In vivo human apolipoprotein E isoform fractional turnover rates in the CNS

Abstract

Apolipoprotein E (ApoE) is the strongest genetic risk factor for Alzheimer's disease and has been implicated in the risk for other neurological disorders. The three common ApoE isoforms (ApoE2, E3, and E4) each differ by a single amino acid, with ApoE4 increasing and ApoE2 decreasing the risk of Alzheimer's disease (AD). Both the isoform and amount of ApoE in the brain modulate AD pathology by altering the extent of amyloid beta (Aβ) peptide deposition. Therefore, quantifying ApoE isoform production and clearance rates may advance our understanding of the role of ApoE in health and disease. To measure the kinetics of ApoE in the central nervous system (CNS), we applied in vivo stable isotope labeling to quantify the fractional turnover rates of ApoE isoforms in 18 cognitively-normal adults and in ApoE3 and ApoE4 targeted-replacement mice. No isoform-specific differences in CNS ApoE3 and ApoE4 turnover rates were observed when measured in human CSF or mouse brain. However, CNS and peripheral ApoE isoform turnover rates differed substantially, which is consistent with previous reports and suggests that the pathways responsible for ApoE metabolism are different in the CNS and the periphery. We also demonstrate a slower turnover rate for CSF ApoE than that for amyloid beta, another molecule critically important in AD pathogenesis.

Figure 1. Plasma ApoE Isoforms have different turnover kinetics(ApoE4>ApoE3>ApoE2).

¹³C₆-leu incorporation into plasma ApoE isoforms was analyzed from a representative individual for each genotype. The ¹³C₆-leu incorporation peaked at 10 h with ApoE4’s maximum reaching 19.2%, ApoE3 9.9% and ApoE2 4.4%. The different isoforms have different clearance rates as indicated by the slope of the Ln plots (A–D insets). A–D : A , E3/3; B, E4/4; C, E3/4; D, E2/4 (Blue: E3/E4 LAVYQAGAR, black: E3/E2 LGADMEDVcGR, red: E4 LGADMEDVR, green: E2 cLAVYQAGAR).

Figure 2. Representative compartmental model analyses of plasma ApoE.

A. Peripheral ApoE compartmental model has 4 adjustable parameters: the plasma ApoE FTR, the rate constants for bi-directional exchange with the non-plasma space, and a scaling factor to account for isotopic dilution. B. ApoE4 peptide LAVYQAGAR from an ApoE2/4 subject. C. ApoE3 peptide LGADMEDVcGR from an ApoE3/3 subject. D, ApoE2 peptide cLAVYQAGAR from the same ApoE2/4 subject as in B. Solid line represents model fit to the data.

Figure 3. ¹³C₆-leucine labeling in CNS-ApoE isoforms in cognitively-normal young individuals.

¹³C₆-leu incorporation into ApoE isoform-specific peptides was quantified by nanoLC/MS/MS. The ratios of the labeled to unlabeled ApoE were normalized to the plasma ¹³C₆-leu precursor levels during the production phase (h0–22) to reduce inter-subject variability due to differential TTR of plasma leucine precursor. Individuals were grouped by genotype and their averages are shown in A–D: A , E3/3 (n = 8); B, E4/4 (n = 2); C, E3/4 (n = 6); D, E2/4 (n = 2) (blue square: LAVYQAGAR, black circle: LGADMEDVcGR, red triangle: LGADMEDVR, green diamond: cLAVYQAGAR.). The linear regression of the means for h4–16 and h28–44 is shown for LAVYQAGAR to demonstrate the time points used for each individual’s FSR and monoexponential slope FCR calculations. E. The averages of the common peptide (LAVYQAGAR) for all four genotypes (n = 18) were compared (green circle: E3/3; red triangle: E4/4; blue square: E3/4; black diamond: E2/4). Error bars represent standard error of the mean (SEM).

Figure 4. CNS kinetic modeling curves.

A, CNS-ApoE compartmental model was used to describe whole-system CNS-ApoE turnover kinetics. The model is based on data from plasma leucine and CSF-ApoE TTRs (solid triangles). The plasma leucine TTR time course for a given subject is used as a “forcing function” to define the tracer availability for ApoE synthesis. The CNS-ApoE system comprises a delay element and a compartment that turns over, and accounts for isotopic dilution of the plasma leucine. The model has 3 adjustable parameters: the shape of the ApoE TTR time course is modified by adjusting the delay time and the rate constant for ApoE turnover, and the magnitude of the ApoE TTR is scaled by varying the degree of isotopic dilution. B–D, A typical compartmental model analysis from a single, representative, ApoE3/3 subject. B, Plasma leucine TTR remains elevated and does not return to baseline enrichment immediately after the tracer infusion is halted. C–D, The ApoE TTR time course exhibits a long time delay and sigmoid rise to a peak enrichment which is well described by the model. C, ApoE3 peptide LAVYQAGAR; D, ApoE3 peptide LGADMEDVcGR. Solid line represents model fit to the data.

Figure 5. Brain ApoE kinetics in ApoE3 and ApoE4 targeted replacement mice.

ApoE was extracted from brains of ApoE3/E3 and ApoE4/E4 mice labeled with ¹³C₆-leucine. Similar kinetics were observed for ApoE3 and ApoE4 mice with monoexponential slopes of 6.2±0.48%/h and 4.8±1.12%/h, respectively (blue: ApoE3, black: ApoE4, n = 3–6 mice per time point, P  = 0.2817, error bars represent SEM).

Figure 6. CNS-ApoE has slower kinetics than CNS-Aβ.

The average of 4 YNC participants’ ApoE and Aβ (total) ¹³C₆-leucine enrichment curves are shown. Aβ reaches a higher TTR than ApoE and clears the ¹³C₆-leucine label 4× faster than ApoE. ApoE (total:LAVYQAGAR, black circle); Aβ (total, red triangles) (n = 4, error bars represent SEM).