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Neuropsychologia 46 (2008) 1688–1697

A� deposits in older non-demented individuals with cognitivedecline are indicative of preclinical Alzheimer’s disease

V.L. Villemagne a,b,c,d,∗, K.E. Pike a,e, D. Darby d,f, P. Maruff d,f, G. Savage e,1,S. Ng a, U. Ackermann a, T.F. Cowie c, J. Currie g, S.G. Chan a, G. Jones a,

H. Tochon-Danguy a, G. O’Keefe a, C.L. Masters b,c,d, C.C. Rowe a,h

a Department of Nuclear Medicine, Centre for PET, Austin Health, 145 Studley Road, Heidelberg, VIC 3084, Australiab The Mental Health Research Institute of Victoria, Parkville, VIC, Australia

c Department of Pathology, University of Melbourne, VIC, Australiad Centre for Neuroscience, University of Melbourne, VIC, Australia

e School of Psychology, Psychiatry and Psychological Medicine, Monash University, Melbourne, VIC, Australiaf Cogstate Pty Ltd., Melbourne, VIC, Australia

g St Vincent’s Hospital, Melbourne, VIC, Australiah Department of Medicine, (Austin Health) University of Melbourne, VIC, Australia

Received 29 July 2007; received in revised form 28 December 2007; accepted 1 February 2008Available online 14 February 2008

bstract

Approximately 30% of healthy persons aged over 75 years show A� deposition at autopsy. It is postulated that this represents preclinicallzheimer’s disease (AD). We evaluated the relationship between A� burden as assessed by PiB PET and cognitive decline in a well-characterized,on-demented, elderly cohort.

PiB PET studies and cognitive tests were performed on 34 elderly participants (age 73 ± 6) from the longitudinal Melbourne Healthy Agingtudy (MHAS). Subjects were classified as being cognitively ‘stable’ or ‘declining’ by an independent behavioural neurologist based on clinicalssessment and serial word-list recall scores from the preceding 6–10 years. Decline was calculated from the slope of the word-list recall scores.� burden was quantified using Standardized Uptake Value normalized to cerebellar cortex.Ten subjects were clinically classified as declining. At the time of the PET scans, three of the declining subjects had mild cognitive impairment,

ne had AD, and six were declining but remained within the normal range for age on cognitive tests. Declining subjects were much more likelyo show cortical PiB binding than stable subjects (70% vs. 17%, respectively). Neocortical A� burden correlated with word-list recall slopesr = −0.78) and memory function (r = −0.85) in the declining group. No correlations were observed in the stable group.

A� burden correlated with incident memory impairment and the rate of memory decline in the non-demented ageing population. These obser-ations suggest that neither memory decline nor A� deposition are part of normal ageing and likely represent preclinical AD. Further longitudinalbservations are required to confirm this hypothesis.

2008 Elsevier Ltd. All rights reserved.

g; PiB

eywords: Brain imaging; Mild cognitive impairment; Amyloid; Normal agein

∗ Corresponding author at: Department of Nuclear Medicine, Centre for PET,ustin Health, 145 Studley Road, Heidelberg, VIC 3084, Australia.el.: +61 3 9496 3321; fax: +61 3 9458 5023.

E-mail address: villemagne@petnm.unimelb.edu.au (V.L. Villemagne).1 Present address: Macquarie Centre for Cognitive Science (MACCS), Mac-uarie University, Sydney, NSW, Australia.

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028-3932/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.neuropsychologia.2008.02.008

; Positron emission tomography

. Introduction

Alzheimer’s disease (AD), the leading cause of dementian the elderly, is an irreversible, progressive neurodegenerativeisorder clinically characterized by memory loss and cognitive

ecline, leading invariably to death (Masters, Cappai, Barnham,

Villemagne, 2006). The progressive nature of neurodegener-tion suggests an age-dependent process that ultimately leadso synaptic failure and neuronal damage in cortical areas of

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he brain essential for memory and higher mental functionsMasters, 2005; Selkoe, 2002). To date no therapy has beenhown to halt or reverse the underlying disease process andreatment is confined to symptomatic palliative interventionsBarrow, 2002).

In the absence of biological markers, direct pathologic exam-nation of brain tissue remains the only definitive method forstablishing diagnosis of AD (Masters & Beyreuther, 2005;elkoe, 2001). The widespread cellular degeneration and neu-onal loss are accompanied by reactive gliosis, and by theresence of the pathological hallmarks of the disease, intracellu-ar neurofibrillary tangles (NFT) and extracellular beta-amyloidA�) plaques (Masters & Beyreuther, 2005; Selkoe, 2001). Allhe available evidence points to the breakdown of A� home-stasis as the key role in AD pathogenesis, leading to synapticysfunction, microgliosis, and neuronal loss (Villemagne et al.,006). This loss of synaptic function seems to be an earlynd critical factor, clinically manifested by memory loss andmpaired cognitive functions (Masters et al., 2006; Selkoe, 2002;illemagne et al., 2006).

A deeper understanding of the molecular mechanism of� formation, degradation, aggregation, and neurotoxicity

s being translated into new neuroimaging and therapeu-ic approaches (Masters et al., 2006; Villemagne, Rowe,

acfarlane, Novakovic, & Masters, 2005). A� imaging withositron emission tomography (PET) permits in vivo assess-ent of A� deposition in the brain, providing an important

ew tool for the evaluation of the causes, diagnosis and, poten-ially, treatment of dementias where A� may play a role.tudies with {N-methyl-[C-11]}2-(4′-methylamino-phenyl)-6-ydroxy-benzothiazole ([C-11]6-OH-BTA-1, also known asPittsburgh Compound-B” or PiB), the most specific and mostidely used PET A� ligand (Klunk et al., 2004), indicate that� imaging may allow earlier diagnosis of Alzheimer’s disease

AD) (Mintun et al., 2006; Rowe et al., 2007) and accurate dif-erential diagnosis of the dementias (Drzezga et al., 2008; Ng,illemagne, Masters, & Rowe, 2007; Rabinovici et al., 2007;owe et al., 2007).

PiB PET studies in human participants have shown a robustifference between the retention pattern in AD patients andealthy controls, with AD cases showing significantly higheretention of PiB in neocortical areas of the brain affected by A�eposition (Buckner et al., 2005; Klunk et al., 2004; Price et al.,005; Rowe et al., 2007; Verhoeff et al., 2004). PiB is also highern people diagnosed with congophilic angiopathy (Johnson etl., 2007), showing a similar distribution to AD and DLB cases,hile is not higher in people with Parkinson’s disease (Johansson

t al., 2007). While in the majority of cases PiB cannot discrimi-ate AD from DLB, it seems to be an ideal technique to reliablyifferentiate AD from FTD (Drzezga et al., 2008; Engler et al.,008; Rabinovici et al., 2007; Rowe et al., 2007). Human PETtudies have also demonstrated a correlation between PiB bind-ng and the rate of cerebral atrophy in AD subjects (Archer et al.,

006), and with decreased CSF A�1–42 in both demented andon-demented subjects (Fagan et al., 2006). Binding also cor-elates with episodic memory impairment in apparently normallderly individuals and in subjects with mild cognitive impair-

arss

ologia 46 (2008) 1688–1697 1689

ent (MCI) (Pike et al., 2007). Increased PiB binding may alsoredict conversion of MCI to AD (Forsberg et al., 2007). About0–25% of cognitively normal elderly subjects evaluated alsoemonstrate higher cortical PiB retention, predominantly in therefrontal and posterior cingulate/precuneus regions, though tolesser degree than AD patients (Mintun et al., 2006; Rowe et

l., 2007). This agrees well with post-mortem reports that 30%f non-demented older persons over the age of 75 present neu-itic plaques in the cerebral cortex (Jorm & Jolley, 1998; Price &

orris, 1999). Furthermore, comparison of the diagnostic util-ty of A� imaging versus FDG demonstrated that PiB imagings more accurate than FDG for the diagnosis of AD (S. Ng et al.,007). Recently, it has been proposed that the research criteriaor the diagnosis of probable AD should be revised so to includehe demonstration of A� with appropriate PET tracers (Duboist al., 2007).

If measurements of cognition are conducted regularly over aufficient period of time it is possible to detect subtle cognitiveeterioration in individuals who do not meet clinical criteria forD or MCI (Collie et al., 2001). To characterize this decline an

xperimental study of the cognitive consequences of aging, theelbourne Healthy Aging Study (MHAS) (Collie et al., 2001;hyte et al., 1997), was established. Recruitment and assess-ent of this cohort began in 1996 and has been described in detail

reviously (Collie et al., 2001; Weaver Cargin, Maruff, Collie,Masters, 2006). Memory performance (delayed verbal recall)

as been observed to show subtle but progressive decline overime in subjects that eventually progress to AD (Collie et al.,001; Maruff et al., 2004; Weaver Cargin et al., 2006). Impor-antly, in relatively young and healthy individuals, the memoryecline is so subtle that very few subjects satisfied clinical cri-eria for MCI (Collie et al., 2001; Maruff et al., 2004; Weaverargin et al., 2006). Hence, when the cognitive data from theHAS group are considered at any single assessment (cogni-

ively declining and non-declining subjects), they are not outsideormal ranges.

The aim of the current study was to determine the relation-hip between A� deposition in the brains of older individualsith known cognitive history. We tested the hypotheses that A�

maging can detect early molecular changes in the brain and that� deposition is associated with a history of cognitive declineespite cognitive performance being within normal limits.

. Methods

.1. Participants

Written informed consent for participation in this study was obtained prioro the scans. Approval for the study was obtained from the Austin Health Humanesearch Ethics Committee.

Thirty-four elderly individuals with well-documented cognitive functionolunteered for the study. All subjects were enrolled in the ongoing experimentaltudy of the cognitive consequences of aging, the MHAS (Collie et al., 2001;

hyte et al., 1997). Participants were originally recruited in 1996 and assessed

t least once a year, with a group median of eight visits (semi-interquartileange 7–9) prior to the PET scan. Out of the 142 participants that by 2005 weretill enrolled in the MHAS, 34 (24%) of them agreed to participate in the PETtudy.

ApoE genotype was determined by direct sequencing.

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.2. Neuropsychological assessment

Screening of participants at entry to the MHAS involved extensive neurolog-cal and general medical assessment to assess each person’s health status. As partf their evaluation for the MHAS, all participants received the Consortium tostablish a Registry for Alzheimer’s Disease (CERAD) battery (Morris, Mohs,ogers, Fillenbaum, & Heyman, 1988), the Cambridge Neuropsychologicalest Automated Battery (CANTAB), the CogState battery, the State-Trait Anx-

ety Inventory and the Centre for Epidemiological Studies depression inventoryCESD) at numerous assessments. The strict exclusion criteria for the study haveeen detailed previously (Collie, Shafiq-Antonacci, Maruff, Tyler, & Currie,999). Briefly, at entry to the study, individuals were required to demonstrateormal cognitive status as by the National Institute of Neurological and Com-unicative Disorders and Stroke-Alzheimer’s Disease and Related Disordersssociation (NINCDS-ADRDA) criteria for dementia (McKhann et al., 1984)

nd a Mini-Mental State Examination (MMSE) score of 28 or higher (Folstein,olstein, & McHugh, 1975). Individuals were excluded if two or more abnor-al neurological signs were present, or if the individual had a past or current

istory of cardiovascular or cerebrovascular disease, endocrine disorders, trau-atic head injury, epilepsy, or psychiatric illness including a major depressive

r major anxiety disorder (see Collie et al., 1999).The inclusion criteria for the current study were therefore the same as those

sed initially in the MHAS with the additional criterion that the volunteers clas-ified as meeting criteria for MCI or AD at any point within the MHAS werexcluded. Data from the MHAS indicated that for the majority of healthy oldereople, cognitive performance had remained stable or even improved slightlyver the study period. For each participant, all available data points (no par-icipant had fewer than six) for the CERAD word-list score were plotted as aunction of time since the initial assessment. A least-squares linear regressionas then fitted to these points, and the slope of this line was calculated.

A board-certified behavioural neurologist with specialization in dementiaDD) reviewed the neuropsychological and medical historical data for all MHASubjects. This clinician had not assessed any of the participants previously andad not been involved directly in the study prior to this assessment. He was alsolind to structural and functional neuroimaging, as well as to ApoE genotypeesults. Each participant, in the company of an informant, then underwent annterview and a neurological evaluation. All available clinical and neuropsycho-ogical data, including the CERAD word-list scores, plus reports made by thearticipants or their informants, were used to categorize participants as cogni-ively stable or declining. On the basis of this assessment, each individual waslso classified as being normal or meeting criteria for MCI. Healthy controlsHC) were then subclassified based on whether their cognitive function wastable or had declined over the previous 6–10 years.

Prior to the PET scans all individuals were assessed again by a neurolo-ist (CCR) and a neuropsychologist (KP) who conducted a separate batteryf neuropsychological tests. Participants underwent a neurological examina-ion and were administered the MMSE (Folstein et al., 1975); 30-item Bostonaming Test (Saxton et al., 2000); Digit Span forwards and backwards, andigit Symbol-Coding from the Wechsler Adult Intelligence Scale-Third edition

WAIS-III; Wechsler, 1997); California Verbal Learning Test-Second editionCVLT-II; Delis, Kramer, Kaplan, & Ober, 2000); Rey Complex Figure TestMeyers, Bayless, & Meyers, 1996); letter fluency (Benton, 1968); and categoryuency tasks (Neuropsychological assessment, 2004).

Based on the results of this evaluation, subjects were re-classified as HC,CI, or AD. By the time of the PET scans, ranging from 3 to 18 months

fter MHAS classification, three subjects met Petersen’s MCI criteria of subjec-ive and objective cognitive difficulties, predominantly affecting memory, in thebsence of dementia or significant functional loss (Petersen et al., 1999), whilene subject met NINCDS-ADRDA criteria for probable AD (McKhann et al.,984). Researchers at the PET centre were blind to participant cognitive sub-lassification from the MHAS study as being cognitively stable or cognitivelyeclining. The blind was broken only at the completion of the current study.

.3. Imaging procedures

All subjects underwent a 3D spoiled gradient echo (SPGR) T1-weighed MRIcquisition for screening and subsequent co-registration with the PET images.

c1Ai

ologia 46 (2008) 1688–1697

roduction of 11C-PiB was performed in the Centre for PET, Austin Hospital,sing the one step 11C-methyl triflate approach (Wilson, Garcia, Chestakova,ung, & Houle, 2004). The average radiochemical yield was 30% after a syn-

hesis time of 45 min with a radiochemical purity of >98% and a specific activityf 30 ± 7.5 GBq/�mol. Each subject received 375 ± 18 MBq 11C-PiB by intra-enous injection over 1 min. Imaging was performed with a Phillips AllegroTM

ET camera. At 35 min after injection of PiB, a rotation transmission sinogramcquisition in 3D mode with a single 137Cs point source was performed forttenuation correction. A 30-min emission acquisition was then performed inD mode starting at 40 min after injection of PiB. Images were reconstructedsing a 3D RAMLA algorithm.

.4. Image analysis

Co-registration of the PET images with the MRI was performed with SPM5Statistical Parametric Mapping, MRC Cognition and Brain Sciences Unit)Friston, Frith, Liddle, & Frackowiak, 1991). A MRI-defined region of inter-st (ROI) template, generated with the aid of an MRI atlas (Yuh et al., 1994),as placed on native space over the co-registered MRI and then transferred to

he PET images. Mean Standardized Uptake Values (SUV) were obtained fromOI for cortical, subcortical and cerebellar regions.

PiB SUV ratios (SUVR) were determined by normalizing the regional SUVo the cerebellar cortex, a region relatively unaffected by amyloid depositionLopresti et al., 2005; Price et al., 2005). Besides regional SUVR values, theean of the SUVR for frontal, cingulate, parietal, lateral temporal and occipital

ortex was calculated and termed the Neocortical SUVR. Based on a prior ROCnalysis comparing HC subjects and AD patients in our laboratory, a SUVRhreshold of 1.60 was selected to define PiB positive (PiB+ve) and PiB negativePiB−ve) values (S. Ng et al., 2007).

.5. Statistical analysis

Because the normality assumption cannot be satisfied given the limited num-er of subjects per group, neuropsychological and relevant clinical measuresere compared between the cognitively stable and declining subgroups usingWilcoxon Signed-Ranks test. Categorical differences were evaluated using

isher’s exact test. To determine the relationship between regional PiB bind-ng and cognitive performance Pearson product-moment correlation analysesere conducted between cortical PiB SUVR and neuropsychological measures.earson’s correlation coefficients were used in correlational analysis betweeniB SUVR and neuropsychological and clinical measures. Statistical signifi-ance was defined as p < 0.05. Multiple comparisons were controlled for withFalse Discovery Rate (Benjamini & Hochberg, 1995). Data are expressed asean ± S.D.

. Results

Demographic data are shown in Table 1. Based on their clin-cal evaluation over the preceding 6–10 years, 10 participantsere classified as cognitively declining. One of these was con-

idered to have an atypical non-amnestic pattern of cognitiveeficits. Despite all participants performing within the expectedange for age and education on neuropsychological tasks overhe same period, the cognitively declining group showed a dropn the delayed verbal recall scores (Table 1).

An ApoE �4 allele was present in 38% of all subjectsTable 1). Fifty-four percent of ApoE �4 carriers were PiB+veompared to only 19% of non-carriers (Fisher Exact test= 0.04). The ApoE �4 allele was associated with signifi-

antly higher PiB binding (Neocortical SUVR 1.88 ± 0.68 vs..36 ± 0.29, p = 0.02), but there was no significant difference inpoE �4 prevalence between the cognitively stable and declin-

ng groups (Fisher Exact test p = 0.45).

V.L. Villemagne et al. / Neuropsychologia 46 (2008) 1688–1697 1691

Table 1Demographics (means (S.D.) for groups by classification)

Stable (n = 24) Declining (n = 10) p value

Age 71.7 (6.7) range 58–83 75.5 (4.4) range 66–82 0.12a

Years of education 13.0 (3.6) 11.8 (3.7) 0.41a

MMSE 29.3 (0.9) 27.8 (1.6) 0.003a

Male/female 12/12 3/7 0.45b

ApoE �4/non-�4 8/16 5/5 0.45b

PiB+ve/PiB−ve 4/20 7/3 0.0005b

C −0.20 (0.25) 0.0007a

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Table 3A� burden in stable and cognitive declining groups (means (S.D.) for groups byclinical classification)

PiB

Stable (n = 24) Declining (n = 10) Effect size

Regional SUVRNeocortexa 1.37 (0.33) 1.99 (0.68)* 1.16Dorsolateral prefrontal 1.27 (0.32) 1.90 (0.72)* 1.13Ventrolateral prefrontal 1.36 (0.39) 2.17 (0.86)* 1.21Orbitofrontal 1.38 (0.37) 2.15 (0.81)* 1.22Gyrus rectus 1.49 (0.49) 2.37 (0.86)* 1.26Post cingulate gyrus 1.48 (0.42) 2.14 (0.76) 1.06Ant cingulate gyrus 1.48 (0.41) 2.12 (0.70) 1.12Superior parietal 1.26 (0.33) 1.81 (0.66)* 1.05Lateral occipital 1.38 (0.17) 1.69 (0.40)* 2.25Lateral temporal 1.36 (0.39) 1.95 (0.67)* 1.08Mesial temporal 1.36 (0.19) 1.54 (0.27) 0.77Caudate nucleus 1.42 (0.30) 2.13 (0.81) 1.16Putamen 1.43 (0.30) 1.89 (0.55)* 1.04Thalamus 1.59 (0.23) 1.87 (0.36) 0.93Midbrain 1.88 (0.21) 1.92 (0.22) 0.19White matter 1.84 (0.30) 1.75 (0.38) 0.26

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a Wilcoxon Signed-Ranks test.b Fisher exact test.

At the time of the PET scans, the cognitively declining grouperformed significantly worse on animal fluency tests, and onemory tasks, particularly on the CVLT II. Based on their neu-

ological and neuropsychological re-assessment, three declinersere diagnosed with amnestic MCI, and one with AD. The other

ix cognitively declining subjects remained within the normalange for age on cognitive tests. Results from their neuropsy-hological re-assessment are shown in Table 2.

Clinical diagnosis of cognitively ‘stable’ carried a negativeredictive value of 87% for A� deposition as measured by PiBET. Out of the 34 subjects evaluated, 11 (32%) presentedith cortical PiB binding. Seven (70%) of the ten cognitivelyeclining subjects were PiB+ve, compared with only 4 (17%)f the 24 cognitively stable subjects. PiB showed significantifferences between cognitively stable and declining partici-ants (Table 3), with a Cohen’s effect size (d) for NeocorticalUVR of 1.16 (Fig. 1). The main regions showing PiB retentionere the ventral prefrontal and posterior cingulate/precuneus

ortices (Fig. 2), following the known pattern of A� depositions described in post-mortem studies (Braak & Braak, 1997). Inddition, the percentage of HC with PiB retention increased withvery decade, with 20%, 35% and 50% of the subjects presentingiB+ve scans in the 61–70, 71–80, and 81+ year-old age groups,espectively, in agreement with post-mortem data (Davies et al.,988) (Fig. 3).

As shown in Table 4, for participants with cognitively stableognition, memory performance and word-recall slopes did notorrelate with A� burden for any brain region. In contrast, in theognitively declining group both regional and global neocortical

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able 2europsychological assessment at time of PET scan (means (S.D.) for groups by clas

Stable (n = 24) Declining (n = 10

ADS anxiety 3.58 (2.02) 4.10 (3.38)ADS depression 1.79 (1.61) 3.47 (3.46)VLT-II delayed recall 12.12 (2.56) 6.1 (4.17)CFT copy 31.67 (2.38) 30.10 (3.77)CFT 30 min delay 16.85 (4.59) 11.00 (5.25)igit span total score 17.91 (3.93) 15.90 (3.63)etter fluency 44.62 (11.47) 38.20 (7.77)NT short form 28.20 (1.77) 28.20 (1.68)nimal fluency 22.83 (6.88) 17.40 (2.50)

ADS = Hospital Anxiety and Depression Scale; CVLT-II = California Verbal Learaming Test.a Wilcoxon Signed-Ranks test.

a Neocortex comprises the average SUVR for frontal, parietal, cingulate,ccipital and lateral temporal cortices.* Wilcoxon Signed-Ranks test (p < 0.05).

� burden was highly correlated with memory impairment and

ith word-recall slopes. The greatest correlation in the cogni-

ively declining group was seen in the parietal cortex (ParietaliB SUVR vs. CVLT II delayed recall: r = −0.93, p < 0.0001,nd vs. word-recall slope: r = −0.81, p = 0.0046) (Table 4). A

sification)

) p valuea Reference

0.88 Zigmond and Snaith (1983)0.11 Zigmond and Snaith (1983)

<0.0002 Delis et al. (2000)0.23 Meyers et al. (1996)0.02 Meyers et al. (1996)0.15 Wechsler (1997)0.08 Benton (1968)0.88 Saxton et al. (2000)0.02 Neuropsychological assessment (2004)

ning Test-Second Edition; RCFT = Rey Complex Figure Test; BNT = Boston

1692 V.L. Villemagne et al. / Neuropsychologia 46 (2008) 1688–1697

Fig. 1. Boxplots reflecting Neocortical A� burden, represented by PiB SUVR,in cognitively stable and cognitively declining individuals. Open circles (©)rba*

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epresent PiB+ve scans, while filled circles (�) represent PiB−ve scans. Rhom-oids (♦) and the square (�) indicate subjects that were classified respectivelys MCI and AD at the time of the PiB PET scan. d = Cohen’s effect size (d).p < 0.01 on Wilcoxon Signed-Ranks test.

igh correlation (r = −0.60, p = 0.0003) was observed between� burden and memory impairment as measured by theVLT II when all participants irrespective of clinical classifi-ation were included (Fig. 4). All correlations were calculated

ig. 2. Representative sagittal (left) and transaxial (right) PET images showingegional 11C-PiB retention, reflecting A� burden in the brain, in asymptomaticge-matched PiB+ve and PiB−ve healthy controls (HC), one PiB+ve MCI sub-ect, and AD patient showing the stages of A� deposition in the human brain,

atching previous neuropathological reports (Braak & Braak, 1997). The twoC subjects belonged to the ‘stable’ group.

Fig. 3. Scatterplots showing neocortical A� burden as measured by PiB SUVR(top) and plaque density as measured by immunohistochemistry (bottom, fromDavies et al., 1988) in different age groups. These studies, performed almost20 years apart and using different approaches for the quantification of aggre-gsp

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ated A�, yielded almost identical results. Thirty-two percent of the individualshowed neocortical PiB retention, while 38% of individuals presented withlaques. Dotted line shows threshold between negative and positive individuals.

or non-demented individuals, with exclusion of the ADatient.

. Discussion

Twenty years ago, in a ground-breaking study, the presencend distribution of plaques in aged individuals was examined forhe first time using antibodies against A� (Davies et al., 1988).oday we revisit the same problem with a new approach. And were faced with the same results. And the same questions “To whategree [are] these prevalence rates [. . .] reflected in clinicallyetectable impairment of higher cortical function”? (Davies etl., 1988).

In the same way neuropathology was boosted by the tech-

iques and dyes introduced by visionary pioneers like Cajal andissl (Villemagne et al., 2006), derivatives of those histologi-

al dyes are now finding their way into emission tomographySair, Doraiswamy, & Petrella, 2004; Villemagne et al., 2005).

V.L. Villemagne et al. / Neuropsychologia 46 (2008) 1688–1697 1693

Table 4A� burden vs. memory performance in non-demented subjects (means (S.D.) for groups by clinical classification)

Stable (n = 24) Declining (n = 9)

Slope word verbal recall CVLT II delayed recall Slope word verbal recall CVLT II delayed recall

PiB SUVRNeocortexa 0.25 −0.03 −0.78* −0.85*

Dorsolateral prefrontal 0.22 −0.02 −0.73* −0.82*

Ventrolateral prefrontal 0.25 −0.03 −0.77* −0.86*

Orbitofrontal 0.24 −0.08 −0.81* −0.85*

Gyrus rectus 0.19 −0.02 −0.73* −0.77*

Post cingulate gyrus 0.28 −0.07 −0.73* −0.80*

Ant cingulate gyrus 0.13 0.02 −0.65 −0.78*

Superior parietal 0.31 0.01 −0.81* −0.93*

Lateral occipital 0.11 −0.01 −0.69 −0.72*

Lateral temporal 0.29 −0.03 −0.82* −0.82*

Mesial temporal 0.04 0.07 −0.71* −0.63Caudate nucleus 0.23 −0.09 −0.64 −0.79*

Putamen 0.08 0.02 −0.74* −0.77*

Thalamus 0.29 −0.12 −0.51 −0.59Midbrain 0.24 −0.04 −0.22 −0.22White matter 0.25 −0.03 −0.30 −0.19

CVLT-II = California Verbal Learning Test-Second Edition.ipital

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a Neocortex comprises the average SUVR for frontal, parietal, cingulate, occ* Pearson correlation (p < 0.05).

hile early neuropathological studies raised questions about theeaning of finding markers of AD in non-demented subjects

Braak & Braak, 1997) these evaluations can now be performedn vivo, and neuroimaging findings correlated with cognitiveerformance obtained on the same day.

Despite the limitations associated with retrospective assess-

ents of cognition, PiB retention was highly correlated witheasures of cognitive decline derived from repeated measures

f cognition over many years as well as with a single assess-ent of memory function in the cognitively declining group.

ig. 4. Correlation between A� burden as measured by PiB PET, and memorys measured by CVLT II Delayed Recall in all non-demented individuals. Openircles (©) represent PiB−ve and open crossed circles (⊕) represent PiB+veubjects in the cognitively stable group, respectively. Filled circles (�) represent

iB+ve and filled dotted circles ( ) represent PiB−ve subjects in the cogni-ively declining group, respectively. Rhomboids (♦) indicate subjects that werelassified as MCI at the time of the PiB PET scan.

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and lateral temporal cortices.

ur results show that it is possible to detect subtle cognitiveeterioration in individuals who do not meet clinical criteria forither AD or MCI (McKhann et al., 1984; Petersen et al., 2001),rovided measurements of cognition are conducted regularlyver a sufficient period of time (Collie et al., 2001). For examplehere is evidence of subtle decline in verbal memory in healthylder people whose cognitive performance has been followed athort re-assessment intervals for over 10 years. In the absence ofxplanatory factors (e.g. other CNS disease, injury or infection)r the indirect effects of other medical conditions (e.g. cardio-ascular disease), progressive worsening of cognitive functionn an older individual is highly likely to reflect early demen-ia. The clinical classification as cognitively declining or stableC, based on their evolution over the previous 6–10 years, waserformed up to 18 months prior to the PiB scans and neuropsy-hological re-assessment, hence it is not surprising that furtherognitive decline had occurred in some subjects. Indeed, by theime of the PET scan, four of the ten subjects characterized asognitively declining had converted to MCI or AD and all wereiB+ve. However, of the 6 remaining cognitively declining sub-

ects, only half were also PiB+ve. Those three PiB−ve subjectsuggest that cognitive decline is not synonymous with prodro-al AD. Indeed, one PiB−ve cognitively declining subject hadclinical profile more suggestive of prodromal frontotemporalementia, in which A� imaging is negative (Drzezga et al., 2008;ngler et al., 2008; Rabinovici et al., 2007; Rowe et al., 2007).owever, the reason for clinically apparent deterioration was notbvious in the two remaining PiB−ve cognitively declining sub-ects. Larger series evaluating cognitive decline prospectively

ay address these concerns.Only 17% of the clinically stable group were PiB+ve. It

emains to be determined whether further follow-up in theseiB+ve cognitively stable participants will be associated with

1 psych

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694 V.L. Villemagne et al. / Neuro

ubsequent onset of decline. Nevertheless, clinically determinedtable cognition appears a reasonable (87%) predictor of nega-ive PiB status and lower risk of AD.

ApoE �4 is the primary genetic risk factor associated withporadic AD (Martins et al., 2006; Ritchie & Dupuy, 1999).nly 19% of the cognitively stable group were ApoE �4 carriers,

nd PiB retention was higher in ApoE e4 carriers, confirmingpoE’s involvement in the economy of A� deposition/removal,anifested in higher A� burdens.FDG PET has been shown to have prognostic value in

resymptomatic AD two or more years before the full demen-ia picture is manifested (Chang & Silverman, 2004; Chetelatt al., 2003; Silverman et al., 2001), and to be more specifichan neuropsychological evaluation in amnestic MCI subjectsChetelat et al., 2005). Comparison of PiB and FDG resultsn our well-characterized population will help elucidate therognostic value of each approach, or the combination of bothpproaches in HC. While the sensitivity and specificity of PiBgainst neuropathology remains unknown, a few cases reportedn the literature showed a high correlation between the PET sig-al and brain A� measured by ELISA (Bacskai et al., 2007;eKosky et al., 2007). Conversely PiB images have a higher

ccuracy than FDG images for distinguishing AD from HC.he sensitivity and specificity of PiB for clinically probableD is very high with a reported accuracy of 90% (S. Ng et

l., 2007) Both PiB+ve MCI and HC subjects showed few oro deficits in FDG in comparison to PiB retention, suggestinghat A� toxicity and deposition precede the failure of com-ensatory mechanisms against oxidative stress that eventuallyeads to synaptic and neuronal dysfunction, manifested as cog-itive impairment and glucose hypometabolism (Eckert et al.,003; Leuner et al., 2007; Suo, Wu, Citron, Wong, & Festoff,004). This may also be associated or attributed to a differentusceptibility/vulnerability to A�, not only at a cellular levelfrontal neurons seems to be more resistant to A� than hip-

ocampal ones (Resende et al., 2007; Roder et al., 2003) – butlso at a individual or personal level, either due to a particu-ar cognitive reserve (Mortimer, 1997; Stern, 2002) or becausen idiosyncratic threshold must be exceeded for synaptic fail-re and neuronal death to ensue (Suo et al., 2004). That wouldxplain why some older individuals with very high A� burdensre cognitively unimpaired while others with lower A� burdensnd no genetic predisposing factors have already developed theull clinical AD phenotype. In HC, the prognostic value of aiB−ve scan may have more profound clinical connotations

han a PiB+ve one, because the absence of cortical deposits of� indicates that the development of AD in the near future isighly unlikely. Longitudinal studies are still necessary to elu-idate the overall positive and negative predictive value of A�maging.

In the cognitively declining group, A� burden was corre-ated not only with word-recall slopes but also with memorympairment in almost every region examined. Though extracel-

ular amyloid plaques are the hallmark brain lesions of AD, theistribution and density of A� plaques at the light microscopicevel have not been consistently shown to correlate with theegree of cognitive impairment (Cummings, Pike, Shankle, &

ote&

ologia 46 (2008) 1688–1697

otman, 1996; Greenberg, Rebeck, Vonsattel, Gomez-Isla, &yman, 1995; McLean et al., 1999; Mega et al., 1999). Theest correlation occurs with soluble levels of A�, measured bio-hemically (Lue et al., 1999; McLean, Beyreuther, & Masters,001; McLean et al., 1999; Naslund et al., 2000; Wang, Dickson,rojanowski, & Lee, 1999). There is also no correlation between

nsoluble A� and either clinical onset of dementia or clinicaliagnosis of AD or MCI (Forman et al., 2007). This is in contrastith the correlation found between episodic memory impair-ent and measures of A� burden obtained through PiB PET

maging in subjects with MCI (Forsberg et al., 2007; Pike etl., 2007). One explanation posits that the time between theast cognitive assessment and the brain examination is suffi-iently protracted to erase or diminish any correlation, but theatest reports have followed subjects very closely until theireaths with comprehensive annual evaluations (Bennett, 2006).mall associations were found in this study, but large cohortsere needed in order to make these associations meaningful

Bennett, 2006). While detectable changes in amyloid burdens measured with PiB PET seems to take a long time, prob-bly reflecting the rate of amyloid deposition (Engler et al.,006; Price & Morris, 1999), the toxic effects of A� oligomersn synapses seems to be much faster (Calabrese et al., 2007;alsh & Selkoe, 2004), and limited by a very short half-life

Cirrito et al., 2003; Ghiso et al., 2004). Furthermore, both pro-esses are at equilibrium so the aggregated A� in the plaqueslso serves as a reservoir for the oligomers (Cirrito et al., 2003;athis, Lopresti, & Klunk, 2007; McLean et al., 1999). Inter-

ction of soluble A� with synaptically released biometals suchs zinc and copper (Qian & Noebels, 2005; Schlief & Gitlin,006) is believed to trigger oxidative stress while promotinghe formation of soluble SDS-resistant oligomers and subse-uent aggregation, leading to neuronal and synaptic toxicity inD (Bush, 2003; Frederickson & Bush, 2001; Maynard, Bush,asters, Cappai, & Li, 2005). Additionally, recent studies have

hown that PiB preferably binds one kind of the N-terminalruncated A�1–42(43) species in senile plaques (Maeda et al.,007). This is relevant because this A� species is associatedith accelerated formation of plaques (Harigaya et al., 2000;chilling et al., 2006). An alternative explanation is that PiBinds to soluble A� oligomers or other misfolded proteins in therain. PIB does not bind to NFTs (Klunk et al., 2003; Lockhartt al., 2007) nor Lewy bodies (Fodero-Tavoletti et al., 2007) athe concentrations achieved during PET studies, so it is unlikelyhey contribute to the PiB PET signal. Similarly, A� solublepecies represent less than 1% of the total brain A� (McLeant al., 1999), and the affinity of PiB for oligomers is lower thanor A� fibrils (Maezawa et al., 2008), rendering negligible theligomers’ contribution to the observed PiB PET signal (Mathist al., 2007).

The demonstration of A� deposits as evidenced by PiB bind-ng in a proportion of healthy control subjects is in agreementith post-mortem reports that at least 25% of non-demented

lder persons over the age of 75 have amyloid plaques, andhat these deposits occur before the onset of dementia (Daviest al., 1988; Forman et al., 2007; Morris & Price, 2001; Price

Morris, 1999). This prevalence of plaques in non-demented

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V.L. Villemagne et al. / Neuro

lder people is also equivalent to the prevalence of dementiat age 85 (Ferri et al., 2005), suggesting that neuropathologicalhanges precede the clinical expression of AD by many yearsBennett et al., 2006; Price & Morris, 1999). Furthermore, aecent report of follow-up of AD patients over 2 years (Englert al., 2006) demonstrated no change in A� burden measured inivo with PET, providing further evidence that A� deposition inatients already diagnosed with AD is either a very slow pro-ess or reaches a plateau. Given the prevalence of AD and itslow and insidious nature, it is highly likely that PiB uptake inon-demented individuals reflects preclinical AD. The presenttudy provides evidence to support this opinion.

Additionally, the findings reported here are extremely rele-ant given that the cognitive evaluation had been in progress foreveral years, highlighting the robust nature of the findings intherwise healthy subjects with unexplained cognitive decline,ecline which was not only reflected in 40% conversion to eitherCI or AD, but also by amyloid deposition in 70% of the indi-

iduals. Furthermore, a clinical diagnosis of ‘stable’ based onepeated longitudinal evaluations, seems to predict the absencef A� deposition. This well characterized group of healthy olderndividuals therefore provides an ideal model for early detectionf AD and, in combination with other biomarkers such as amy-oid imaging, may assist in the evaluation of disease-modifyingrugs.

In conclusion, declining subjects are much more likely tohow cortical PiB retention than stable subjects. A� burden asssessed by PiB PET correlates with memory impairment andhe rate of memory decline in the non-demented ageing pop-lation. These observations suggest that A� deposition is notart of normal ageing and is likely to represent preclinical AD.urther longitudinal observations are required to confirm thisypothesis.

cknowledgements

Supported in part by funds from the Austin Hospital Medicalesearch Foundation, Neurosciences Victoria, and the Univer-

ity of Melbourne.We thank Jessica Sagona, Kunthi Pathmaraj, Tim Saunder,

lare Smith, Bridget Chappell, and Jason Bradley for their cru-ial role during PET examinations and image processing.

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