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

Jacob M. Basak, Jungsu Kim, Yuriy Pyatkivskyy, Kristin R. Wildsmith, Hong Jiang, Maia Parsadanian, Bruce W. Patterson, Randall J. Bateman, David M. Holtzman

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Article number14
JournalMolecular Neurodegeneration
Volume7
Issue number1
DOIs
StatePublished - 2012
Externally publishedYes

Fingerprint

Isotope Labeling
Apolipoproteins
Amyloid
Apolipoproteins E
Adenosine Triphosphate
Brain
Neurodegenerative Diseases
Proteins
Mass Spectrometry
Apolipoproteins A
LDL Receptors
Leucine
Alzheimer Disease
Costs and Cost Analysis

Keywords

  • ABCA1
  • Amyloid beta
  • Apolipoprotein E
  • Kinetics
  • LDLR
  • Multiple reaction monitoring mass spectrometry
  • Protein turnover
  • Stable isotope

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience
  • Clinical Neurology
  • Molecular Biology

Cite this

Basak, J. M., Kim, J., Pyatkivskyy, Y., Wildsmith, K. R., Jiang, H., Parsadanian, M., ... Holtzman, D. M. (2012). Measurement of apolipoprotein e and amyloid β clearance rates in the mouse brain using bolus stable isotope labeling. Molecular Neurodegeneration, 7(1), [14]. https://doi.org/10.1186/1750-1326-7-14

Measurement of apolipoprotein e and amyloid β clearance rates in the mouse brain using bolus stable isotope labeling. / Basak, Jacob M.; Kim, Jungsu; Pyatkivskyy, Yuriy; Wildsmith, Kristin R.; Jiang, Hong; Parsadanian, Maia; Patterson, Bruce W.; Bateman, Randall J.; Holtzman, David M.

In: Molecular Neurodegeneration, Vol. 7, No. 1, 14, 2012.

Research output: Contribution to journalArticle

Basak, JM, Kim, J, Pyatkivskyy, Y, Wildsmith, KR, Jiang, H, Parsadanian, M, Patterson, BW, Bateman, RJ & Holtzman, DM 2012, 'Measurement of apolipoprotein e and amyloid β clearance rates in the mouse brain using bolus stable isotope labeling', Molecular Neurodegeneration, vol. 7, no. 1, 14. https://doi.org/10.1186/1750-1326-7-14
Basak, Jacob M. ; Kim, Jungsu ; Pyatkivskyy, Yuriy ; Wildsmith, Kristin R. ; Jiang, Hong ; Parsadanian, Maia ; Patterson, Bruce W. ; Bateman, Randall J. ; Holtzman, David M. / Measurement of apolipoprotein e and amyloid β clearance rates in the mouse brain using bolus stable isotope labeling. In: Molecular Neurodegeneration. 2012 ; Vol. 7, No. 1.
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