Human apoE isoforms differentially regulate brain amyloid-β peptide clearance

Abstract

The apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for late-onset, sporadic Alzheimer's disease (AD). The APOE ε4 allele markedly increases AD risk and decreases age of onset, likely through its strong effect on the accumulation of amyloid-β (Aβ) peptide. In contrast, the APOE ε2 allele appears to decrease AD risk. Most rare, early-onset forms of familial AD are caused by autosomal dominant mutations that often lead to overproduction of Aβ(42) peptide. However, the mechanism by which APOE alleles differentially modulate Aβ accumulation in sporadic, late-onset AD is less clear. In a cohort of cognitively normal individuals, we report that reliable molecular and neuroimaging biomarkers of cerebral Aβ deposition vary in an apoE isoform-dependent manner. We hypothesized that human apoE isoforms differentially affect Aβ clearance or synthesis in vivo, resulting in an apoE isoform-dependent pattern of Aβ accumulation later in life. Performing in vivo microdialysis in a mouse model of Aβ-amyloidosis expressing human apoE isoforms (PDAPP/TRE), we find that the concentration and clearance of soluble Aβ in the brain interstitial fluid depends on the isoform of apoE expressed. This pattern parallels the extent of Aβ deposition observed in aged PDAPP/TRE mice. ApoE isoform-dependent differences in soluble Aβ metabolism are observed not only in aged but also in young PDAPP/TRE mice well before the onset of Aβ deposition in amyloid plaques in the brain. Additionally, amyloidogenic processing of amyloid precursor protein and Aβ 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 contribute to AD risk by differentially regulating clearance of Aβ from the brain, suggesting that Aβ clearance pathways may be useful therapeutic targets for AD prevention.

Fig. 1. Biomarkers of amyloid differ according to APOE genotype in cognitively normal individuals

(A) Percentage of individuals (n=283) with [CSF Aβ42] < 500 pg/mL according to the following APOE genotypes: ε2/ε3, ε3/ε3, ε3/ε4, and ε4/ε4. Number in parentheses indicates number of individuals for each group. (B) Percentage of PIB+ individuals (n=153) according to APOE genotype: ε2/ε3, ε3/ε3, ε3/ε4, and ε4/ε4. Individuals with mean cortical binding potential for Pittsburgh compound B (MCBP) > 0.18 were considered PIB+. Number in parentheses indicates number of individuals for each group. χ² analyses for proportions in (A) (χ²(3)=22.1785, P=5.99 × 10⁻⁵) and (B) (χ²(3)=14.4735, P=2.33 × 10⁻³) were performed; follow-up χ² tests for pairwise comparisons of proportions were performed using Benjamini and Hochberg’s linear step-up adjustment to control for type I error. *P<0.05,**P<0.01,***P<0.001.

Fig. 2. Aβ/amyloid deposition varies according to apoE isoform in old PDAPP/TRE mice

(A-C) Representative coronal brain sections from 20- to 21-month-old, sex-matched PDAPP/E2 (A), PDAPP/E3 (B), and PDAPP/E4 (C) mice. Aβ immunostaining was performed using anti-Aβ antibody (biotinylated-3D6). Scale bars, 50 μm. (D) Quantification of the area of the hippocampus occupied by Aβ immunostaining (n=7 mice/group). *P<0.05, **P<0.01. (E-G) Representative coronal brain sections from 20- to 21-month-old PDAPP/E2 (E), PDAPP/E3 (F), and PDAPP/E4 (G) mice. Amyloid was detected using the congophilic fluorescent dye, X-34. Scale bars, 50 μm. (H) Quantification of the area of hippocampus occupied by X-34 staining (n=7 mice/group). When one-way ANOVA was significant, differences among groups were assessed using Tukey’s post hoc test for multiple comparisons *P<0.05, ***P<0.001. Values represent means ± SEM.

Fig. 3. Soluble Aβ concentration and clearance in the brain ISF of old mice is human apoE isoform-dependent

(A) Mean steady state concentrations of eAβ_1-X (exchangeable Aβ) from sampling hippocampal ISF in old, sex-matched PDAPP/E2, PDAPP/E3, and PDAPP/E4 mice, measured by enzyme-linked immunosorbent assay (ELISA) (n=6 to 7 mice per group; 20 to 21 months old). (B) Schematic diagram of a typical clearance experiment in which a stable baseline period is obtained, followed by intraperitoneal (i.p.) injection of LY411,575 (10mg/kg) to halt Aβ production. Aβ concentrations during the elimination phase are transformed with the common logarithm. Log-transformed values are fit with a linear regression, allowing calculation of slope, k’. eAβ t_1/2 = 0.693/k, where k = 2.303k’. (C) eAβ t_1/2 from clearance experiments performed with the mice in (A) after stable baseline measurement of eAβ_1-x. When one-way ANOVA was significant, differences among groups were assessed using Tukey’s post hoc test for multiple comparisons (*P<0.05). Values represent means ± SEM.

Fig. 4. ApoE isoform-dependent differences in soluble Aβ concentration and clearance exist prior to the onset of Aβ deposition

(A) An exponential decay regression was used to fit the concentrations of eAβ_1-x measured by ELISA at each flow rate for individual mice from groups of young, sex-matched PDAPP/TRE mice (n=6 mice per group; 3 to 4 months old). The equations from the individual regressions were used to calculate [eAβ_1-x] at x=0 for each mouse, representing the in vivo concentration of eAβ_1-x recoverable by microdialysis. (B) Mean in vivo concentrations of eAβ_1-x (pg/mL) calculated from the method in (A). (C) Mean concentrations of Aβ_x-42 (pg/mL) collected from the hippocampal ISF of young, sex-matched PDAPP/TRE mice using a flow rate of 0.3μl/min (n=8 mice per group; 3-4 months old). (D) eAβ t_1/2 from clearance experiments in young, sex-matched PDAPP/TRE mice after stable baseline measurement of eAβ_1-x (n=10 to 11 mice per group; 3 to 4 months old). When one-way ANOVA was significant, differences among groups were assessed using Tukey’s post hoc test for multiple comparisons *P<0.05, **P<0.01, ***P<0.001. Values represent means ± SEM.

Fig. 5. Amyloidogenic processing of APP does not vary according to human apoE isoform

(A) Representative Western blot of the proximal amyloidogenic metabolite, C99, from hippocampal homogenates (extracted with RIPA buffer) from young, sex-matched PDAPP/TRE mice. C99 was detected using 82E1 antibody. All bands were normalized to α-tubulin band intensity (n=9 mice per group; 3 to 4 months old). (B) Quantification of C99 levels after normalizing each band’s intensity to α-tubulin band intensity. (C) Quantification of β-secretase activity in hippocampal homogenates from young PDAPP/TRE mice using a sensitive FRET assay. Homogenates were incubated with fluorescent APP substrate, resulting in β-cleavage that could be followed by fluorescence increase (emission, 585 nm). The interval over which kinetics were linear was used for quantification of reaction velocity [relative fluorescence units (RFU)/min] for each sample. One-way ANOVA revealed no significant differences among groups. Values represent means ± SEM.

Fig. 6. Rates of Aβ synthesis do not differ according to human apoE isoform in PDAPP/TRE mice

(A) Aβ detection in hippocampal lysates from young PDAPP/TRE mice by TSQ Vantage triple quadrupole mass spectrometry. Left, representative total ion count multiple reaction monitoring (MRM) peak of the unlabeled Aβ tryptic peptide, LVFFAEDVGSNK, (m/z = 663.340). Right, MRM peak for [¹³C₆]-leucine labeled Aβ (m/z = 666.350). (B) Standard curve generated with known quantity of [¹³C₆]-leucine labeled and unlabeled Aβ. Aβ secreted from H4-APP695ΔNL neuroglioma cells incubated with labeled/unlabeled leucine was immunoprecipitated with HJ5.2 antibody (anti-Aβ_13-28), followed by trypsin digestion. Aβ_17-28 fragments were analyzed on a TSQ Vantage mass spectrometer. The expected percentage of labeled Aβ versus measured percentage was fit by linear regression. Variance is reported with 95% confidence interval. (C) Relative FSRs of Aβ from hippocampi of PDAPP/TRE mice intraperitoneally injected with [¹³C₆]-leucine (200mg/kg) (n=5 to 6 mice per group; 4 to 5 months old). Relative FSRs of Aβ were calculated from the ratio of [¹³C₆]-leucine labeled to unlabeled Aβ. [¹³C₆]/[¹²C₆]-Aβ ratio was normalized to the ratio of labeled to unlabeled free leucine in plasma (determined by GC-MS). Mass spectrometry data were normalized with the media standard curve in (B). One-way ANCOVA (analysis of covariance) revealed no significant differences among groups. Values represent means ± SEM.