Author Affiliations: Laboratory of Personality and Cognition, National Institute on Aging, National Institutes of Health (Drs Thambisetty, Beason-Held, and Resnick), MedStar Research Institute (Mr An), and Department of Radiology and Radiological Sciences, Johns Hopkins University School of Medicine (Dr Kraut), Baltimore, Maryland.
To study differences in longitudinal changes in regional cerebral blood flow (rCBF) between apolipoprotein E (APOE) ε4 carriers and noncarriers in nondemented older adults from the Baltimore Longitudinal Study of Aging using positron emission tomography in order to determine whether there are regionally specific longitudinal changes in rCBF in APOE ε4 carriers that might be related to its well-established role as a genetic risk factor for Alzheimer disease.
Design, Setting, and Participants
Using oxygen 15 ([15O])–labeled water positron emission tomography and voxel-based analysis, we compared changes in rCBF over an 8-year period between 29 nondemented APOE ε4 carriers and 65 noncarriers older than 55 years. Serial neuropsychological data were collected for all participants.
Widespread differences were observed in longitudinal change in rCBF between ε4 carriers and noncarriers. The predominant pattern was greater rCBF decline in ε4 carriers. These differences were observed in the frontal, parietal, and temporal cortices. The affected brain regions were those especially vulnerable to pathological changes in Alzheimer disease. Both ε4 carriers and noncarriers remained free of clinical diagnoses of dementia or mild cognitive impairment during the course of the study.
Our findings suggest that APOE ε4–mediated risk for Alzheimer disease is associated with widespread decline in rCBF over time that precedes the onset of dementia. Accelerated rates of decline in brain function in APOE ε4 carriers may contribute to an increased risk for Alzheimer disease and a younger age at onset.
Numerous epidemiological studies as well as recent genome-wide association studies have established that the apolipoprotein E (APOE [GenBank AF261279]) ε4 allele is a risk factor for Alzheimer disease (AD), increasing the risk by 3- to 8-fold and lowering the age at onset by 7 to 15 years in a dose-dependent manner.1- 3 The ε4 allele is associated with integral features of AD neuropathology, including neuritic plaques and neurofibrillary tangles.4 Moreover, it confers a greater risk of conversion to AD in subjects with mild cognitive impairment.5 However, despite overwhelming evidence linking it with both incipient and established AD, the precise mechanism by which APOE ε4 mediates increased risk for AD is poorly understood.
Functional neuroimaging methods have been applied to study changes in brain function in APOE ε4 carriers. Cross-sectional studies in healthy young, middle-aged, and elderly APOE ε4 carriers have reported reductions in both regional cerebral blood flow (rCBF) and regional cerebral glucose metabolism in several brain regions, including those especially vulnerable to pathological changes in AD.6 Cross-sectional studies, however, are limited in their ability to address the effects of an AD risk factor over time. Longitudinal functional neuroimaging studies in healthy elderly individuals are especially advantageous as they examine brain function at 2 or more discrete times and can thereby address the role, if any, of the ε4 allele in increasing disease risk over time. However, results from such studies to date in cognitively normal ε4 carriers have been somewhat inconsistent. These inconsistent findings may be due to the fact that some of these studies have been carried out in middle-aged subjects7 and that in the limited number of longitudinal studies carried out in elderly subjects, ε4-related effects were observed in only relatively few subjects with accompanying cognitive decline and/or age-associated memory impairment.8,9
Longitudinal neuroimaging studies on the influence of APOE genotype on changes in brain function in cognitively normal elderly individuals are valuable as differences in brain metabolism or rCBF have the potential to be useful surrogate markers of AD in preclinical studies. Such predictive biomarkers of AD have considerable potential value in clinical practice as well as research, where they may help accelerate the development of novel disease-modifying treatments. In both the United States and Europe, public and private consortia have been formed to conduct trials to discover such antecedent biomarkers.10,11
Here we report associations between APOE genotype and longitudinal changes in rCBF in nondemented older adults in the Baltimore Longitudinal Study of Aging. We hypothesized that APOE ε4 carriers will exhibit significant changes in rCBF over time in brain regions vulnerable to AD pathological changes in comparison with noncarriers.
We used positron emission tomographic (PET) data from 94 participants in the neuroimaging substudy12 of the Baltimore Longitudinal Study of Aging.13 We excluded individuals with intracranial tumors and clinical strokes. Data from participants who fulfilled consensus criteria (National Institute of Neurological and Communicative Disorders and Stroke–Alzheimer's Disease and Related Disorders Association criteria) for AD14 and those with mild cognitive impairment were excluded from the time of diagnosis. Neuropsychological and PET data from these participants were included from baseline through the time preceding the diagnosis of AD or mild cognitive impairment. A diagnosis of mild cognitive impairment was assigned by consensus conference if a participant either had deficits in a single cognitive domain (usually memory) or had more than 1 cognitive deficit but did not have functional loss in activities of daily living.
To examine the cardiovascular risk profile of participants in the 2 groups, we calculated the Framingham risk score for each subject at baseline to derive a 10-year risk profile for coronary heart disease.15 This composite score of cardiovascular risk was based on the presence of the following specific risk factors: age, total serum cholesterol concentration, hypertension, diabetes mellitus, and smoking. A positive family history of dementia was defined as 1 or more first-degree relatives with a clinical diagnosis of dementia at the time of entry into the study.
This study was approved by the local institutional review board. All participants provided written informed consent prior to each assessment.
APOE genotype analysis was performed on DNA extracted from fresh blood by restriction enzyme isoform genotyping in all participants.16 The 2 groups were defined as APOE ε4 carriers (both heterozygous [n = 22] and homozygous [ie, ε4/ε4; n = 7]) and noncarriers (n = 65). Annual neuropsychological data were available for all participants. Data from imaging at baseline (year 1) and at the last available follow-up were used in the analysis. The mean (SD) follow-up interval was 7.8 (1.1) years. The demographic characteristics of the participants are detailed in Table 1.
During each neuroimaging visit, participants completed a battery including 12 neuropsychological tests evaluating 6 cognitive domains. Memory was assessed using the California Verbal Learning Test and the Benton Visual Retention Test. Word knowledge and verbal ability were measured using Primary Mental Abilities vocabulary test. Verbal fluency was assessed by letter (ie, FAS) and category fluency tests. Attention and working memory were measured by the Digit Span Test of the Wechsler Adult Intelligence Scale–Revised and the Trail Making Test. Digits backward, Trail Making Test part B, and verbal fluency (categories and letters) assessed executive function. The Card Rotation Test assessed visuospatial function. Data from evaluations at baseline and the last follow-up were used to examine differences in change in performance over time between the 2 groups.
Participants underwent PET scans at baseline (year 1) and up to 8 annual follow-ups. Each imaging session included a resting scan in which participants were instructed to keep their eyes open and focused on a computer screen covered by a black cloth. Participants also underwent PET scans during verbal and figural recognition memory tasks. Scan order was counterbalanced but remained constant over repeated assessments. For comparability with previous literature, we present analyses of the resting state PET data.
The PET measurements of rCBF were obtained using oxygen 15 ([15O])–labeled water. For each scan, 75 mCi of [15O]water was injected as a bolus (to convert to becquerel, multiply by 3.7 × 107). Scans were performed on a GE 4096 Plus scanner (GE Healthcare, Waukesha, Wisconsin), which provides 15 slices of 6.5-mm thickness. Images were acquired for 60 seconds from the time at which the total radioactivity counts in the brain reached threshold level. Attenuation correction was performed using a transmission scan acquired prior to the emission scans.
A 3-dimensional spoiled gradient refocused magnetic resonance imaging scan (124 slices; 256 × 256 matrix; 0.94 × 0.94-mm voxel size; 1.5-mm slice thickness) was obtained on a 1.5-T GE Signa scanner (GE Healthcare) at each imaging visit.
Data from PET scans obtained at baseline and the last available follow-up were used in the analyses. The mean interval between PET scans at baseline and the last follow-up did not differ significantly between ε4 carriers (7.8 years) and noncarriers (7.7 years). The PET scans were realigned and spatially normalized into standard stereotactic space and smoothed to full width at half maximum of 12 × 12 × 12 mm in the x, y, and z planes. To control for variability in global flow, rCBF values at each voxel were ratio adjusted to the mean global flow and scaled to 50 mL/100 g/min for each image. The image data were analyzed using Statistical Parametric Mapping 2 software (Wellcome Department of Cognitive Neurology, London, England).
To examine the differences in longitudinal rCBF change between ε4 carriers and noncarriers, voxel-based group × time interactions for rCBF change between scans at baseline and the last follow-up were determined. Age at baseline and sex were included as covariates, and significant effects for each contrast were based on the magnitude of activation (P ≤ .005) and spatial extent (>50 voxels). For those regions showing differences in longitudinal changes in rCBF between ε4 carriers and noncarriers, a region-of-interest analysis was performed to quantify both baseline rCBF as well as the magnitude of change in rCBF over time. The region-of-interest analysis was performed by extracting the adjusted rCBF values at the local maxima for each region using a 4-mm spherical search area.
Additional analyses were performed to control for the effects of potential differences in tissue volume between the 2 groups that could account for differences in rCBF changes. The magnetic resonance imaging scans were segmented into gray matter, white matter, and cerebrospinal fluid and spatially normalized into stereotactic space using a high-dimensional elastic warping method and a volume-preserving transformation.17 For each participant, binary maps of the regions showing significant differences in rCBF over time between ε4 carriers and noncarriers were registered with the magnetic resonance image. Total volumes of gray + white matter were subsequently calculated for each region. The PET data were then reanalyzed using the total tissue volumes (gray + white matter) of these regions as additional covariates. Total gray + white matter volume could not be calculated for 1 region in the right inferior temporal cortex owing to a registration error.
APOE ε4 carriers tended to be slightly younger than noncarriers (P = .06). There were no significant differences in sex distribution between the 2 groups. There were also no significant differences between the 2 groups in educational status or Mini-Mental State Examination scores at either baseline or the last follow-up (Table 1). There was no significant difference in the cardiovascular risk profile between ε4 carriers and noncarriers as determined by the composite Framingham risk score for each group. There was no significant intergroup difference in the proportion of subjects with a family history of dementia. APOE ε4 carriers tended to perform slightly worse than noncarriers in the Trail Making Test part A cognitive task after controlling for age and sex (mean [SD], 35.17 [1.88] vs 30.59 [1.26] seconds, respectively; P = .05). There were no significant differences at baseline between the groups on any other cognitive task.
For each task or cognitive domain, a linear mixed-effects model adjusting for sex and baseline age was used to compare changes in performance from baseline to the last follow-up for ε4 carriers vs noncarriers (Table 2). APOE ε4 noncarriers showed significantly greater decline than carriers in category fluency over time (P = .02). There were no significant intergroup differences in performance over time on any other cognitive task.
Analysis of group differences in longitudinal change from baseline to the last follow-up revealed several regions of rCBF declines that differed significantly between ε4 carriers and noncarriers (Table 3 and Figure). The brain regions exhibiting significantly greater rCBF declines over time in ε4 carriers included the right orbitofrontal cortex (Brodmann area [BA] 47), left middle (BA 10) and inferior (BA 47) frontal gyri, left middle (BA 20) and superior (BA 22) temporal gyri, right inferior temporal cortex (BA 20), and right superior parietal cortex (BA 7). A significantly greater increase in rCBF in the right insula was observed in ε4 carriers relative to noncarriers. The region-of-interest analysis in these regions showed that the magnitude of the change in rCBF over time in ε4 carriers ranged from 2% to 6% relative to baseline. We also observed significantly greater baseline rCBF in APOE ε4 carriers in each of these regions (Table 3).
Figure. Differences in longitudinal change in regional cerebral blood flow between APOE ε4–positive and APOE ε4–negative groups. Blue areas indicate significantly greater longitudinal decreases in regional cerebral blood flow in APOE ε4 carriers; green areas, greater longitudinal increases in regional cerebral blood flow in APOE ε4 carriers; red line, the z level of the representative slice shown; R, right; and L, left.
These longitudinal differences in rCBF between the 2 groups were largely preserved after correcting for tissue volume in each of the regions noted, with the exception of the left superior temporal gyrus. The observed decline in rCBF in this region in ε4 carriers compared with noncarriers was no longer significant after correcting for the corresponding regional tissue volume.
Our principal objective was to examine differences in changes in rCBF over time in ε4 carriers vs noncarriers in nondemented older individuals. Consistent with our hypothesis, longitudinal changes in rCBF differed between ε4 carriers and noncarriers in several brain regions that are especially vulnerable to pathological changes in AD. Carriers of the APOE ε4 allele showed greater longitudinal declines in rCBF in the frontal, parietal, and temporal cortices. These results, based on the largest sample of older adults studied to date, demonstrate that there indeed are widespread longitudinal differences in rCBF change between older ε4 carriers and noncarriers. Furthermore, we demonstrate that most of these differences in brain function over time were evident even after adjusting for differential loss of brain tissue.
Studies in middle-aged individuals have indicated accelerated decline in regional cerebral glucose metabolism in APOE ε4 carriers.7 For example, Reiman et al7 used fluorodeoxyglucose F18 PET in late- to middle-aged subjects (10 ε4 heterozygotes and 15 ε4 noncarriers) with at least 1 first-degree relative with probable AD and showed significantly greater decline in regional cerebral glucose metabolism over 2 years in ε4 carriers. The regions showing the greatest declines in ε4 carriers were bilateral temporal, occipitotemporal, and prefrontal cortices as well as the right hippocampus and left fusiform gyrus.
However, previous longitudinal PET studies on the effect of APOE genotype on cerebral metabolism in older adults have been inconclusive.8,9 In the few studies that have demonstrated APOE genotype–related changes in regional cerebral glucose metabolism over time in older people, such effects were observed in only relatively few subjects with a family history of AD or in those who underwent significant cognitive decline over the follow-up period. Using fluorodeoxyglucose F18 PET, de Leon et al8 studied 12 individuals older than 70 years who underwent significant cognitive decline over a period of 3 years. Using a region-of-interest approach, they observed a significantly greater decline in regional cerebral glucose metabolism in the lateral temporal lobe (averaged across hemispheres) in ε4 carriers compared with noncarriers. However, using a similar approach in an older sample of 10 ε4 carriers and 10 ε4 noncarriers with both age-associated memory impairment and a family history of AD, Small et al9 found no significant group differences in longitudinal change in regional cerebral glucose metabolism.
The predominant pattern of differences in rCBF over time in our study is one of greater decline in ε4 carriers. We also confirmed that ε4-associated changes in rCBF were largely independent of changes in tissue volume between the 2 groups. Previous PET studies on the effect of APOE genotype on longitudinal changes in brain function have not corrected for differences in tissue volume in brain regions showing significant changes in ε4 carriers.7- 9 These additional volumetric analyses are especially important in light of previous data showing atrophic changes in s everal brain regions in cognitively normal ε4 carriers.18- 20 Although our study does not address the precise mechanisms underlying the observed APOE genotype–associated changes in rCBF over time, we suggest that the widespread decline in rCBF in ε4 carriers might represent regional patterns of neuronal vulnerability in subjects at risk for AD. It is likely that these changes reflect declining brain function resulting from multiple biological effects mediated by the APOE ε4 allele. These may include impaired brain repair mechanisms in ε4 carriers21 rendering specific regions differentially vulnerable to the deleterious effects of environmental risk factors. It is interesting that we observed higher baseline rCBF values in APOE ε4 carriers in regions exhibiting significant longitudinal declines in rCBF. It is plausible that these increments in baseline rCBF represent compensatory mechanisms in APOE ε4 carriers. These findings are in partial agreement with earlier studies using [15O]water PET to study rCBF in young adult APOE ε4 carriers.22 The observed longitudinal changes in rCBF in this study may also reflect, to a certain extent, early neuropathological changes such as β-amyloid deposition23 or neurofibrillary degeneration24 in ε4 carriers. Some ε4-associated changes in rCBF observed in our study are distributed in brain regions that are particularly vulnerable to pathological changes in AD such as temporal cortex and bilateral frontal lobes. Moreover, the patterns of rCBF decline show some regional overlap with the distribution of β-amyloid deposition measured with carbon 11 ([11C])–labeled Pittsburgh Compound B PET in both normal aging and AD.25
It is important to note that in this relatively healthy sample excluding individuals with cognitive impairment, we did not detect significant differences in memory decline in ε4 carriers compared with noncarriers. APOE ε4–associated cognitive decline in nondemented subjects is reported to be especially prominent in those with low educational levels.26 As the Baltimore Longitudinal Study of Aging comprises a volunteer sample with an unusually high level of education,27 it is possible that cognitive reserve protects them from objective memory problems until later in the course of a preclinical disease process. Furthermore, participants are rigorously evaluated during study visits to detect both clinical and occult cardiovascular disease.28 Many participants in our study undergo early treatment and lifestyle-based interventions against established vascular risk factors for dementia such as diabetes, hypertension, and hyperlipidemia. Taken together, these factors may have contributed to the absence of cognitive decline in ε4 carriers in our study, perhaps by modifying the preclinical course of disease.
In summary, we used [15O]water PET to demonstrate greater longitudinal decline in rCBF in cognitively intact, older subjects carrying the APOE ε4 allele compared with noncarriers. These results suggest that APOE ε4–mediated risk for AD is associated with widespread changes in rCBF over time and that these changes precede the development of cognitive decline. These findings have potential for utility as neuroimaging markers of longitudinal changes in brain function in subjects at risk for AD as well as for monitoring response to novel disease-modifying treatments in clinical trials.
Correspondence: Madhav Thambisetty, MD, PhD, Laboratory of Personality and Cognition, National Institute on Aging, National Institutes of Health, 251 Bayview Blvd, 4B-311, Baltimore, MD 21224-6825 (firstname.lastname@example.org).
Accepted for Publication: May 21, 2009.
Author Contributions:Study concept and design: Thambisetty, Beason-Held, and Resnick. Acquisition of data: Thambisetty, Beason-Held, and Resnick. Analysis and interpretation of data: Thambisetty, Beason-Held, An, Kraut, and Resnick. Drafting of the manuscript: Thambisetty, Beason-Held, An, and Resnick. Critical revision of the manuscript for important intellectual content: Thambisetty, Kraut, and Resnick. Statistical analysis: Beason-Held, An, and Resnick. Obtained funding: Kraut and Resnick. Administrative, technical, and material support: Resnick. Study supervision: Thambisetty and Resnick.
Financial Disclosure: None reported.
Funding/Support: This work was supported in part by research and development contract N01-AG-3-2124 from the Intramural Research Program, National Institute on Aging, National Institutes of Health and by a research and development contract with MedStar Research Institute.
Additional Contributions: We are grateful to the Baltimore Longitudinal Study of Aging participants and neuroimaging staff for their dedication to these studies and the staff of the Johns Hopkins University PET facility for their assistance.
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