Measurement of apolipoprotein E and amyloid β clearance rates in the mouse brain using bolus stable isotope labeling

BACKGROUND: Abnormal proteostasis due to alterations in protein turnover has been postulated to play a central role in several neurodegenerative diseases. Therefore, the development of techniques to quantify protein turnover in the brain is critical for understanding the pathogenic mechanisms of these diseases. We have developed a bolus stable isotope-labeling kinetics (SILK) technique coupled with multiple reaction monitoring mass spectrometry to measure the clearance of proteins in the mouse brain. RESULTS: Cohorts of mice were pulse labeled with (13) C(6)-leucine and the brains were isolated after pre-determined time points. The extent of label incorporation was measured over time using mass spectrometry to measure the ratio of labeled to unlabeled apolipoprotein E (apoE) and amyloid β (Aβ). The fractional clearance rate (FCR) was then calculated by analyzing the time course of disappearance for the labeled protein species. To validate the technique, apoE clearance was measured in mice that overexpress the low-density lipoprotein receptor (LDLR). The FCR in these mice was 2.7-fold faster than wild-type mice. To demonstrate the potential of this technique for understanding the pathogenesis of neurodegenerative disease, we applied our SILK technique to determine the effect of ATP binding cassette A1 (ABCA1) on both apoE and Aβ clearance. ABCA1 had previously been shown to regulate both the amount of apoE in the brain, along with the extent of Aβ deposition, and represents a potential molecular target for lowering brain amyloid levels in Alzheimer's disease patients. The FCR of apoE was increased by 1.9- and 1.5-fold in mice that either lacked or overexpressed ABCA1, respectively. However, ABCA1 had no effect on the FCR of Aβ, suggesting that ABCA1 does not regulate Aβ metabolism in the brain. CONCLUSIONS: Our SILK strategy represents a straightforward, cost-effective, and efficient method to measure the clearance of proteins in the mouse brain. We expect that this technique will be applicable to the study of protein dynamics in the pathogenesis of several neurodegenerative diseases, and could aid in the evaluation of novel therapeutic agents

In Vivo Human Apolipoprotein E Isoform Fractional Turnover Rates in the CNS

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

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

<p>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 <b>A–D</b>: <b><i>A</i></b><i>,</i> E3/3 (n = 8); <b><i>B</i></b>, E4/4 (n = 2); <b><i>C</i></b>, E3/4 (n = 6); <b><i>D</i></b>, 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. <b>E.</b> 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).</p

Peripheral Plasma ApoE Kinetic Parameters.

<p>Peripheral Plasma ApoE Kinetic Parameters.</p

Brain ApoE kinetics in ApoE3 and ApoE4 targeted replacement mice.

<p>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).</p

CNS-ApoE Kinetic Parameters.

*<p>Linear fits with r²<0.8 were excluded from mean calculations.</p