ORIGINAL CONTRIBUTION ␣7 Nicotinic Receptor Up-regulation in Cholinergic Basal Forebrain Neurons in Alzheimer Disease Scott E. Counts, PhD; Bin He, MD; Shaoli Che, MD, PhD; Milos D. Ikonomovic, MD; Steven T. DeKosky, MD; Stephen D. Ginsberg, PhD; Elliott J. Mufson, PhD Background: Dysfunction of basocortical cholinergic pro- Participants: Participants were members of the Rush jection neurons of the nucleus basalis (NB) correlates with Religious Orders Study cohort. cognitive deficits in Alzheimer disease (AD). Nucleus ba- Main Outcome Measures: Real-time quantitative poly- salis neurons receive cholinergic inputs and express nico- merase chain reaction was performed to validate micro- tinic acetylcholine receptors (nAChRs) and muscarinic array findings. AChRs (mAChRs), which may regulate NB neuron activ- ity in AD. Although alterations in these AChRs occur in Results: Cholinergic NB neurons displayed a statisti- the AD cortex, there is little information detailing whether cally significant up-regulation of ␣7 nAChR messenger defects in nAChR and mAChR gene expression occur in RNA expression in subjects with mild to moderate AD cholinergic NB neurons during disease progression. compared with those with NCI and MCI (PϽ.001). No differences were found for other nAChR and mAChR sub- types across the cohort. Expression levels of ␣7 nAChRs Objective: To determine whether nAChR and mAChR were inversely associated with Global Cognitive Score and gene expression is altered in cholinergic NB neurons dur- with Mini-Mental State Examination performance. ing the progression of AD. Conclusions: Up-regulation of ␣7 nAChRs may signal Design: Individual NB neurons from subjects diag- a compensatory response to maintain basocortical cho- nosed ante mortem as having no cognitive impairment linergic activity during AD progression. Alternatively, pu- ␤ (NCI), mild cognitive impairment (MCI), or mild to mod- tative competitive interactions of this receptor with -amy- loid may provide a pathogenic mechanism for NB erate AD were analyzed by single-cell AChR expression ␣ profiling via custom-designed microarrays. dysfunction. Increasing NB 7 nAChR expression may serve as a marker for the progression of AD. Setting: Academic research. Arch Neurol. 2007;64(12):1771-1776 ORTICAL PROJECTION NEU- nAChRs,9 suggesting that the cholinergic rons of the cholinergic tone of NB projection neurons in the AD Author Affiliations: basal forebrain nucleus ba- brain may be regulated by their expres- Department of Neurological salis (NB) play a key role sion of AChRs. To investigate whether Sciences, Rush University in memory and atten- AChR gene expression is altered during the Medical Center, Chicago, tion1 and undergo selective degeneration in progression of AD, we performed single- Illinois (Drs Counts, He, and C Mufson); Center for Dementia Alzheimer disease (AD) that correlates with cell genetic profiling to generate mAChR Research, Nathan Kline disease duration and degree of cognitive im- and nAChR expression profiles for indi- 2 Institute and Department of pairment. Cholinergic neurotransmis- vidual cholinergic NB neurons microaspi- Psychiatry (Drs Che and sion is mediated by nicotinic acetylcho- rated in tissue samples from individuals Ginsberg), and Department of line receptors (nAChRs) and muscarinic clinically diagnosed ante mortem as hav- Physiology and Neuroscience AChRs (mAChRs) through ligand-gated ing no cognitive impairment (NCI), mild (Dr Ginsberg), New York Ca2ϩ influx and G protein–coupled recep- cognitive impairment (MCI) (a putative University School of Medicine, tor-mediated pathways, respectively.3,4 preclinical AD stage10), or mild to moder- Orangeburg; and Departments Binding of nAChRs is reduced in the AD ate AD. We also examined whether changes of Psychiatry and Neurology 5,6 (Drs Ikonomovic and cortex, indicating a loss of cortical cho- in AChR messenger RNA (mRNA) levels DeKosky), University of linergic activity as the disease progresses. were associated with antemortem cogni- Pittsburgh Medical Center, Intriguingly, NB neurons receive cholin- tive performance or with postmortem neu- Pittsburgh, Pennsylvania. ergic afferents7 and express mAChRs8 and ropathological variables. (REPRINTED) ARCH NEUROL / VOL 64 (NO. 12), DEC 2007 WWW.ARCHNEUROL.COM 1771 ©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 Table. Case Demographicsa Clinical Diagnosis Group NCI MCI AD Total Comparison Pairwise Variable (n=12) (n=10) (n=12) (N=34) P Value Comparisonsb Age, y 81.0±9.1 81.9±4.3 84.5±6.9 82.4±7.1 .34 . Male sex 6 (50) 3 (30) 5 (42) 14 (41) .68c ... Education, y 17.5±4.8 18.8±2.3 16.3±4.1 17.6±3.9 .41 . PMI, h 12.4±10.7 7.8±4.7 6.9±3.2 7.4±3.2 .70 . Mini-Mental State Examination score 27.6±1.5 26.6±2.8 14.0±9.7 22.4±8.8 Ͻ.001 (NCI, MCI)ϾAD Global Cognitive Score 0.5±0.3 0.2±0.2 −0.9±0.5 0.0±0.7 Ͻ.001 (NCI, MCI)ϾAD APOE ε4 allele 2 (17) 4 (40) 6 (50) 12 (35) .13c ... Braak score .02 NCIϽ(MCI, AD) 01001 I/II 5 0 1 6 III/IV 6 8 4 18 V/VI 0 2 7 9 National Institute on Aging and Reagan Institute .004 NCIϽAD Working Group diagnosis No 1 0 0 1 ... ... Low 8 3 1 12 ... ... Intermediate 3 7 7 17 ... ... High 0 0 4 4 ... ... Abbreviations: AD, mild to moderate Alzheimer disease; MCI, mild cognitive impairment; NCI, no cognitive impairment; PMI, postmortem interval. a Data are given as mean±SD, number, or as percentages unless otherwise indicated. b Bonferroni-type correction. c Fisher exact test. Kruskal-Wallis test for all other values in this column. METHODS IMMUNOHISTOCHEMISTRY Ribonuclease-free precautions were used throughout the experi- CLINICAL AND PATHOLOGICAL EVALUATION mental procedures. The presence of intact RNA in the tissue sec- tions was confirmed by acridine orange histofluorescence and bio- Details of the annual clinical and neuropsychological evalua- analysis (Agilent, Santa Clara, California).17,18 Tissue sections were tions of the Rush Religious Orders Study cohort have been pub- processed using a monoclonal antibody raised against the hu- lished.11 Cognitive testing was performed under the auspices of man p75 neurotrophin receptor, which co-localizes with approxi- a neuropsychologist, and scores were available within the last year mately 95% of all cholinergic basal forebrain neurons within the prior to death. A board-certified neurologist with expertise in the human NB.19 Tissue sections were incubated for 1 hour in a phos- evaluation of older persons made a clinical diagnosis for each par- phate-buffered saline (pH 7.2) solution containing 0.3% Triton ticipant based on review of all clinical data and physical exami- X-100, 3% normal horse serum, and 2% bovine serum albumin. nation findings. Subjects were categorized as having NCI (n=12), Primary antibody (human p75 neurotrophin receptor, 1:60 000; MCI (n=10), or mild to moderate AD (n=12) (Table). The MCI Neomarkers, Fremont, California) was applied for 4 hours at ap- population was defined as subjects who exhibited impaired neu- proximately 25°C in phosphate-buffered saline containing 0.4% ropsychological test scores but did not meet the clinical criteria Triton X-100, 1% normal horse serum, and 1% bovine serum al- for AD recommended by the Joint Working Group of the Na- bumin. Sections were processed (ABC kit; Vector Laboratories, tional Institute of Neurological and Communicative Disorders and Burlingame, California) and developed with 2.5% nickel II sul- Stroke and the Alzheimer’s Disease and Related Disorders Asso- fate, 0.05% 3,3-diaminobenzidine (Sigma-Aldrich Inc, St Louis, ciation.12 These criteria are compatible with those used by ex- Missouri), and 0.005% hydrogen peroxide.13 Immunostained tis- perts in the field to describe subjects who are not cognitively nor- sue sections were slide mounted but not coverslipped and were mal but do not meet established criteria for dementia.10 None of stored in phosphate buffer at 4°C. the subjects examined reported a history of smoking or cholin- esterase inhibitor therapy on entry into the cohort. At autopsy, brains were removed from the calvarium and SINGLE-CELL MICROASPIRATION, RNA were cut into 0.5-cm-thick slabs using a transparent thermo- AMPLIFICATION, AND ARRAY HYBRIDIZATION plastic brain-cutting apparatus (produced by the University of Illinois, Chicago). The slabs were hemisected, and 1 hemi- Immunopositive neurons from the anterior NB subfield were sphere was immersion fixed in a 4% paraformaldehyde solu- aspirated using a micromanipulator and microcontrolled vacuum tion in 0.1M phosphate buffer (pH 7.2), cryoprotected, and cut source (Eppendorf, Westbury, New York) attached to an in- frozen into 40-µm-thick sections.13 Neuropathological assess- verted microscope (Nikon TE2000; Fryer, Huntley, Illinois).13,20,21 ments were performed by a neuropathologist blinded to clini- RNA amplification from NB neurons was performed using ter- cal diagnosis. Cases were classified based on the National In- minal continuation (TC) RNA amplification methods17,18 (http: stitute on Aging and the Reagan Institute Working Group14 and //cdr.rfmh.org/pages/ginsberglabpage.html). The final ampli- Consortium to Establish a Registry for Alzheimer’s Disease cri- fication step used complementary DNA (cDNA) made from teria,15 as well as by Braak stage.16 Slabs from the opposite hemi- individual neuronal mRNA as a template for in vitro transcrip- sphere were frozen at −80°C. tion in the presence of T7 RNA polymerase and phosphorus P (REPRINTED) ARCH NEUROL / VOL 64 (NO. 12), DEC 2007 WWW.ARCHNEUROL.COM 1772 ©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 ∗ CHRM1 CHRM2 CHRM3 CHRM4 CHRM5 CHRNA1 CHRNA2 CHRNA3 CHRNA4 CHRNA5 CHRNA6 CHRNA7 CHRNB1 CHRNB2 CHRNB3 CHRNB4 AD MCI NCI 048 Figure 1. Dendrogram illustrating relative expression levels of muscarinic acetylcholine receptors (CHRM1-5) and nicotinic acetylcholine receptors (CHRNA1-7 and CHRNB1-4) within individual nucleus basalis neurons from subjects with mild to moderate Alzheimer disease (AD), mild cognitive impairment (MCI), or no cognitive impairment (NCI).