ORIGINAL CONTRIBUTION ␣7 Nicotinic Receptor Up-regulation in 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 receptors (nAChRs) and muscarinic array findings. AChRs (mAChRs), which may regulate NB 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 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 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 –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.

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©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 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

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©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). *PϽ.001, AD compared with NCI and MCI.

33 uridine triphosphate. Radiolabeled TC RNA probes were then human postmortem brain RNA. For graphical representation, hybridized to custom-designed cDNA arrays. Arrays were hy- cycle threshold values were converted to signal intensity values bridized overnight at 42°C in a rotisserie oven and were washed using commercially available software (Easy Engine, Bio-Rad). sequentially in 2ϫ saline sodium citrate (SSC) buffer–0.1% so- dium dodecyl sulfate (SDS), 1ϫ SSC–0.1% SDS, and 0.5ϫ SSC– 0.1% SDS for 20 minutes each at 42°C.13,20,21 Arrays were placed STATISTICAL ANALYSIS in a phosphor screen for 24 hours and were developed on a phos- phor imager (GE Healthcare, Piscataway, New Jersey). Demographic variables (Table) were compared among clinical di- agnostic groups using a Kruskal-Wallis test or Fisher exact test, CUSTOM-DESIGNED cDNA ARRAY PLATFORMS with Bonferroni-type correction for pairwise comparisons. Ex- AND DATA COLLECTION pression levels of AChR mRNAs were clustered and displayed using a bioinformatics and graphics software package (GeneLinker Gold; 13,20,21 Array platforms consisted of 1 µg of linearized cDNA purified from Predictive Patterns, Kingston, Ontario, Canada). A false dis- 25 plasmid preparations adhered to high-density nitrocellulose (Hy- covery rate-controlling procedure was used to reduce type I er- bond XL, GE Healthcare). Approximately 576 cDNAs and/or ex- rors due to the large number of analyzed simultaneously. pressed sequence tags (ESTs) were used on the array platform. The Relative changes in hybridization signal intensity of individual human ESTs encoding specific nAChRs and mAChRs were selected mRNAs and qPCR were analyzed using 1-way analysis of vari- using the UniGene database (http://www.ncbi.nlm.nih.gov/sites ance with post hoc Newman-Keuls analysis. The association be- /?db=unigene) to identify individual subunits with mini- tween gene expression levels and clinical or neuropathological mal cross-hybridization based on to the origi- variables was evaluated via mixed-models repeated-measures analy- 24 Ͻ nal cDNAs from rodents22,23 as described previously.21,24 Each AChR ses. The level of significance was set at P .01. EST was specific for the respective individual subunit, and each EST is sequence verified to be 100% identical to the wild-type RESULTS cDNA clone before inclusion on the custom-designed array plat- form.18 Hybridization signal intensity is quantified by subtract- ing background using empty vector (pBluescript; Stratagene, La A total of 174 single cholinergic NB neurons were analyzed Jolla, California). Expression of TC-amplified RNA bound to each from 34 postmortem brains, with a mean of 5 to 6 cells per linearized cDNA was expressed as a ratio of the total hybridiza- subject measured in triplicate on independent arrays. The tion signal intensity of the array (ie, global normalization).18 Data arrays contained cDNAs encoding mAChR subtypes m1 analysis generated an expression profile of relative changes in through m5 (UniGene and National Center for Biotechni- mRNA levels among cholinergic NB neurons isolated from dif- cal Information annotation CHRM1-5) and nAChR subunits ferent clinical groups. ␣1 through ␣7 (CHRNA1-7) and ␤1 through ␤4 (CHRNB1- 4). Genes at the limit of resolution within individual neu- REAL-TIME QUANTITATIVE POLYMERASE rons included CHRM4, CHRNA5, CHRNB2, and CHRNB3 CHAIN REACTION and did not vary to any substantial degree across the cohort. There was no difference in the relative expression levels of Real-time quantitative polymerase chain reaction (qPCR) was the mAChR subtype mRNAs among NB neurons isolated performed on unfixed microdissected frozen anterior NB and from NCI, MCI, and mild to moderate AD cases (Figure 1). caudate tissue from 11 NCI cases, 9 MCI cases, and 10 mild to ␣ ␣ In contrast, there was a statistically significant selective 60% moderate AD cases using PCR primers specific for human 4 increaseintheexpressionof␣7nAChRsubunitmRNAwithin and ␣7 nAChRs and the housekeeping gene glyceraldehyde-3- NB neurons from the mild to moderate AD cases compared phosphate dehydrogenase (GAPDH) (SuperArray, Frederick, Ͻ Maryland). Samples were loaded in triplicate on 96-well plates with the NCI and MCI cases (F2,33=12.673, P .001). A sta- and were analyzed using SYBR green reporter dye on a DNA en- tistically significant association was found between increas- gine (Opticon 2; Bio-Rad, Hercules, California). Standard curves ing␣7nAChRmRNAlevelsandlowercognitivetestingscores and cycle threshold were measured using standards obtained from ontheMini-MentalStateExaminationandacompositeGlob-

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 mRNA levels were statistically significantly increased in A NB samples from mild to moderate AD cases relative to 2.5 Ͻ NCI and MCI cases (F2,29=5.396, P .01) (Figure 2A), whereas ␣4 nAChR mRNA levels were similar among the 2.0 ∗ diagnostic groups (Figure 2B). In the caudate nucleus, an area enriched in cholinergic neurons but unaffected 4 1.5 in AD, ␣7 nAChR mRNA levels were unchanged in mild

GAPDH to moderate AD cases compared with NCI and MCI cases (Figure 2C); ␣4 nAChR mRNA was not detected. The 1.0 ␣ CHRNA7/ 7 nAChR primer set revealed no cross-reactivity with ␣4 nAChRs (cycle threshold Ͻ10 using 10 ng of ␣7 0.5 nAChR plasmid as input cDNA; cycle threshold Ͼ40 using 10 ng of ␣4 nAChR plasmid as input cDNA). 0.0 NCI MCI AD

B COMMENT 1.5 We report the novel finding that individual cholinergic NB neurons up-regulate the expression of ␣7 nAChR sub- 1.0 unit mRNA in mild to moderate AD but not in MCI. These subunits are likely expressed as homomeric ␣7 nAChR GAPDH assemblies, which combined with heteromeric ␣4␤2 as- semblies constitute the 2 major nAChR subtypes ex-

CHRNA4/ 0.5 pressed in brain.3 Subunit expression for the ␣4␤2 sub- type was unchanged across clinical diagnoses. A principal strength of the present single-cell profiling approach is

0.0 that the target AChR cDNA sequences on the custom ar- NCI MCI AD rays are nonoverlapping. This provides optimal mAChR subtype and nAChR subunit specificity and sensitivity C and avoids potential confounds inherent in studies in 0.75 which, for instance, nAChRs are detected by radioli- gands that cannot discriminate between individual subunits.3 ␣ 0.50 The probable increase in 7 nAChRs (as indexed by increased message) in AD may regulate basocortical GAPDH cholinergic tone through presynaptic or postsynaptic mechanisms within NB neurons before their frank

CHRNA7/ 0.25 degeneration in the later stages of AD. In human brain, ␣7 nAChR–specific hybridization probes and iodine I 125–␣-bungarotoxin label NB neurons,9 suggesting that ␣ 0.0 7 nAChRs within cholinergic cells are localized to NCI MCI AD postsynaptic somatodendritic compartments. Choliner- gic inputs to cholinergic NB perikarya have been identi- Figure 2. Quantitative polymerase chain reaction validation of CHRNA7 and fied in primate brain,7 while and nicotinic ago- CHRNA4 expression profiling. A, CHRNA7 messenger RNA in anterior Ͻ nists depolarize these cells in the presence of nucleus basalis (NB). *P .01, mild to moderate Alzheimer disease (AD) 26,27 ␣ compared with no cognitive impairment (NCI) and mild cognitive impairment tetrodotoxin. Hence, increased postsynaptic 7 (MCI). B, CHRNA4 in anterior NB. C, CHRNA7 in striatum. CHRNA4 and nAChR expression may facilitate feed-forward choliner- CHRNA7 expression levels were normalized to glyceraldehyde-3-phosphate gic activity within NB cortical projection neurons in dehydrogenase gene (GAPDH) expression levels for quantitative analysis. CHRNA4 and CHRNA7 expression levels were normalized to GAPDH AD. Increased nicotinic transmission may also result in expression levels for quantitative analysis. altered gene expression through the transcription factor cyclic adenosine monophosphate–dependent binding protein (CREB),28 which promotes cholinergic neuron al Cognitive Score (PϽ.001 for both). A statistically signifi- survival.29 Alternatively, whereas the presence of ␣7 cantassociationwasalsofoundbetweenincreasing␣7nAChR nAChRs on cholinergic within the cortex mRNA levels and a higher likelihood of AD by the National remains controversial,30,31 presynaptic ␣7 nAChRs have Institute on Aging and the Reagan Institute Working Group been shown to stimulate ACh release.32 This suggests criteria(P=.01).Noassociationwasfoundbetween␣7nAChR that increased NB neuron ␣7 nAChR expression could levels and Braak stage (P=.11). stimulate autoreceptor-mediated ACh release within Array data were validated by qPCR analysis of ␣4 and cortical projection sites during AD progression in an ␣7 nAChR mRNA levels in frozen anterior NB and cau- effort to increase a failing cholinergic signal. Since the date nucleus tissue from the same cases. Similar to the anterior NB cholinergic neurons project to the frontal single NB neuron expression profiling studies, ␣7 nAChR cortex,33 an increase in presynaptic ␣7 nAChRs in these

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 neurons may account for the stability of ␣7 nAChR lev- tia severity in Alzheimer’s disease: relative importance of the cholinergic deficits. els in the frontal cortex compared with its decrease in J Neurochem. 1995;64(2):749-760. 5 3. Gotti C, Fornasari D, Clementi F. Human neuronal nicotinic receptors. Prog other cortical areas in AD. This putative plasticity Neurobiol. 1997;53(2):199-237. response in NB neurons is reminiscent of the increases 4. Levey AI. Muscarinic expression in memory circuits: im- in ␣7 nAChRs observed in other limbic structures, plications for treatment of Alzheimer disease. Proc Natl Acad Sci U S A. 1996; including the entorhinal cortex and ,34,35 93(24):13541-13546. as well as in peripheral leukocytes36 in AD. This latter 5. Rei RT, Sabbagh MN, Corey-Bloom J, Tiraboschi P, Thal LJ. Nicotinic receptor losses in dementia with Lewy bodies: comparisons with Alzheimer’s disease. Neu- observation, combined with our present data showing robiol Aging. 2000;21(5):741-746. that NB ␣7 nAChR mRNA expression is inversely asso- 6. Whitehouse PJ, Martino AM, Marcus KA, et al. Reductions in acetylcholine and ciated with Global Cognitive Score and Mini-Mental nicotine binding in several degenerative diseases. Arch Neurol. 1988;45(7): State Examination score, suggests that increases in this 722-724. 7. Smiley JF, Mesulam MM. Cholinergic neurons of the nucleus basalis of Meynert receptor may serve as a marker for AD progression. receive cholinergic, catecholaminergic and GABAergic synapses: an electron Despite potential beneficial roles for increased NB neu- microscopic investigation in the monkey. Neuroscience. 1999;88(1): ronal ␣7 nAChR expression in AD, evidence suggests that 241-255. increased ␣7 nAChR expression contributes to cellular 8. Mufson EJ, Jaffar S, Levey AI. m2 Muscarinic acetylcholine receptor– degeneration. Notably, ␣7 nAChRs bind37 or interact38 immunoreactive neurons are not reduced within the nucleus basalis in Alzhei- ␤ ␤ ␤ mer’s disease: relationship with cholinergic and galaninergic perikarya. J Comp with -amyloid (A ) precursor protein and A pep- Neurol. 1998;392(3):313-329. 39 tides, which form the nidus of senile plaques in AD. Al- 9. Breese CR, Adams C, Logel J, et al. Comparison of the regional expression of though ␣7 nAChR activation can be protective against nicotinic acetylcholine receptor ␣7 mRNA and [125I]-␣-bungarotoxin binding in A␤ neurotoxicity,40,41 competitive A␤ binding to ␣7 human postmortem brain. J Comp Neurol. 1997;387(3):385-398. nAChRs may inhibit the action of this receptor in pro- 10. Petersen RC, Doody R, Kurz A, et al. Current concepts in mild cognitive impairment. 42 43 Arch Neurol. 2001;58(12):1985-1992. moting long-term potentiation or activation of CREB. 11. Bennett DA, Wilson RS, Schneider JA, et al. Natural history of mild cognitive im- Furthermore, cell surface A␤–␣7 nAChR interactions may pairment in older persons. Neurology. 2002;59(2):198-205. promote A␤ endocytosis, producing intraneuronal A␤ ac- 12. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical cumulation, with possible subsequent plaque forma- diagnosis of Alzheimer’s disease: report of the NINCDS-ADRDA Work Group un- tion.39 Therefore, increased NB neuronal ␣7 nAChR ex- der the auspices of Department of Health and Human Services Task Force on Alzheimer’s Disease. Neurology. 1984;34(7):939-944. pression may arise as a compensatory neuroprotective 13. Mufson EJ, Counts SE, Ginsberg SD. Gene expression profiles of cholinergic nucleus response that is offset by aberrant A␤–␣7 nAChR inter- basalis neurons in Alzheimer’s disease. Neurochem Res. 2002;27(10):1035- actions, leading to cholinergic dysfunction. 1048. 14. Hyman BT, Trojanowski JQ. Consensus recommendations for the postmortem diagnosis of Alzheimer disease from the National Institute on Aging and the Reagan Accepted for Publication: April 14, 2007. Institute Working Group on diagnostic criteria for the neuropathological assess- Correspondence: Elliott J. Mufson, PhD, Department of ment of Alzheimer disease. J Neuropathol Exp Neurol. 1997;56(10):1095-1097. Neurological Sciences, Rush University Medical Cen- 15. Mirra SS, Heyman A, McKeel D, et al. The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD), part II: standardization of the neuropathologic as- ter, 1735 W Harrison St, Ste 300, Chicago, IL 60612 sessment of Alzheimer’s disease. Neurology. 1991;41(4):479-486. ([email protected]). 16. Braak H, Braak E. Neuropathological stageing of Alzheimer-related changes. Acta Author Contributions: Study concept and design: Counts, Neuropathol (Berl). 1991;82(4):239-259. Che, DeKosky, Ginsberg, and Mufson. Acquisition of data: 17. Che S, Ginsberg SD. Amplification of RNA transcripts using terminal continuation. Counts, He, Che, and Ginsberg. Analysis and interpreta- Lab Invest. 2004;84(1):131-137. 18. Ginsberg SD. RNA amplification strategies for small sample populations. Methods. tion of data: Counts, Ikonomovic, DeKosky, Ginsberg, and 2005;37(3):229-237. Mufson. Drafting of the manuscript: Counts, Ginsberg, and 19. Mufson EJ, Bothwell M, Hersh LB, Kordower JH. receptor Mufson. Critical revision of the manuscript for important immunoreactive profiles in the normal, aged human basal forebrain: colocaliza- intellectual content: He, Che, Ikonomovic, DeKosky, and tion with cholinergic neurons. J Comp Neurol. 1989;285(2):196-217. Ginsberg. Statistical analysis: Counts. Obtained funding: 20. Counts SE, Chen EY, Che S, et al. Galanin fiber hypertrophy within the choliner- gic nucleus basalis during the progression of Alzheimer’s disease. Dement Geriatr Counts, Ikonomovic, DeKosky, Ginsberg, and Mufson. Cogn Disord. 2006;21(4):205-211. Administrative, technical, and material support: Counts, He, 21. Ginsberg SD, Che S, Counts SE, Mufson EJ. Shift in the ratio of three-repeat tau Che, DeKosky, Ginsberg, and Mufson. Study supervi- and four-repeat tau mRNAs in individual cholinergic basal forebrain neurons in sion: Counts, Ginsberg, and Mufson. mild cognitive impairment and Alzheimer’s disease. J Neurochem. 2006;96 Financial Disclosure: None reported. (5):1401-1408. 22. Heinemann S, Boulter J, Deneris E, et al. The brain nicotinic acetylcholine re- Funding/Support: This study was supportd by grants ceptor gene family. Prog Brain Res. 1990;86:195-203. AG26032 (Dr Counts), AG01533 (Dr DeKosky), 23. Patrick J, Boulter J, Deneris E, et al. Structure and function of neuronal nicotinic AG17617 (Dr Ginsberg), NS48447 (Dr Ginsberg), acetylcholine receptors deduced from cDNA clones. Prog Brain Res. 1989; AG14449 (Dr Mufson), AG09466 (Dr Mufson), and 79:27-33. 24. Ginsberg SD, Che S, Wuu J, Counts SE, Mufson EJ. Down regulation of trk but AG10161 (Dr Mufson), from the National Institutes of not p75NTR gene expression in single cholinergic basal forebrain neurons mark Health, the Illinois Department of Public Health (Dr the progression of Alzheimer’s disease. J Neurochem. 2006;97(2):475-487. Counts), and the Alzheimer’s Association (Dr Ginsberg). 25. Reiner A, Yekutieli D, Benjamini Y. Identifying differentially expressed genes using false discovery rate controlling procedures. Bioinformatics. 2003;19(3): 368-375. REFERENCES 26. Khateb A, Fort P, Williams S, Serafin M, Jones BE, Muhlethaler M. Modulation of cholinergic nucleus basalis neurons by acetylcholine and N-methyl-D-aspartate. 1. Bartus RT. On neurodegenerative diseases, models, and treatment strategies: Neuroscience. 1997;81(1):47-55. lessons learned and lessons forgotten a generation following the cholinergic 27. Lamour Y, Dutar P, Rascol O, Jobert A. Basal forebrain neurons projecting to hypothesis. 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Call for Papers

Archives Express

The Archives launched a new Archives Express section in the September 2000 issue. This section will enable the editors to publish highly selected papers within approxi- mately 2 months of acceptance. We will consider only the most significant research, the top 1% of accepted pa- pers, on new important insights into the pathogenesis of disease, brain function, and therapy. We encourage authors to send their most exceptional clinical or basic research, designating in the cover letter a request for ex- pedited Archives Express review. We look forward to pub- lishing your important new research in this accelerated manner. Roger N. Rosenberg, MD

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