Human apoE Isoforms Differentially Regulate Brain Amyloid- Peptide Clearance

DOI: 10.1126/scitranslmed.3002156, 89ra57 (2011);3 Sci Transl Med

, et al.Joseph M. CastellanoClearance

PeptideβHuman apoE Isoforms Differentially Regulate Brain Amyloid-

Editor's Summary

developing drugs to slow or even halt the accumulation of amyloid plaques in patients with AD. clearance pathway as a new target forβstudy sheds new light on how apoE4 is implicated in AD and highlights the A

but not its synthesis. This thoroughβgenotype, lending credence to the hypothesis that apoE4 affects clearance of A peptide did not vary according toβshowed that processing of the amyloid precursor protein and generation of the A

than those with the apoE2 or apoE3 isoforms. The researchersβhuman apoE4 isoform were less able to clear A from the interstitial fluid of young mice and showed that those with theβplaques. They then measured clearance of A

genotypes was present in young as well as aged mice, suggesting that it predates the appearance of amyloid APOE concentration between the mice carrying different βE2 isoforms. They discovered that this difference in A

fluid and the hippocampus in mice expressing the human apoE4 isoform than in animals expressing either the E3 or concentrations in both interstitialβlooked at stained hippocampal sections from these mice. They found greater A

concentrations in the interstitial fluid of these mice using in vivo microdialysis and thenβisoforms. They measured Agenotypes. They then moved to a mouse model of AD in which the mice expressed one of the three human apoE

APOE4 genotype bound more dye than did those with the other ε4/εbrain and showed that those individuals with the researchers analyzed imaging data using a dye called Pittsburgh compound B that binds to amyloid plaques in the

peptide is accumulating in the brain and thus is not moving into the CSF. Next, theβpg/ml is an indication that A concentration of less than 500β3 genotype. A CSF Aε2/ε concentration than did those with the protective βCSF A

4 genotype had a much lowerε4/ε genotypes. They found that those with the APOEunder age 70 carrying different concentration in the cerebrospinal fluid (CSF) of cognitively normal individualsβFirst, the authors looked at the A

synthesis but by affecting its clearance.β accumulation in the brain not by affecting Aβcontributes to Astudy in humans and mice, Holtzman and his team at Washington University in St. Louis now show that apoE4plaques in the brain that contribute to neuronal death and are the characteristic hallmark of AD. In a tour de force

), which forms amyloidβ (Aβthat the apoE4 isoform might somehow help to drive accumulation of the peptide amyloid-important for lipoprotein metabolism, but how it might be involved in AD has remained unclear. It has been suggested

isAPOE2 allele decreases the risk of developing this neurodegenerative disorder. ε15 years. In contrast, the 4 alleles can lower the age of onset of AD by 10 toε4 allele encoding apolipoprotein E4 (apoE4). Two εold age is the

The strongest risk factor for developing the common sporadic form of Alzheimer's disease (AD) that occurs in

Clearing the Debris in Alzheimer's Disease

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R E S EARCH ART I C L E

ALZHE IMER ’S D I S EASE

Human apoE Isoforms Differentially Regulate BrainAmyloid-b Peptide ClearanceJoseph M. Castellano,1,2,3* Jungsu Kim,1,2,3* Floy R. Stewart,1,2,3 Hong Jiang,1,2,3

Ronald B. DeMattos,4 Bruce W. Patterson,5 Anne M. Fagan,1,2,3 John C. Morris,1,3

Kwasi G. Mawuenyega,1,2,3 Carlos Cruchaga,2,3,6 Alison M. Goate,1,2,3,6 Kelly R. Bales,7

Steven M. Paul,8 Randall J. Bateman,1,2,3 David M. Holtzman1,2,3,9†

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The apolipoprotein E (APOE) e4 allele is the strongest genetic risk factor for late-onset, sporadic Alzheimer’sdisease (AD). The APOE e4 allele markedly increases AD risk and decreases age of onset, likely through itsstrong effect on the accumulation of amyloid-b (Ab) peptide. In contrast, the APOE e2 allele appears to decreaseAD risk. Most rare, early-onset forms of familial AD are caused by autosomal dominant mutations that oftenlead to overproduction of Ab42 peptide. However, the mechanism by which APOE alleles differentially modulateAb accumulation in sporadic, late-onset AD is less clear. In a cohort of cognitively normal individuals, we reportthat reliable molecular and neuroimaging biomarkers of cerebral Ab deposition vary in an apoE isoform–dependent manner. We hypothesized that human apoE isoforms differentially affect Ab clearance or synthesisin vivo, resulting in an apoE isoform–dependent pattern of Ab accumulation later in life. Performing in vivomicrodialysis in a mouse model of Ab-amyloidosis expressing human apoE isoforms (PDAPP/TRE), we find thatthe concentration and clearance of soluble Ab in the brain interstitial fluid depends on the isoform of apoEexpressed. This pattern parallels the extent of Ab deposition observed in aged PDAPP/TRE mice. ApoE isoform–dependent differences in soluble Ab metabolism are observed not only in aged but also in young PDAPP/TREmice well before the onset of Ab deposition in amyloid plaques in the brain. Additionally, amyloidogenic pro-cessing of amyloid precursor protein and Ab synthesis, as assessed by in vivo stable isotopic labeling kinetics,do not vary according to apoE isoform in young PDAPP/TRE mice. Our results suggest that APOE alleles con-tribute to AD risk by differentially regulating clearance of Ab from the brain, suggesting that Ab clearancepathways may be useful therapeutic targets for AD prevention.

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INTRODUCTION

Alzheimer’s disease (AD) is the leading cause of dementia in the el-derly, with an estimated prevalence of 26 million cases worldwide.Because the number of cases and associated costs are projected toincrease markedly, effective strategies aimed at prevention and pre-clinical intervention will likely depend on our understanding of howmajor risk factors contribute to the disease process. The prevailinghypothesis of AD pathogenesis posits that accumulation of brainamyloid-b (Ab) peptide initiates a pathogenic cascade that culminatesin neurodegeneration and dementia (1). The Ab peptide is generatedthrough sequential proteolytic processing of the amyloid precursorprotein (APP) by b- and g-secretases. Strong biochemical and geneticevidence has demonstrated that most rare, early-onset forms of famil-

1Department of Neurology, Washington University School of Medicine, St. Louis, MO63110, USA. 2Hope Center for Neurological Disorders, Washington University Schoolof Medicine, St. Louis, MO 63110, USA. 3Charles F. and Joanne Knight Alzheimer’sDisease Research Center, Washington University School of Medicine, St. Louis, MO63110, USA. 4Eli Lilly and Co., Lilly Research Labs, Indianapolis, IN 46285, USA.5Department of Internal Medicine, Washington University School of Medicine, St. Louis,MO 63110, USA. 6Department of Psychiatry, Washington University School of Medicine,St. Louis, MO 63110, USA. 7Neuroscience Research Unit, Pfizer Global Research &Development, Groton, CT 06430, USA. 8Appel Alzheimer’s Disease Research Institute,Weill Cornell Medical College, Cornell University, New York, NY 10065, USA. 9De-partment of Developmental Biology, Washington University School of Medicine, St. Louis,MO 63110, USA.*These authors contributed equally to this work.†To whom all correspondence should be addressed. E-mail: [email protected]

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ial AD are caused by autosomal dominant mutations that result inabnormal processing of APP, leading to overproduction of Ab or anincrease in the ratio of Ab42 to Ab40. Much less is known about thefactors that initiate or modulate the onset of brain Ab accumulation inthe more common (>99%) sporadic, late-onset form of AD. The bestestablished genetic risk factor for sporadic, late-onset AD is the apo-lipoprotein E (APOE) e4 allele, the presence of which markedly increasesrisk for developing AD and decreases age of onset by 10 to 15 years; incontrast, the APOE e2 allele confers protection against developing AD(2–5). APOE status has been found to modulate the onset of extra-cellular amyloid plaque deposition, one of the key pathognomonicfeatures of the disease (6, 7). Strong evidence demonstrating accel-erated onset of amyloid deposition in APOE e4 carriers has led to thehypothesis that APOE genotype differentially modulates AD risk andonset through effects on Ab metabolism (6–9). Consistent with thishypothesis, we and others have reported human apoE isoform–dependentdifferences in amyloid plaque deposition in APP-transgenic mice(E4 > E3 > E2) (10–14). Although it has been hypothesized that apoEisoforms differentially modulate Ab accumulation through effects onAb clearance, direct in vivo evidence demonstrating apoE isoform–dependent differences in brain Ab clearance or synthesis has beenlacking. Here, we provide in vivo evidence that apoE isoforms differ-entially modulate brain Ab burden in a manner that corresponds toearly apoE isoform–dependent differences in Ab clearance. Specifical-ly, we used in vivo microdialysis to measure the concentration of sol-uble Ab and its clearance from the brain interstitial fluid (ISF) of young

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and aged PDAPP/TRE mice. This mouse model of Ab-amyloidosisoverexpresses human APP carrying an autosomal dominant familialAD-linked mutation (V717F) and also expresses one of the three hu-man apoE isoforms under the control of mouse apoE regulatory

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elements. We found that the soluble Ab concentration in ISF andits clearance depends on the human apoE isoform expressed in a man-ner that parallels the pattern of Ab deposition in old PDAPP/TRE mice.Finally, using an in vivo stable isotopic labeling kinetics technique, wefound no differences in fractional synthesis rates (FSRs) of Ab amongPDAPP/TRE mice, consistent with biochemical evidence suggesting noapoE isoform–dependent changes in amyloidogenic processing of APP.Together, our results provide direct in vivo evidence for a mechanismwhereby apoE isoform–dependent differences in Ab clearance modulatethe onset of Ab accumulation in transgenic mice and in humans.

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RESULTS

Biomarkers of cerebral Ab deposition differ according toAPOE genotype in humansSeveral groups have now validated molecular and neuroimaging bio-markers of the neuropathological hallmarks of AD (15–19). In par-ticular, low concentrations of Ab42 in the cerebrospinal fluid (CSF)reflect the presence of cerebral Ab deposition, likely as a result ofAb42 being sequestered into amyloid plaques, changing the equilibri-um between the brain and CSF pools of Ab (20–22). Additionally, the[11C]benzothiazole radiotracer, Pittsburgh compound B (PIB), as wellas other tracers, can bind to fibrillar Ab plaques, allowing for visual-ization of brain amyloid in individuals during positron emission to-mography (PET) (23–25). A preponderance of evidence supports theinterpretation that PIB uptake and CSF Ab42 are reliable surrogatemarkers of amyloid plaque pathology in living subjects (15–19). Arecent study revealed that CSF and neuroimaging biomarkers of am-yloid pathology are more prevalent in cognitively normal APOE e4carriers relative to individuals who have no APOE e4 alleles (that is,have the APOE e3 and/or APOE e2 alleles) (7). Additionally, the APOEe4 allele increases brain amyloid burden assessed by PIB-PET imaging ina gene dose–dependent manner (6). To study the impact of APOE e2, e3,and e4 alleles on the development of cerebral Ab deposition in the

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absence of AD dementia, we analyzed acohort of cognitively normal individualsyounger than age 70, an age after whichthe presence of AD dementia may con-found the analyses. From this cohort,we analyzed the concentration of Ab42in CSF from APOE e4/e4, e3/e4, ande3/e3 individuals; because APOE e2homozygous individuals are exceptional-ly rare, CSF Ab42 was analyzed in e2/e3individuals. Although various demo-graphic features of our cohort, such asage, sex, and education level, did notdiffer by APOE genotype, the meanconcentration of CSF Ab42 was signifi-cantly lower in APOE e4/e4 individualscompared to individuals of all otherAPOE genotypes in the cohort (Table 1).Given that a CSF Ab42 concentrationlower than 500 pg/ml has been usedas a reliable threshold for the presenceof cerebral Ab deposition in humans(7, 19, 22, 26), we determined the pro-
Table 1. Demographic characteristics and biomarker information forcognitively normal individuals according to APOE genotype. Values repre-sent means ± SD. When one-way ANOVA was significant, pair-wise com-parisons of APOE genotypes were made with Tukey’s post hoc test; onlysignificant differences were indicated (‡P < 0.05; **P < 0.01; ***P < 0.001).‡ denotes significant difference compared to APOE e3/e4. ** or *** de-notes significant difference compared to APOE e4/e4. MMSE, Mini MentalState Examination from 0 to 30; LP, lumbar puncture.

APOE genotype

e2/e3
e3/e3 e3/e4 e4/e4
n
32 151 81 19
Female (%)
62.50 64.24 67.90 63.16
Caucasian (%)
87.50 92.05 90.12 84.21
Age at LP,years (SD)

59.28(7.13)

60.72(7.17)

60.51(7.80)

57.58(8.85)

MMSE
29.28(0.96)
29.37(0.94)

29.38(0.96)

29.32(1.16)

Education,years (SD)

15.69(2.76)

15.92(2.62)

15.83(2.31)

16.53(3.42)

Ab42, pg/ml (SD)
755.65‡,***
(212.85)
695.58***
(243.87)
619.58**
(193.79)
437.39(183.53)
Tau, pg/ml (SD)
253.33(114.68)
248.72(126.48)

267.54(131.89)

244.13(90.58)

pTau, pg/ml (SD)
50.72(17.76)
48.18(19.71)

55.86(28.83)

51.45(13.62)

Fig. 1. Biomarkers of amyloid differ according to APOE genotype in cognitively normal individuals. (A)Percentage of individuals (n = 283) with [CSF Ab ] < 500 pg/ml according to the following APOE geno-
42
types: e2/e3, e3/e3,e3/e4, and e4/e4. Number in parentheses indicates number of individuals for eachgroup.(B) Percentage of PIB+ individuals (n = 153) according to APOE genotype: e2/e3, e3/e3, e3/e4, and e4/e4.Individuals with mean cortical binding potential (MCBP) for Pittsburgh compound B >0.18 were consideredPIB+. Number in parentheses indicates number of individuals for each group. c2 analyses for proportions in(A) [c2(3) = 22.1785, P = 5.99 × 10−5) and (B) [c2(3) = 14.4735, P = 2.33 × 10−3] were performed; follow-up c2

tests for pairwise comparisons of proportions were performedwith Benjamini andHochberg’s linear step-upadjustment to control for type I error. *P < 0.05; **P < 0.01; ***P < 0.001.

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portion of individuals in each genotype with CSF Ab42 lower than500 pg/ml. We found that there was a significantly greater proportion ofAPOE e4/e4 individuals with CSF Ab42 lower than 500 pg/ml comparedto APOE e3/e4, e3/e3, and e2/e3 individuals (Fig. 1A). We next identi-fied individuals in the cohort who had received PIB-PET scans within 2years of lumbar puncture for CSF analysis. On the basis of previous

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studies (7, 27), individuals with mean cortical binding potential (MCBP)for PIB >0.18 were considered PIB-positive (PIB+). We found that theproportion of PIB+ individuals also follows a strong APOE allele–dependent pattern (Fig. 1B). These results demonstrate a clear APOEallele–dependent difference in the relative frequency at which individ-uals exhibit molecular and neuroimaging correlates of amyloid pathology.


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Ab and amyloid deposition inold PDAPP mice is humanapoE isoform–dependentTo further investigate the role of apoEisoforms in differentially modulatingAb metabolism, we used PDAPP micein which human apoE isoforms are ex-pressed under control of the mouse reg-ulatory elements (PDAPP/TRE) (14).After allowing each cohort of mice toage to 20 to 21 months, we immuno-stained brain sections using an anti-Abantibody (3D6) and quantified the ex-tent of Ab deposition covering the hip-pocampus. Consistent with a previousreport (14), we observed marked differ-ences in Ab deposition depending onthe isoform of apoE expressed (Fig. 2,A to C). Quantification revealed thathippocampal Ab burden in 20- to 21-month-old PDAPP/E4 mice was about2- and 4.6-fold higher than in PDAPP/E3and PDAPP/E2 mice, respectively (Fig.2D). ApoE is strongly associated withthe amount of fibrillar amyloid that de-posits into plaques (28). Thus, we nextcharacterized amyloid plaque load inthe context of human apoE by stainingadjacent brain sections from these micewith X-34, a congophilic dye that bindsto amyloid. Consistent with the Ab im-munostaining pattern observed, wefound that hippocampal amyloid plaqueload varied according to apoE isoform(Fig. 2, E to H). Together, these resultsprovide clear evidence that apolipo-protein E4 (apoE4) increases Ab deposi-tion relative to apoE3 and apoE2 in amanner that closely recapitulates the hu-man biomarker findings reported in Fig. 1.

Soluble Ab concentration andclearance in brain ISF of old mice ishuman apoE isoform–dependentTo investigate the mechanism by whichAb accumulation in the brain variesaccording to apoE isoform in PDAPP/TREmice, we used in vivo microdialysis todynamically assess ISF Ab metabolismin the contralateral hippocampus ofPDAPP/TRE mice before harvesting

Fig. 2. Ab/amyloid deposition varies according to apoE isoform in old PDAPP/TRE mice. (A to C) Repre-sentative coronal brain sections from 20- to 21-month-old, sex-matched PDAPP/E2 (A), PDAPP/E3 (B),and PDAPP/E4 (C)mice. Ab immunostainingwas performedwith anti-Ab antibody (biotinylated 3D6). Scalebars, 50 mm. (D) Quantification of the area of the hippocampus occupied by Ab immunostaining (n = 7miceper group). *P < 0.05; **P < 0.01. (E to G) Representative coronal brain sections from 20- to 21-month-oldPDAPP/E2 (E), PDAPP/E3 (F), and PDAPP/E4 (G) mice. Amyloid was detected with the congophilic fluores-cent dye X-34. Scale bars, 50 mm. (H) Quantification of the area of hippocampus occupied by X-34 stain-ing (n = 7mice per group). When one-way ANOVAwas significant, differences among groupswere assessedwith Tukey’s post hoc test for multiple comparisons. *P < 0.05; ***P < 0.001. Values represent means ± SEM.

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for pathological analysis. The concentration of soluble Ab in the ISFthroughout life has been shown to be closely associated with theamount of Ab that ultimately deposits in the extracellular space ofthe brain (29–31). Because soluble ISF Ab has been shown to closelyreflect extracellular pools of Ab (29–31), we hypothesized that the con-centration of soluble Ab in the ISF would closely follow the pattern ofAb deposition analyzed from the same mice in Fig. 2. HippocampalISF was sampled in PDAPP/TRE mice for a stable baseline periodduring which mice were able to freely behave for the duration ofthe experiment. We found that the steady-state concentration of ISFAb1–x (Ab species containing the N terminus through the central do-main of Ab) varied according to apoE isoform (Fig. 3A). Specifically,the brains of PDAPP/E4 mice had significantly more Ab in the ISFpool, about 2- and 3.8-fold more than PDAPP/E3 and PDAPP/E2mice, respectively. To understand whether the apoE isoform–dependent differences in soluble Ab concentration may be the result

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of altered Ab clearance from the ISF, weperformed clearance microdialysis ex-periments by analyzing the eliminationkinetics of Ab after halting Ab produc-tion with a potent g-secretase inhibitor(29) (Fig. 3B). We found that the half-life (t1/2) of ISF Ab in the hippocampusof PDAPP/E4 mice was 1.1 hours, com-pared to 0.71 and 0.56 hours, measuredfrom PDAPP/E3 and PDAPP/E2 mice,respectively (Fig. 3C). These results dem-onstrate that the clearance of endogenousAb from brain ISF is impaired in oldPDAPP/E4 mice relative to PDAPP/E3and PDAPP/E2 mice.

ApoE isoform–dependentdifferences in Ab concentrationand clearance exist beforeAb depositionBecause changes in apoE and Ab me-tabolism in the brain ISF early in lifecan markedly alter Ab deposition laterin life (31, 32), we next asked whetherthe Ab deposition pattern observed inold PDAPP/TRE mice may be a resultof early apoE isoform–dependent differ-ences in ISF Ab metabolism. To test thishypothesis, we performed in vivo micro-dialysis in young PDAPP/TRE miceusing a sensitive zero flow extrapolationmethod. Theoretically, the maximum invivo steady-state concentration of ananalyte being dialyzed exists at thepoint at which there is no flow of theperfusion buffer (31, 33). To obtainthis value in the hippocampal ISF ofPDAPP/TRE mice, we used several flowrates during microdialysis to extrapolateto the point of zero flow for each mouse(Fig. 4A). As shown in Fig. 4B, themean in vivo steady-state concentration

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of soluble ISF Ab was highest in PDAPP/E4 mice compared toPDAPP/E3 and PDAPP/E2 mice. The concentration of soluble ISFAb at each flow rate also varied strongly according to apoE isoform(fig. S1A). To address the possibility that microdialysis probe functionmay differ in the context of different human apoE isoforms, wedetermined the percent recovery at each flow rate, which revealedno significant differences among PDAPP/TRE mice (fig. S1B). Be-cause the metabolite urea has been used as an independent measureof probe function and recovery in both human and animal brain mi-crodialysis studies (34–37), we measured the concentration of urea inthe brain ISF of PDAPP/TRE mice. The concentration of urea did notdiffer among groups, suggesting that probe function was equivalentacross experiments in PDAPP/TRE mice (fig. S1C). Neither the levelsof phosphate-buffered saline (PBS)–soluble Ab40 nor the levels of mu-rine APP in hippocampal lysates from young apoE knock-in miceexpressing murine APP differed according to apoE isoform, suggesting

Fig. 3. Soluble Ab concentration and clearance in thebrain ISF of oldmice is humanapoE isoform–dependent.(A) Mean steady-state concentrations of eAb1–x (ex-changeable Ab) from sampling hippocampal ISF in old,sex-matched PDAPP/E2, PDAPP/E3, and PDAPP/E4mice,measured by enzyme-linked immunosorbent assay(ELISA) (n = 6 to 7 mice per group; 20 to 21 monthsold). (B) Schematic diagram of a typical clearance exper-iment in which a stable baseline period is obtained,followed by intraperitoneal (i.p.) injection of LY411,575(10mg/kg) tohalt Ab production. Ab concentrationsdur-ing the elimination phase are transformedwith the com-

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mon logarithm. Log-transformed values are fit with a linear regression, allowing calculation of slope, k′. eAbt1/2 = 0.693/k, where k = 2.303k′. (C) eAb t1/2 from clearance experiments performedwith themice in (A) afterstable baseline measurement of eAb1–x. When one-way ANOVA was significant, differences among groupswere assessedwith Tukey’s post hoc test for multiple comparisons. *P < 0.05. Values representmeans ± SEM.

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1, 2

012

that regulation of Ab concentration by human apoE may depend onthe human Ab sequence, which differs from murine Ab by threeamino acids (fig. S2, A and B). We next asked whether the concentra-tion of the more aggregation-prone Ab42 species varies according tohuman apoE isoform in young mice in the ISF pool, the site of Abdeposition in old PDAPP/TRE mice. We found that the concentrationof soluble Ab42 was highest in young PDAPP/E4 mice compared toPDAPP/E3 or PDAPP/E2 mice (Fig. 4C). We also measured levels ofPBS-soluble and PBS-insoluble Ab40 and Ab42 after sequential extrac-tion of hippocampi from young PDAPP/TRE mice (table S1). Al-though the overall pattern was similar to what we observed in theISF, the effects were of lesser magnitude or nonsignificant trends wereevident, perhaps suggesting that the extracted pools we measured donot completely reflect the ISF pool of Ab (31, 38).

To test the hypothesis that human apoE isoforms differentially reg-ulate the concentration of soluble Ab in the ISF of young PDAPP/TREmice through effects on Ab clearance, we performed clearance micro-dialysis experiments in young PDAPP/TRE mice. As shown in Fig.4D, Ab t1/2 measured in the hippocampal ISF of PDAPP/E4 mice is

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significantly longer compared to PDAPP/E3 and PDAPP/E2 mice, re-spectively. We next assessed Ab clearance in PS1DE9/APPswe/TREmice, a mouse model of Ab-amyloidosis based on autosomal domi-nant AD-linked mutations in PS1 and APP that also expresses oneof the three human apoE isoforms. We also observed a similar patternof apoE isoform–dependent Ab clearance in these mice (fig. S3), sug-gesting that the clearance impairment in the context of apoE4 is notan artifact of the PDAPP transgene. Together, these results stronglysuggest that the reduced clearance of Ab from the brain ISF ofPDAPP/E4 mice contributes to the increased concentration of Abin the ISF, likely resulting in earlier Ab/amyloid plaque deposition.Several studies have indicated that apoE concentration varies by hu-man apoE isoform (39), raising the possibility that altered apoE con-centration may be an endophenotype among APOE genotypes thatregulates APOE allele–dependent Ab metabolism. We analyzed indi-viduals in our cohort whose CSF had been analyzed by multianalyteprofiling, as previously described (40), which revealed that the pres-ence of one e2 allele of APOE was associated with significantlyincreased concentrations of apoE relative to other APOE genotypes


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(fig. S4A). The concentration of apoEin the CSF was also significantly lowerin APOE e4 carriers (individuals withone or two copies of APOE e4) com-pared to APOE e3/e3 individuals, butthe concentration of apoE did not differbetween those who were APOE e3/e3and those who were APOE e4/e4.Whereas apoE levels from brain homog-enates were higher in PDAPP/E2 micecompared to PDAPP/E3 or PDAPP/E4mice, levels did not differ betweenPDAPP/E3 and PDAPP/E4 mice (fig.S4B). Together, these results suggest thatwhereas higher apoE concentration inthe context of apoE2 may underlie morerapid Ab clearance relative to apoE4,apoE concentration is unlikely to underlieAb clearance differences observed in thecontext of apoE3 versus apoE4. More-over, because the extent of apoE lipida-tion may also play a role in modulatingAb accumulation (41), we assessed thesize of lipidated apoE particles from theCSF of young and old PDAPP/TRE miceby native polyacrylamide gel electro-phoresis (PAGE)/Western blot analysis.Regardless of apoE isoform or age, apoEparticles were between 12.2 nm andslightly larger than 17 nm in size (fig. S4C).

Amyloidogenic processing ofAPP does not vary according tohuman apoE isoformIn the amyloidogenic pathway of APPprocessing, b-secretase (BACE1) cleavesAPP N-terminally at the Ab domain,leading to the generation of sAPPb andC99, the latter of which ultimately gives

Fig. 4. ApoE isoform–dependent differences in soluble Ab concentration and clearance exist before theonset of Ab deposition. (A) An exponential decay regression was used to fit the concentrations of eAb1–xmeasured by ELISA at each flow rate for individual mice from groups of young, sex-matched PDAPP/TREmice (n = 6 mice per group; 3 to 4 months old). The equations from the individual regressions were usedto calculate [eAb1–x] at x = 0 for eachmouse, representing the in vivo concentration of eAb1–x recoverable bymicrodialysis. (B) Mean in vivo concentrations of eAb1–x (pg/ml) calculated from themethod in (A). (C) Meanconcentrations of Abx–42 (pg/ml) collected from the hippocampal ISF of young, sex-matched PDAPP/TREmice with a flow rate of 0.3 ml/min (n = 8 mice per group; 3 to 4 months old). (D) eAb t1/2 from clearanceexperiments in young, sex-matched PDAPP/TREmice after stable baselinemeasurement of eAb1–x (n = 10 to11 mice per group; 3 to 4 months old). When one-way ANOVA was significant, differences among groupswere assessed with Tukey’s post hoc test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001. Valuesrepresent means ± SEM.

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rise to the Ab peptide. In the context of different human apoE isoforms,amyloidogenic processing of APP may vary according to apoE isoform,contributing to the differences in the concentration of ISF Ab observedin Fig. 4B. To begin to address this possibility, we compared levels of theamyloidogenic metabolite C99 in hippocampal homogenates fromyoung PDAPP/TRE mice. As shown in a representative Western blotprobed with 82E1 antibody (anti-Ab1–16), which recognizes C99, rela-tive levels of C99 did not differ among PDAPP/TRE mice (Fig. 5A).Quantification revealed that C99 levels did not vary significantlyaccording to human apoE isoform (Fig. 5B). Additionally, full-lengthAPP levels did not appear to vary significantly among groups (fig. S5,A and B). To further assess whether amyloidogenic processing differsaccording to apoE isoform, we measured b-secretase activity in hippo-campal homogenates from young PDAPP/TRE mice. b-Secretase activ-ity was measured by monitoring the fluorescence increase that resultsfrom cleavage of a peptide based on the b-cleavage site of APP. On thebasis of the quantification shown in Fig. 5C, there were no significantdifferences in reaction velocity among PDAPP/TRE mice, suggestingthat apoE isoform–dependent differences in b-secretase activity are un-likely to account for differences in soluble ISF Ab concentration inyoung PDAPP/TRE mice. Overall, our results in PDAPP/TRE miceare consistent with a previous in vitro study showing no effect of apoEisoforms on APP processing (42).

Rates of Ab synthesis do not differ according tohuman apoE isoformTo sensitively assess the rates of Ab synthesis in the context of hu-man apoE isoforms in PDAPP/TRE mice, we adapted an in vivostable isotopic labeling kinetics technique previously described inhumans (43). Briefly, young PDAPP/TRE mice were intraperi-toneally injected with the stable isotope–labeled amino acid[13C6]leucine, which crosses the blood-brain barrier and incorpo-rates into newly synthesized APP/Ab during normal protein syn-thesis in the central nervous system. We next sacrificed mice at20 and 40 min after the injection and immunoprecipitated totalAb from brain lysates using HJ5.2, an anti-Ab13–28 antibody. Aftertrypsin digestion of immunoprecipitated Ab, samples weresubmitted to liquid chromatography–mass spectrometry (LC-MS),allowing quantification of the relative abundance of labeled to un-labeled Ab by analyzing mass shifts of predicted MS/MS ions in thespectra (Fig. 6A). To accurately quantify and calibrate the mass spec-trometry signals from mouse brain samples, we used cell-secreted Abto generate a standard curve based on a known quantity of Ab labeledwith [13C6]leucine (Fig. 6B). We next calculated FSRs of Ab based onthe rate of increase in the amount of labeled to unlabeled Ab between20 and 40 min after injection, normalized to the average enrichmentof plasma leucine. We found no significant differences in Ab FSRsamong young PDAPP/TRE mice (Fig. 6C), strongly suggesting thatapoE isoforms do not differentially modulate Ab synthesis in vivo.

DISCUSSION

Despite significant advances in our understanding of the patholog-ical events leading to AD, the causes of Ab accumulation are onlyreasonably well understood for a small subset of individuals withAD who have autosomal dominant mutations, resulting in early-onset, familial AD. Most AD cases are sporadic, and in these indi-

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viduals, the factors leading to Ab accumulation are not well under-stood. Because effective AD treatments will likely depend on interveningduring the preclinical (presymptomatic) phase of AD (44, 45),

Fig. 5. Amyloidogenic processing of APP does not vary according to humanapoE isoform. (A) Representative Western blot of the proximal amyloido-genicmetabolite, C99, fromhippocampal homogenates (extractedwithRIPAbuffer) from young, sex-matched PDAPP/TRE mice. C99 was detected with82E1 antibody. All bands were normalized to a-tubulin band intensity (n = 9mice per group; 3 to 4months old). (B) Quantification of C99 levels after nor-malizing each band’s intensity to a-tubulin band intensity. (C) Quantificationof b-secretase activity in hippocampal homogenates fromyoungPDAPP/TREmicewith a sensitive FRET assay. Homogenateswere incubatedwith fluores-cent APP substrate, resulting in b-cleavage that could be followed by fluores-cence increase (emission, 585 nm). The interval over which kinetics werelinear was used for quantification of reaction velocity [relative fluorescenceunits (RFU)/min] for each sample. One-way ANOVA revealed no significantdifferences among groups. Values represent means ± SEM.




understanding how environmental and genetic risk factors modulatepathological hallmarks of the disease will be critical. The strongestgenetic risk factor for late-onset, sporadic AD is the APOE e4 allele,which markedly increases risk and reduces the age of onset (46), like-ly by accelerating the onset of brain Ab accumulation (6–9). Indeed,we showed in a cohort of cognitively normal individuals less than 70years of age that biomarkers of brain amyloid accumulation werepresent at a relative frequency that corresponded to APOE genotype,that is, e4 > e3 > e2. Consistent with this observation, we found thatold PDAPP/TRE mice developed Ab/amyloid deposition in an apoEisoform–dependent pattern, that is, E4 > E3 > E2, a finding thatextends previous reports of apoE isoform–dependent Ab depositionin various mouse models (10, 11, 13, 14). Because the concentrationof Ab in the extracellular space of the brain reflects a balance betweenits synthesis and clearance rates, we hypothesized that APOE geno-

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type differentially modulates Ab accumulation through effects on Abclearance and/or synthesis. To test this hypothesis, we used novel invivo methodologies to measure endogenous brain Ab clearance andsynthesis in PDAPP mice expressing human apoE isoforms undercontrol of the endogenous mouse APOE promoter. Using in vivo mi-crodialysis, we found that the concentration of ISF Ab in the hip-pocampus of young and old PDAPP/E4 mice was greater than inPDAPP/E3 or PDAPP/E2 mice, likely as a result of reduced Ab clear-ance in PDAPP/E4 mice. ApoE isoform–dependent Ab clearance wasalso observed in PDAPP/TRE mice before the onset of Ab accumula-tion. To investigate the impact of apoE isoforms on Ab synthesis, wedeveloped a sensitive method to measure the FSR of brain Ab in vivo,adapted from the stable isotopic labeling kinetics technique recentlyused by our group in humans (43). Using this technique, we foundthat the fractional rates of brain Ab synthesis from young PDAPP/TRE


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mice did not differ according to the hu-man apoE isoform expressed, consistentwith our biochemical results showingthat amyloidogenic processing of APPdid not vary by human apoE isoform.Our results strongly suggest that APOEgenotype differentially modulates the onsetof Ab accumulation via differential regula-tion of Ab clearance, although the cellularand molecular mechanisms underlyingthis regulation remain unclear.

Once the link between APOE geno-type and AD risk had been described,several groups focused on characteriz-ing the putative apoE/Ab interactionand the extent to which this interac-tion influenced the aggregation of Abin vitro. Although our results suggestthat apoE isoforms differentially regulateAb accumulation via effects on Ab clear-ance, we cannot exclude the possibilitythat apoE isoforms also modulate Ab ac-cumulation by directly facilitating Ab fi-brillization. For example, lipid-free apoE4was found to facilitate Ab fibrillization invitro to a greater degree compared toapoE3 (47, 48) [see (39) for a review]. Per-haps due to differences in experimentalconditions, others have reported that apoEisoforms inhibit the process of Ab aggre-gation (39, 49, 50), making it difficult tointerpret whether they differentially mod-ulate Ab accumulation in vivo. Several invitro studies have demonstrated thatlipidated apoE2 and apoE3 bind Abwith greater affinity compared to apoE4(39, 51–54). This observation has promptedsome to hypothesize that the stronger in-teraction between Ab and apoE2 or apoE3relative to apoE4 may result in greater Abclearance, consistent with the clearancepattern we observed in vivo in the currentstudy. Indeed, several studies have demon-

Fig. 6. Rates of Ab synthesis do not differ according to human apoE isoform in PDAPP/TRE mice. (A) Abdetection in hippocampal lysates fromyoungPDAPP/TREmice by TSQVantage triple quadrupolemass spec-
trometry. Left, representative total ion count multiple reaction monitoring (MRM) peak of the unlabeled Abtryptic peptide, LVFFAEDVGSNK [mass/charge ratio (m/z) = 663.340]. Right, MRM peak for [13C6]leucine-labeled Ab (m/z = 666.350). (B) Standard curve generated with known quantity of [13C6]leucine-labeledand [13C6]leucine-unlabeled Ab. Ab secreted from H4-APP695DNL neuroglioma cells incubated withlabeled/unlabeled leucine was immunoprecipitated with HJ5.2 antibody (anti-Ab13–28), followed by trypsindigestion. Ab17–28 fragments were analyzed on a TSQ Vantagemass spectrometer. The expected percentageof labeled Ab versus measured percentage was fit by linear regression. Variance is reported with 95% con-fidence interval. (C) Relative FSRs of Ab from hippocampi of PDAPP/TRE mice intraperitoneally injected with[13C6]leucine (200mg/kg) (n = 5 to 6mice per group; 4 to 5months old). Relative FSRs of Abwere calculatedfrom the ratio of [13C6]leucine-labeled to [13C6]leucine-unlabeled Ab. [13C6]/[
12C6]Ab ratio was normalized tothe ratio of labeled to unlabeled free leucine in plasma (determinedbyGC-MS).Mass spectrometry datawerenormalized with the media standard curve in (B). One-way ANCOVA (analysis of covariance) revealed nosignificant differences among groups. Values represent means ± SEM.

29 June 2011 Vol 3 Issue 89 89ra57 7



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strated that Ab transport from brain into blood is altered whencomplexed to human apoE (55, 56). A recent study wherein apoE/Abcomplexes were microinjected into wild-type mouse brain revealedthat Ab bound to apoE4 is cleared more slowly than when Ab iscomplexed to apoE3 or apoE2 (56). One study found that antagoniz-ing the apoE/Ab interaction with a small peptide decreased Ab pathol-ogy in the mouse brain, further suggesting that the apoE/Ab interactionmay be relevant to Ab clearance in vivo (57). Additional studies areneeded to characterize the extent of the apoE/Ab interaction undermore physiological conditions and whether differential apoE/Ab inter-actions may underlie our current in vivo results. Aside from Ab egressfrom brain to blood, in vitro studies have suggested that cellularuptake and degradation of Ab may also represent clearance mecha-nisms that are regulated by human apoE (58–60). One recent in vitrostudy found that human apoE isoforms differed in their ability to fa-cilitate neprilysin-mediated degradation of Ab42 within microglia, withapoE4 being the least effective in facilitating Ab degradation comparedto apoE2 or apoE3 (60).

Although there are some conflicting studies (61), several groupshave reported that the concentration of apoE in the brains of humanapoE knock-in mice varies in an apoE isoform–dependent manner,that is, E2 > E3 > E4 (14, 62, 63). Together with our present results,the isoform-dependent pattern of apoE concentration in humans andin mice raises the possibility that apoE concentration alone may playa role in the pattern of Ab clearance and subsequent Ab accumula-tion, though apoE concentration differences are unlikely to complete-ly account for Ab metabolism differences in the setting of apoE3versus apoE4 (14). The impact of structural differences among apoEisoforms (64), especially differences in relative affinities for variousapoE receptors, may also contribute to differences in Ab clearance.Future studies delineating the precise contribution of both apoE con-centration and isoform may directly bear on therapeutic strategiesaimed at targeting apoE. Using a mouse model of human apoE-dependent b-amyloidosis, our present results may be directly relevantto human studies. For example, using in vivo stable isotopic labelingkinetics, our group recently reported that CSF Ab clearance and notsynthesis is impaired in a small cohort of late-onset AD patients, al-though the effect of APOE genotype was not assessed (65). Coupledwith this recent finding, our present results motivate further investi-gation into whether Ab clearance in humans is modulated by APOEgenotype. These findings further motivate the development of thera-pies that increase brain Ab clearance.

MATERIALS AND METHODS

CSF Ab42, apoE, and PIB-PET assessment in humansParticipants were cognitively normal volunteers (between 43 and 70years of age at time of participation) for a longitudinal memory andaging study at the Washington University Alzheimer’s Disease Re-search Center. Cognitive status was assessed by clinical evaluationbased on whether intraindividual decline existed in performance oftypical activity (as a result of loss of cognitive function). “Cognitive-ly normal” corresponds to a “0” on the Clinical Dementia Rating(CDR) scale. TaqMan assays (Applied Biosystems) for both rs429358(ABI#C_3084793_20) and rs7412 (ABI#C_904973_10) were usedfor APOE genotyping. The allelic discrimination analysis module ofABI Sequence Detection Software was used for allele calling. Positive

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controls for the six possible APOE genotypes were included on thegenotyping plate. Individuals with confirmed causative mutationswere excluded. CSF Ab42 was measured with the Innotest Ab42 ELISA(enzyme-linked immunosorbent assay) kit (Innogenetics) according toprevious procedures (7). CSF apoE concentration in individuals fromour cohort for whom CSF had been analyzed by the company Rules-Based Medicine was quantified with multianalyte profiling (40). PIB-PET assessment, performed within 2 years of lumbar puncture to collectCSF, was performed as reported previously (27). All procedures wereapproved by Washington University’s Human Protection Office, andwritten informed consent was obtained from all participants beforestudy entry.

AnimalsHomozygous PDAPP (APPV717F) mice lacking apoE on a mixedbackground composed of DBA/2J, C57BL/6J, and Swiss Websterwere crossed with mice expressing APOE e2, e3, and e4 under con-trol of mouse regulatory elements on a C57BL/6J background (giftfrom P. Sullivan at Duke University) (14). Resulting mice were in-tercrossed to generate homozygous PDAPP/TRE mice, which werethen maintained via a vertical breeding strategy. Male and femalePDAPP/TRE mice were used throughout experiments. For exper-iments involving TRE mice with murine APP, 2.5-month-old malelittermates on a C57BL/6J background from each APOE genotypewere purchased from Taconic. All animal procedures were per-formed according to protocols accepted by the Animal StudiesCommittee at Washington University School of Medicine.

Tissue preparation and quantification of Ab/amyloid burdenIn vivo microdialysis was performed in the left hemisphere of 20- to21-month-old mice, after which mice were immediately perfusedtranscardially, fixing brains in 4% paraformaldehyde overnight. Afterbrains were placed in 30% sucrose, the contralateral (noncannulated)hemisphere was sectioned on a freezing-sliding microtome. Serial 50-mmcoronal sections were taken from the rostral anterior commissure throughthe caudal extent of the hippocampus, staining sections with biotinylated3D6 antibody (anti-Ab1–5) for Ab immunostaining quantification andX-34 dye for amyloid load quantification. Slides were scanned in batchmode with the NanoZoomer slide scanner system (Hamamatsu Photonics),capturing images in bright-field mode (Ab immunostaining) or fluo-rescent mode (X-34). NDP viewer software was used to export imagesfrom slides before quantitative analysis with ImageJ software [NationalInstitutes of Health (NIH)]. Using three sections per mouse separatedeach by 300 mm (corresponding to bregma −1.7, −2.0, and −2.3 mm inmouse brain atlas), we determined the percentage of area occupied byimmunoreactive Ab or amyloid (X-34–positive signal) in a blinded fash-ion, thresholding each slide to minimize false-positive signal, as de-scribed (31).

In vivo microdialysisIn vivo microdialysis in 20- to 21-month-old and 3- to 4-month-oldPDAPP/TRE mice was performed essentially as described to assesssteady-state concentrations of various analytes in the hippocampalISF with a 38-kD cutoff dialysis probe (Bioanalytical Systems Inc.)(29, 32). ISF exchangeable Ab1–x (eAb1–x) was collected with a flowrate of 1.0 ml/min, whereas ISF eAbx–42 and urea were collected with aflow rate of 0.3 ml/min. For clearance experiments, a stable baseline ofISF eAb1–x concentration was obtained with a constant flow rate of




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1.0 ml/min before intraperitoneally injecting each mouse with 10 mg/kgof a selective g-secretase inhibitor (LY411,575), which was prepared bydissolving in dimethyl sulfoxide (DMSO)/PBS/propylene glycol. Theelimination of eAb1–x from the ISF followed first-order kinetics; there-fore, for each mouse, t1/2 for eAb was calculated with the slope, k′, ofthe linear regression that included all fractions until the concentrationof eAb stopped decreasing (t1/2 = 0.693/k, where k = 2.303k′). Micro-dialysis using the zero flow extrapolated method was performed byvarying the flow rates from 0.3 to 1.6 ml/min, as described (31). Zeroflow data for each mouse were fit with an exponential decay regressionwith GraphPad Prism 5.0 software (33).

Quantitative measurements of ISF eAbQuantitative measurements of Ab collected from in vivo microdialysisfractions were performed with sensitive sandwich ELISAs. For humanAb1–x quantification, ELISA plates were coated with m266 antibody(anti-Ab13–28), and biotinylated 3D6 antibody (anti-Ab1–5) was usedfor detection. For Abx–42 ELISAs, HJ7.4 (anti-Ab35–42) antibodywas used to capture, followed by biotinylated HJ5.1 antibody to detect(anti-Ab13–28).

Biochemical analyses of hippocampal homogenates fromyoung PDAPP/TRE miceAfter transcardial perfusion with heparinized PBS, brain tissue wasmicrodissected and immediately frozen at −80°C. Hippocampal tissuewas manually Dounce-homogenized with 75 strokes in radioimmu-noprecipitation assay (RIPA) buffer [50 mM tris-HCl (pH 7.4), 150 mMNaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA] containing acocktail of protease inhibitors (Roche). Total protein concentration inhippocampal homogenates was determined with a BCA protein assaykit (Pierce). Equivalent amounts of protein (50 mg) were loaded on 4to 12% bis-tris gels (Invitrogen) for SDS-PAGE before transferring pro-tein to 0.2-mmnitrocellulose membranes. Immediately after transfer, blotswere boiled for 10 min before blocking and incubation with 82E1 anti-body (anti-Ab1–16; IBL) to detect C99. Loading was normalized bystripping blots and reprobing with a-tubulin antibody (Sigma). Normal-ized band intensities were quantified with ImageJ software (NIH).

b-Secretase activity in hippocampal lysates was assessed with a com-mercially available kit (#P2985; Invitrogen) that relies on fluorescenceresonance energy transfer (FRET) that results from b-secretase cleav-age of a fluorescent peptide based on the APP sequence (Rhodamine-EVNLDAEFK-Quencher). Briefly, 5 mg of protein per sample wasmixed with sample buffer and b-secretase substrate, monitoring flu-orescence signal every minute for 120 min with a Synergy2 BioTek(BioTek Instruments Inc.) plate reader (excitation, 545 nm/emission,585 nm). Because the kinetics of the reaction for all samples werereliably linear in the 20- to 60-min interval, reaction velocity [relativefluorescence units (RFUs) per minute] was calculated and reportedover this interval for all samples. Specificity of b-secretase activitywas validated with a commercially available b-secretase inhibitor.

In vivo stable isotopic labeling kineticsFSRs of Ab were measured in hippocampal lysates from youngPDAPP/TRE mice with a method adapted from the in vivo stableisotopic labeling kinetics technique we have previously described inhumans (43) (detailed Materials and Methods available in the Supple-mentary Material). Briefly, after mice were injected intraperitoneallywith [13C6]leucine (200 mg/kg), brain tissue harvesting and plasma

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collection were performed 20 and 40 min after injection. Whole hip-pocampus was lysed with 1% Triton X-100 lysis buffer containingprotease inhibitors, and Ab in the extracts was immunoprecipitatedwith HJ5.2 antibody (anti-Ab13–28). After trypsin digestion of immu-noprecipitated Ab, LC-MS was performed to measure the relativeabundance of labeled to unlabeled tryptic Ab peptide, which was cal-ibrated with a standard curve of Ab secreted from H4 APP695DNLneuroglioma cells. FSR curves were then generated based on theamount of labeled to unlabeled Ab present 20 and 40 min after[13C6]leucine injection, normalized to the amount of free leucine inthe plasma, which was measured by gas chromatography (GC)–MS.

Statistical analysisUnless indicated otherwise, differences among group means wereassessed with a one-way analysis of variance (ANOVA) followedby Tukey’s post hoc test for multiple comparisons when the ANOVAwas significant. Levels of significance were indicated as follows: *P < 0.05;**P < 0.01; ***P < 0.001. Analyses were performed with GraphPadPrism 5.0 software; human data were analyzed with SAS 9.2 software.

SUPPLEMENTARY MATERIAL

www.sciencetranslationalmedicine.org/cgi/content/full/3/89/89ra57/DC1Materials and MethodsFig. S1. eAb1–x concentration measured with equivalent microdialysis probe function differsaccording to apoE isoform.Fig. S2. PBS-soluble Ab40 levels and APP levels do not vary according to apoE isoform in thecontext of murine APP/Ab.Fig. S3. Soluble Ab clearance from the ISF is apoE isoform–dependent in young PSAPP/TREmice.Fig. S4. ApoE concentration is higher in the context of apoE2 in both humans and PDAPP/TREmice.Fig. S5. Full-length total APP levels do not differ according to apoE isoform.Table S1. Serial extraction of Ab40 and Ab42 from hippocampi of young PDAPP/TRE mice.References

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66. Acknowledgments: We acknowledge investigators and staff of the Charles F. andJoanne Knight ADRC’s clinical and genetics cores and the Adult Children Study’s BiomarkerCore for CSF Ab42. LY411,575 was a gift from P. C. May (Eli Lilly and Co.). Funding: This workwas supported by NIH grants AG13956 (D.M.H.), NS034467 (D.M.H.), AG034004 (J.M.C.), andP30NS069329 (J.K.); the American Health Assistance Foundation (J.K. and D.M.H.); DK56341(B.W.P., Nutrition Obesity Research Center); NIH grants AG05681 (J.C.M.), AG03991 (J.C.M.),AG026276 (J.C.M.), P30NS069329-01 (C.C.), and K23-AG03094601 (R.J.B.); NIH NeuroscienceBlueprint Center Core Grant P30-NS057105; and Eli Lilly and Pfizer to Washington University(D.M.H.). Author contributions: J.M.C. wrote and edited the manuscript with critical eval-uation/interpretation from co-authors. J.M.C., F.R.S., H.J., and D.M.H. performed/designedresearch in Figs. 1 to 6 and the Supplementary Material. A.M.F. and J.C.M. provided hu-man subject data for Fig. 1 and Table 1. C.C., A.M.G., and K.R.B. provided human data forfig. S3A. J.K., J.M.C., K.G.M., and R.J.B. designed/performed experiments for Fig. 6. J.M.C.,J.K., and D.M.H. analyzed the data. R.B.D. and S.M.P. contributed valuable reagents.Competing interests: D.M.H. and R.J.B. are scientific advisors to C2N Diagnostics, whichuses the SILK methodology in human studies and are co-inventors on U.S. patent7,892,845 “Methods for measuring the metabolism of neurally derived biomolecules invivo.” Washington University, with D.M.H. and R.J.B. as co-inventors, has also submittedthe U.S. nonprovisional patent application “Methods for measuring the metabolism ofCNS derived biomolecules in vivo,” serial #12/267,974. D.M.H. is also on the scientific ad-visory boards of Satori, En Vivo. D.M.H., J.C.M., and A.M.G. consult for Pfizer and BristolMyers Squibb. D.M.H. also consults for Innogenetics. J.C.M. also consults for AstraZeneca,Eisai, Janssen, Genentech, Eli Lilly, Merck, Novartis, Otsuka, and Schering Plough. A.M.G.also consults for AstraZeneca and Genentech. All other authors declare no competinginterests.

Submitted 18 January 2011Accepted 10 June 2011Published 29 June 201110.1126/scitranslmed.3002156

Citation: J. M. Castellano, J. Kim, F. R. Stewart, H. Jiang, R. B. DeMattos, B. W. Patterson,A. M. Fagan, J. C. Morris, K. G. Mawuenyega, C. Cruchaga, A. M. Goate, K. R. Bales,S. M. Paul, R. J. Bateman, D. M. Holtzman, Human apoE isoforms differentially regulate brainamyloid-b peptide clearance. Sci. Transl. Med. 3, 89ra57 (2011).

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Human apoE Isoforms Differentially Regulate Brain Amyloid- Peptide Clearance