In the proteomics era, significant effort has been devoted to developing techniques that accurately and efficiently determine differences in protein amounts under normal physiological conditions and disease states . However, quantifying protein turnover rates at both a cellular and systemic level is also necessary for a complete understanding of the mechanisms dictating changes in protein levels . Several neurodegenerative diseases are characterized by the accumulation of protein aggregates in the brain, including Alzheimer’s disease (AD) , Parkinson’s disease , Huntington’s disease , and frontotemporal dementia. Although the underlying cause of protein aggregation in these diseases remains unclear, it is likely due to abnormal proteostasis caused by alterations in protein production or clearance [7, 8]. Therefore, the development of techniques that can assess protein dynamics in the brain are fundamental for advancing our understanding of these disease processes and aiding the conception of innovative therapeutics.
Stable isotope tracers have been in use for many years to facilitate the analysis of protein turnover in cells and whole organisms . Mass spectrometry (MS) has proven an effective tool for the analysis of stable isotope incorporation into individual proteins . Liquid chromatography-mass spectrometry (LC-MS) analysis allows for the comparison of the relative abundance of labeled to unlabeled peptides due to their mass separation. Coupling stable isotope amino acid labeling with LC-MS has been applied to quantify protein synthesis and degradation in yeast , mammalian cell lines [12, 13], and small animals [14, 15]. However, protein turnover studies in animals have been limited due to issues with MS detection sensitivity and accurate label quantification, along with difficulties in achieving cost-effective and practical methods for tracer administration. Recently, Bateman et al. have developed a method to measure the dynamics of low abundance proteins in the cerebral spinal fluid (CSF) of humans . In this technique, 13 C6-leucine is injected intravenously into research participants and samples of the lumbar CSF are serially collected over a predetermined time period. The synthesis and clearance rates of proteins are then measured by quantifying the appearance and disappearance of the 13 C6-leucine in proteins over time via LC-MS [16, 17]. The value of this technique has specifically been highlighted for the amyloid β (Aβ) peptide, which accumulates in the brains of AD patients and has been implicated in the disease pathogenesis . Application of stable isotope labeling to studies of Aβ dynamics have demonstrated impaired Aβ clearance in individuals with AD and the ability of a gamma secretase inhibitor to decrease Aβ synthesis in the CNS [8, 16, 18].
Apolipoprotein E (apoE) plays a central role in the transport of cholesterol by functioning as a ligand for the receptor-mediated endocytosis of lipoprotein particles into cells . In humans, three common apoE isoforms exist (apoE2, apoE3, and apoE4) that differ by amino acids at positions 112 and 158. ApoE4 is currently the strongest known genetic risk factor for late-onset AD, and as a result significant effort has been devoted to understanding apoE’s physiological function in the brain along with its role in AD pathogenesis . A major hypothesis for how apoE4 affects the onset of AD contends that apoE promotes the aggregation of Aβ into amyloid plaques in the brain, either through impairing Aβ clearance [21, 22], directly regulating the propensity of Aβ to form amyloid fibrils [23, 24], or both mechanisms. Independent of apoE isoform, the amount of apoE in the brain appears to be critical for determining the extent of amyloid deposition [25, 26]. Therefore, finding proteins and molecular pathways that regulate apoE levels in the brain has been the focus of significant attention in the AD research community.
Increasing the endocytosis of apoE via the low-density lipoprotein receptor (LDLR) has been shown to decrease apoE levels in the mouse brain, likely through increased apoE clearance . Brain levels of the cholesterol transporter ATP binding cassette A1 (ABCA1) also alter the amount of apoE and the extent of apoE lipidation in mice. Both the overexpression and deletion of ABCA1 in the mouse brain resulted in a decrease in apoE protein level [28–30]. In transgenic mice that overexpress the human amyloid precursor protein (APP), increasing ABCA1 levels caused a significant decrease in amyloid deposition in the brain . Therefore, it has been hypothesized that ABCA1 regulates amyloid levels in the brain by altering Aβ clearance, and this could occur through the effect of ABCA1 on either apoE lipidation or total apoE levels. However, no study has yet addressed this hypothesis formally by studying apoE and Aβ metabolism in the brain of mice with altered ABCA1 levels.
Herein, we describe a novel method to study protein clearance in the mouse brain. We show that following a bolus injection of 13 C6-leucine into mice, LC-MS analysis of brain tissue can be used to measure the fractional clearance rate (FCR) of individual proteins. We validate our technique by analyzing changes in the clearance of apolipoprotein E (apoE) in mice that have been genetically engineered to overexpress LDLR. Finally, we use 13 C6-leucine labeling coupled to LC-MS analysis to measure the clearance of apoE and Aβ in APP transgenic mice that either overexpress or lack ABCA1. Both overexpression and deletion of ABCA1 resulted in an increased fractional clearance rate of apoE. However, ABCA1 levels did not alter the clearance rate of Aβ in the mouse brain, suggesting ABCA1 acts via another pathway, such as directly influencing Aβ aggregation, to regulate amyloid deposition. These results highlight the power of our stable isotope labeling technique in elucidating mechanisms of protein clearance from the brain, and suggest that future studies could use this technique to study the clearance pathways of other proteins implicated in neurodegenerative disease.