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Age and Apolipoprotein E*4 Allele Effects on Cerebrospinal Fluid -Amyloid 42 in Adults With Normal Cognition

Elaine R. Peskind, MD; Ge Li, PhD, MD; Jane Shofer, MS; Joseph F. Quinn, MD; Jeffrey A. Kaye, MD; Chris M. Clark, MD; Martin R. Farlow, MD; Charles DeCarli, MD; Murray A. Raskind, MD; Gerard D. Schellenberg, PhD; Virginia M.-Y. Lee, PhD; Douglas R. Galasko, MD

Arch Neurol. 2006;63:936-939.

ABSTRACT
Background  Decreased cerebrospinal fluid (CSF) -amyloid 42 (A₄₂) concentration, but not A₄₀ concentration, is a biomarker for Alzheimer disease. This A₄₂ concentration decrease in CSF likely reflects precipitation of A₄₂ in amyloid plaques in brain parenchyma. This pathogenic plaque deposition begins years before the clinical expression of dementia in Alzheimer disease. Normal aging and the presence of the apolipoprotein E (APOE*4) allele are the most important known risk factors for Alzheimer disease.

Objective  To estimate the interactive effects of normal aging and presence of the APOE*4 allele on CSF A₄₂ concentration in adults with normal cognition across the life span.

Design  The CSF was collected in the morning after an overnight fast using Sprotte 24-g atraumatic spinal needles. The CSF A₄₂ and A₄₀ concentrations were measured in the 10th milliliter of CSF collected by sandwich enzyme-linked immunosorbent assay. The APOE genotype was determined by a restriction digest method.

Subjects  One hundred eighty-four community volunteers with normal cognition aged 21 to 88 years.

Results  The CSF A₄₂, but not the A₄₀, concentration decreased significantly with age. There was a sharp decrease in CSF A₄₂ concentration beginning in the sixth decade in subjects with the APOE*4 allele. This age-associated decrease in CSF A₄₂ concentration was significantly and substantially greater in subjects with the APOE*4 allele compared with those without the APOE*4 allele.

Conclusion  These CSF A₄₂ findings are consistent with acceleration by the APOE*4 allele of pathogenic A₄₂ brain deposition starting in later middle age in persons with normal cognition.

INTRODUCTION

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Lower concentration of a long form of the -amyloid protein ending at amino acid 42 (A₄₂) in cerebrospinal fluid (CSF) is a consistent biomarker of Alzheimer disease (AD).^1-2 In contrast, CSF A₄₀ concentration is unaffected by AD. Aging and presence of theapolipoprotein E*4 (APOE*4) allele are the 2 strongest risk factors for AD. The presence of the APOE*4 allele interacts with aging to lower by 10 to 15 years the age of clinical dementia onset.³ Neuropathological studies have demonstrated that years before the onset of clinical dementia, plaques of insoluble aggregated A protein, the neuropathological hallmark of AD, begin to accumulate in brain tissue.⁴ The A peptide is generated by proteolytic processing of the amyloid precursor protein into a series of fragments 38 to 42 amino acids long; A₄₂ represents about 10% of synthesized A but is by far the earliest and predominant A species deposited in plaques in AD.⁵ Decreased concentration of A₄₂ in CSF is a consistent biomarker of AD^1-2 and likely reflects deposition of A₄₂ in plaques.⁶ This A deposition is considered central to the pathogenesis of AD. In transgenic mice that overexpress mutant amyloid precursor protein, A₄₂ deposition in the brain increases with age and parallels a decrease in CSF A₄₂ concentration.⁷ Animal and human neuropathology studies suggest that the presence of the APOE*4 allele hastens AD onset by modulating the deposition and clearance of A to favor the formation of plaques.⁸

Herein, we estimated in adults with normal cognition the effects of age and APOE genotype on biomarkers related to the deposition in brain of A. Specifically, we determined CSF A₄₂ concentration and APOE genotype in 184 healthy adults without dementia across a broad age range. We also measured CSF A₄₀ concentration to determine whether effects on CSF A₄₂ levels are selective. We hypothesized that CSF A₄₂ concentration would begin to decline years before the age at which clinical AD commonly presents; that this decline would be substantially accentuated by the presence of the APOE*4 allele; and that neither age nor presence of the APOE*4 allele would affect CSF A₄₀ concentrations.

METHODS

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All procedures were approved by the institutional review boards of the participating institutions; all subjects provided written informed consent. Ninety-four men and 90 women (age range, 21-88 years [mean ± SD age, 50 ± 20 years]) underwent detailed clinical and laboratory evaluation and had no clinically significant abnormalities. Subjects had Mini-Mental State Examination⁹ scores between 26 and 30 (mean ± SD, 28.8 ± 1.5), Clinical Dementia Rating Scale¹⁰ scores of 0, and no evidence or history of cognitive or functional decline. For subjects older than 50 years, scores on delayed recall were higher than a cutoff of 1.5 SD lower than age-adjusted means (for both the Logical Memory¹¹ and New York University paragraph tests¹²). We collected CSF in the morning after an overnight fast using Sprotte 24-g atraumatic spinal needles. Samples with more than 500 red blood cells per milliliter were excluded. Samples were frozen immediately on dry ice and stored at –80°C until assay. The A₄₂ and A₄₀ concentrations were measured in the 10th milliliter of CSF collected using sensitive, well-validated sandwich enzyme-linked immunosorbent assays.¹³ The APOE genotypes were performed by a restriction digest method.^14-15

We examined the relationship between A₄₂ (or A₄₀) concentration and age and APOE genotype using a linear regression model of A₄₂ (or A₄₀) concentration on age and APOE*4 status. Age was modeled as an orthogonal quadratic polynomial to separate out linear from quadratic trends in the A peptide–age relationship. Interaction with APOE*4 status was also modeled to determine if trends differed by presence or absence of the APOE*4 allele. To assess selectivity of changes in A₄₂ concentration, we also examined if the ratio of A₄₂-A₄₀ concentration was influenced by age and the APOE*4 allele. The ratio was modeled directly as a dependent variable on age and APOE*4 status and, indirectly, using a regression of A₄₂ concentration on age and APOE*4 status with A₄₀ concentration as an additional covariate. Because A₄₀ and A₄₂ are both produced by cleavage from amyloid precursor protein by and secretase, there should be a fixed ratio of secretion of these forms of A into CSF. We hypothesized that different disposition of these molecules would be demonstrated as changes in the ratio of A₄₂-A₄₀ concentration.

RESULTS

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Lower levels of A₄₂ were associated with older age and with presence of the APOE *4 allele. There was a significant difference in the relationship between age and A₄₂ concentration by APOE*4 allele status (interaction between age and APOE*4 status, P = .01). The relationship for APOE*4-positive subjects was strongly linear with A₄₂ concentration decreasing as age increased. This linear relationship was absent for APOE*4-negative subjects. The quadratic component of the age term indicated that there was a change in the trend in the relationship between age and A₄₂ concentration for both groups but the shape of the curves differed markedly (Figure, A). For APOE*4-negative subjects, mean A₄₂ concentration rose slightly until approximately age 50 years, then fell slightly with increasing age. In contrast, for APOE*4-positive subjects, A₄₂ concentration declined slightly in younger subjects, then declined rapidly beginning between ages 50 and 60 years. Graphical assessment confirms that changes in trend occurred between ages 50 to 60 years for both APOE*4-positive and APOE*4-negative subjects, but the slope of subsequent CSF A₄₂ concentration decline was markedly greater in the APOE*4-positive subjects.

View larger version (26K): [in this window] [in a new window] [as a PowerPoint slide]   Figure. Cerebrospinal fluid (CSF) -amyloid 42 (A42) (A) and A40 (B) concentrations by age and apolipoprotein E (APOE*4) allele status in 184 normal adults aged 21 to 88 years. Closed triangles represent APOE*4-positive subjects; A = Loess-fitted line for APOE*4-positive subjects. Open circles represent APOE*4-negative subjects; B = Loess-fitted line for APOE*4-negative subjects.

There was also a significant interaction between age and APOE*4 in the regression model of A₄₀ concentration (P = .02). In contrast to A₄₂ concentration, A₄₀ concentration did not change with age in APOE*4-positive subjects (P = .68) and increased linearly with age in APOE*4-negative subjects (P<.001), with neither relationship having a significant quadratic component (Figure, B). Regression of the ratio of A₄₂-A₄₀ concentration showed a significant trend with age (P<.001); the ratio values were stable until about age 50 years, then the ratio decreased sharply as age increased (data not shown). This decrease was stronger for APOE*4-positive subjects, but this difference was not quite statistically significant (interaction between age and APOE*4 status, P = .13). These results were confirmed by a regression of A₄₂ concentration on age and APOE*4 status, adjusting for A₄₀ concentration.

We also examined whether CSF A₄₂ levels were related to scores on delayed recall tests and on the Mini-Mental State Examination, using partial correlation controlling for age. None of these test scores showed a significant association at P<.05.

COMMENT

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A previous study that measured CSF A₄₂ concentration found a nonlinear relationship with age,¹⁶ while another showed no relationship with age.¹⁷ However, in these previous studies, APOE genotypes were not determined and characterization to assure that subjects were cognitively normal was minimal. Studies in which APOE genotyping was performed have demonstrated decreased CSF A₄₂ concentration in APOE*4-positive vs APOE*4-negative subjects. No age x APOE*4 interactions were seen, but the age ranges of subjects in these studies were limited (mean ± SD age, 59 ± 8 years¹⁸ and 58 ± 7 years¹⁹) and young subjects were not included. To our knowledge, the present study is the only study that examined effects of the APOE*4 allele across a broad age range. It is also the only study, to our knowledge, that measured both A₄₀ as well as A₄₂ concentrations to assess the specificity of these effects on the more pathogenic 42 amino acid–length A species. Our results suggest that the APOE*4 allele is associated with selective alteration of the fate of A₄₂, but not A₄₀, that results in decreasing concentration of A₄₂ in CSF during the normal adult life span. In persons with the APOE*4 allele, decline in CSF A₄₂ concentration possibly begins in young adulthood, followed by marked acceleration of this decline beginning in midlife—decades before clinical manifestations of AD. -amyloid 40, although also found in amyloid plaques, did not show the same association of decreased CSF concentration with age and presence of the APOE*4 allele. Because A₄₂ and A₄₀ are produced by the same secretases, the concentrations of these forms of A are initially in equilibrium. Our results are therefore consistent with differential aggregation and deposition in the brain, clearance, or binding to carrier molecules that selectively affects A₄₂. These results do suggest that sequestration of A₄₂ in the brain occurs early in APOE*4 allele carriers, bolstering evidence for this as a key initiating factor in AD pathogenesis. Measuring A concentration in CSF provides an indirect estimation of the net effect of production, clearance, aggregation, and deposition; therefore, we cannot determine which of these factors is most important. Our results are consistent with the findings of a recent study of asymptomatic adult carriers of presenilin mutations who had low levels of CSF A₄₂, lending further support to a decrease in CSF A₄₂ concentration as a preclinical biomarker for AD.²⁰

CONCLUSIONS

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These findings have implications for the preclinical diagnosis of AD, as well as for treatment. Follow-up of cohorts such as ours will be important to affirm our cross-sectional findings and to assess whether subjects who fall in the lowest part of the age- and APOE-adjusted range for CSF A₄₂ concentration have the highest risk of developing AD. It will also be important to correlate CSF A₄₂ concentration with methods of assessing brain amyloid deposition in vivo, such as positron emission tomographic scanning with the Pittsburgh Compound-B.²¹ Therapeutic strategies aimed at prevention of AD may need to be applied in early midlife or even younger ages to have maximal effect on amyloid deposition. Primary prevention trials for AD targeting elderly persons may be too late to affect the early stages of disease pathology.

AUTHOR INFORMATION

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Correspondence: Elaine R. Peskind, MD, VA Puget Sound Health Care System, S-116MIRECC, 1660 S Columbian Way, Seattle, WA 98108 (peskind{at}u.washington.edu).

Accepted for Publication: December 23, 2005.

Author Contributions: Study concept and design: Peskind, Farlow, Raskind, and Galasko. Acquisition of data: Peskind, Quinn, Kaye, Clark, Farlow, DeCarli, Raskind, Schellenberg, Lee, and Galasko. Analysis and interpretation of data: Peskind, Li, Shofer, Kaye, Farlow, Raskind, Lee, and Galasko. Drafting of the manuscript: Peskind, Li, Shofer, Farlow, Raskind, Lee, and Galasko. Critical revision of the manuscript for important intellectual content: Peskind, Li, Shofer, Quinn, Kaye, Clark, Farlow, DeCarli, Raskind, Schellenberg, and Galasko. Statistical analysis: Li and Shofer. Obtained funding: Peskind, Raskind, and Galasko. Administrative, technical, and material support: Peskind, Kaye, Farlow, Raskind, Schellenberg, Lee, and Galasko. Study supervision: Peskind, Kaye, Farlow, and Lee.

Funding/Support: This study was supported by grants AG05136, AG08419, AG08017, AG10124, AG10133, AG23185, and M01 RR00034 from the US National Institute on Aging; the National Alzheimer's Coordinating Center; Friends of Alzheimer's Research; Alzheimer's Association of Western and Central Washington; and the Department of Veterans Affairs.

Role of the Sponsor: None of the funding sources had a role in study design; collection, analysis, and interpretation of data; writing of the report; or the decision to submit the paper for publication.

Author Affiliations: VA Puget Sound Health Care System, Mental Illness Research, Education, and Clinical Center (Drs Peskind, Li, and Raskind and Ms Shofer), Geriatric Research, Education, and Clinical Center (Dr Schellenberg), and Departments of Psychiatry and Behavioral Sciences (Drs Peskind, Li, and Raskind) and Medicine (Dr Schellenberg), University of Washington School of Medicine, Seattle; Department of Neurology, Oregon Health and Science University (Drs Quinn and Kaye) and Portland VA Medical Center (Dr Quinn), Portland; Department of Neurology, Institute on Aging, University of Pennsylvania, Philadelphia (Drs Clark and Lee); Department of Neurology, Indiana University School of Medicine, Indianapolis (Dr Farlow); Department of Neurology, University of California at Davis, Sacramento (Dr DeCarli); Department of Neurosciences, University of California at San Diego and VA Medical Center, San Diego (Dr Galasko).

REFERENCES

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