samples containing both soluble and formic acid–extracted insoluble Aβ peptides. The supernatant was neutralized with 1M Tris and 0.5M sodium phosphate, and the samples were assayed using a fluorescent-based ELISA (BioSource, Carlsbad, California) following the kit's instructions, with a capture antibody specific for the amino terminus of Aβ (amino acids 1-16) and detection antibodies specific for Aβ₄₀ and Aβ₄₂ peptides. Values were determined from standard curves using synthetic Aβ_1-40 and Aβ_1-42 peptides (BioSource) and expressed as picomoles per gram of wet brain tissue. "Total" Aβ values represent a sum of Aβ_1-40 and Aβ_1-42 peptide values.

STATISTICAL ANALYSIS

β-amyloid levels were log-transformed because of data skewness. Comparisons of demographic characteristics, MLA binding, and Aβ levels between clinically or neuropathologically defined groups were performed using the Wilcoxon rank sum test, Kruskal-Wallis test, or the Fisher exact test, as appropriate. The association between demographic characteristics and MLA binding or Aβ levels was assessed by Spearman rank correlation or Wilcoxon rank sum test. Partial correlation was used for additional analyses adjusting for age. The correlation between MLA binding and Aβ levels was assessed by Spearman correlation. The level of statistical significance was set at .05 (2-sided).

RESULTS

The 3 clinical groups differed in Mini-Mental State Examination (MMSE) scores (P < .001), with the mAD group performing worse than both the NCI and MCI groups (Table 1). Clinical diagnostic groups also differed in CERAD diagnosis (P = .04), Braak staging (P = .04), and the NIA-Reagan diagnosis (P = .02), with the mAD subjects being more advanced neuropathologically compared with both the MCI and NCI groups (Table 1).

Subjects with a CERAD diagnosis of AD (CERAD score < 4; possible, probable, or definite AD; positive for cortical plaques) had lower MMSE scores (P = .06) and more advanced Braak stages and NIA-Reagan neuropathologic diagnoses (P = .004 and P < .001) (Table 1) than the not AD group (CERAD score = 4; no cortical plaques).

MLA BINDING AND Aβ CONCENTRATIONS ACROSS CLINICAL AND NEUROPATHOLOGICAL CATEGORIES

The MLA binding levels were slightly higher in mAD subjects; however, the difference was not statistically significant (Table 2). Clinical groups differed in total Aβ (combined Aβ₄₂ and Aβ₄₀; P = .02) (Figure) and Aβ₄₂ (P = .02), but not Aβ₄₀, concentrations, with mAD subjects having the highest levels.

View this table: [in this window] [in a new window] [as a PowerPoint slide]   Table 2. Superior Frontal Cortex MLA Binding and Aβ ELISA Levels by Diagnostic Group

View larger version (17K): [in this window] [in a new window] [as a PowerPoint slide]   Figure. 7 Nicotinic acetylcholine receptor binding (A and B) and total β-amyloid (Aβ) peptide concentrations (C and D) in the frontal cortex from subjects categorized into clinical diagnostic groups of no cognitive impairment (NCI), mild cognitive impairment (MCI), and Alzheimer disease (AD) (A and C) or into neuropathological groups of not AD and AD (possible, probable, or definite) by modified Consortium to Establish a Registry for Alzheimer's Disease (CERAD) criteria (B and D). MLA indicates [3H]methyllycaconitine.

Subjects with a CERAD diagnosis of AD had higher MLA binding (P = .08) (Figure) and higher concentrations of Aβ₄₂ (P = .02), Aβ₄₀ (P = .06), and total Aβ (P = .02) (Table 2) (Figure) in SFC when compared with those with a CERAD diagnosis of not AD.

MLA BINDING AND Aβ CONCENTRATION: ASSOCIATION WITH CLINICAL-NEUROPATHOLOGICAL FACTORS

There was no association of MLA binding with any of the demographic or clinical variables examined. Higher MLA binding levels correlated with greater likelihood of AD by the NIA-Reagan diagnosis (r = –0.47; P = .02) (Table 3) and weakly with Braak staging. There was an association of higher Aβ concentrations with more advanced age (r = 0.48-0.56; P = .01-.03; Table 3), but not with sex, education, the presence of APOE 4 allele, or postmortem interval. Higher concentrations of total Aβ and Aβ₄₂, but not Aβ₄₀, correlated with lower MMSE scores (r = –0.62 for both; P = .003 and .004) (Table 3). In addition, higher total Aβ and Aβ₄₂ concentrations correlated with worse neuropathological scores (r = 0.62-0.70; P < .01), as did Aβ₄₀ concentrations, although to a lesser extent (Table 3). Adjusting for age, partial correlation showed similar results, although it yielded smaller correlation coefficients. There was no correlation between MLA binding and Aβ protein concentrations.

View this table: [in this window] [in a new window] [as a PowerPoint slide]   Table 3. Association Between Clinical/Neuropathological Variables and Superior Frontal Cortex MLA Binding and Aβ ELISA Levels

COMMENT

The apparent stability of 7 nAChRs across clinical categories of NCI, MCI, and mAD, and the lack of an association with MMSE scores, could be explained by the presence of plaques in all clinical groups. Cortical plaques were present in more than half of our NCI cases (Table 1), in agreement with previous reports of a substantial AD pathology in cognitively normal aged individuals.^{28, 36-40} Although these studies would benefit from examining larger numbers of cognitively intact subjects free of any Aβ pathology, such individuals are rare, as Aβ plaques are a common feature in brains of elderly individuals.^36-37,39-40 Furthermore, the pathological burden of Aβ includes not only insoluble fibrils in plaques, but also soluble Aβ oligomers.⁴¹ The impact of these distinct pools of Aβ on 7 nAChR binding in preclinical, early/moderate, and severe end-stage AD cases will be an important question to answer in future studies.

There are several possible explanations for the observed association between Aβ plaques and increased 7 nAChR binding. Plaques may serve as reservoirs of soluble Aβ species,¹ which can bind with high affinity to neuronal 7 receptors^4-5 into a complex that is subsequently internalized.¹² This may result in a compensatory increase in expression of 7 nAChR on the cell surface. Additionally, excessive intracellular accumulation of Aβ₄₂ and subsequent neuronal lysis may contribute to plaque pathology,¹² potentially creating a cycle of neuronal degeneration and Aβ plaque deposition in AD. High concentrations of fibrillar Aβ in plaques, or soluble Aβ in the vicinity of these structures, may also influence the upregulation of 7 nAChR by reactive astrocytes. Astrocytes proliferate and display increased 7 nAChR density in the presence of Aβ plaques^{35, 42-43} and upregulate nAChR messenger RNA expression and protein levels when exposed to Aβ in vitro.⁴⁴ Receptor binding assays cannot differentiate the relative contribution of different cell types to the overall regional expression of 7 nAChRs detected in tissue homogenates. Adding to this complexity are the postsynaptic and presynaptic sites of expression of 7 nAChRs, involving both local neuronal circuitry and afferent projections from distant neuronal cell populations. In this regard, a recent single-cell expression profiling study demonstrated upregulation of 7 nAChR messenger RNA in cortical-projecting basal forebrain cholinergic neurons in mAD subjects,⁴⁵ suggesting that changes in cortical 7 protein levels involve presynaptic elements on an important cholinergic afferent system. Collectively, these studies suggest that in SFC, 7 nAChR levels reported herein reflect changes both in cortical-projecting cholinergic basal forebrain neurons and regional cell-specific expression of these receptors in response to Aβ pathology.

In conclusion, the present findings demonstrate that cognitive decline in mAD is not associated with detectable changes in cortical 7 nAChR binding levels. In contrast, Aβ concentrations increased in mAD and correlated with cognitive impairment, in accord with reported associations of increased Aβ load with cognitive decline in AD.^46-47 The observed trend for increased SFC 7 in subjects with plaques is in agreement with a previously reported positive correlation between -bungarotoxin binding and Aβ plaque density³⁴ and warrants further investigation. These changes are in contrast with reports of reduced cortical 4 nAChR immunoreactivity with increased Aβ plaque densities and a loss of epibatidine binding with increased Aβ₄₂ concentrations.³⁴ Thus, 7 and non-7 nAChRs may be differentially affected by Aβ pathology. In vivo positron emission tomography imaging techniques using radiolabeled probes for early detection of Aβ plaques^48-49 and changes in select nAChRs⁵⁰ may act as early biomarkers for AD and will enable the timely implementation of appropriate therapies.

AUTHOR INFORMATION

Accepted for Publication: November 5, 2008.

Author Contributions: Study concept and design: Ikonomovic, Wecker, Abrahamson, Ginsberg, Mufson, and DeKosky. Acquisition of data: Ikonomovic, Wecker, and DeKosky. Analysis and interpretation of data: Ikonomovic, Wecker, Abrahamson, Wuu, Counts, Ginsberg, Mufson, and DeKosky. Drafting of the manuscript: Ikonomovic, Wecker, Abrahamson, Wuu, and Mufson. Critical revision of the manuscript for important intellectual content: Ikonomovic, Wecker, Abrahamson, Wuu, Counts, Ginsberg, Mufson, and DeKosky. Statistical analysis: Wuu. Obtained funding: Ikonomovic, Ginsberg, Mufson, and DeKosky. Administrative, technical, and material support: Ikonomovic, Wecker, Ginsberg, and DeKosky. Study supervision: Ikonomovic.

Financial Disclosure: None reported.

Funding/Support: This work was supported by NIA grants AG14449 and AG10610.

Additional Contributions: Theresa Landers-Concatelli, MS, and William R. Paljug, MS, provided expert technical assistance. We are indebted to the support of the participants in the Religious Orders Study; for a list of participating groups, see http://www.rush.edu/rumc/page-R12394.html.