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

Basak JM, Kim J, Pyatkivskyy Y, Wildsmith KR, Jiang H, Parsadanian M, Patterson BW, Bateman RJ, Holtzman DM - Mol Neurodegener (2012)

Bottom Line: 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.However, ABCA1 had no effect on the FCR of Aβ, suggesting that ABCA1 does not regulate Aβ metabolism in the brain.Our SILK strategy represents a straightforward, cost-effective, and efficient method to measure the clearance of proteins in the mouse brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Saint Louis, Missouri 63110, USA.

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 13C6-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.

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Related in: MedlinePlus

Experimental schematic for stable isotope labeling and isolation of mouse brain proteins. (A) Cohorts of mice were pulse-labeled with 13 C6-leucine via a bolus intraperitoneal injection (200 mg/kg of body weight). After a pre-determined time following the 13 C6-leucine administration, the mice were euthanized and the brains were removed. The brain tissue was then lysed using a 1% Triton X-100 lysis buffer, and the protein of interest was immunoprecipitated from the brain lysate (apoE is shown as an example). The precipitated proteins were then eluted from the antibody beads and subjected to trypsin digestion. The resulting peptide mixture was separated and analyzed via ultra performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) (yellow = apoE, blue = sepharose bead, red = trypsin). (B) To observe the bioavailability of the 13 C6-leucine, plasma samples were collected at sequential time points following the bolus injection and subjected to GC-MS analysis. The tracer-to-tracee ratio (TTR, shown as labeled/unlabeled leucine) was then measured by quantifying the relative amounts of 13 C6-leucine and dividing by the amount of unlabeled leucine in each sample. Each time point in the graph represents the average value from 5–6 individual mice
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Figure 1: Experimental schematic for stable isotope labeling and isolation of mouse brain proteins. (A) Cohorts of mice were pulse-labeled with 13 C6-leucine via a bolus intraperitoneal injection (200 mg/kg of body weight). After a pre-determined time following the 13 C6-leucine administration, the mice were euthanized and the brains were removed. The brain tissue was then lysed using a 1% Triton X-100 lysis buffer, and the protein of interest was immunoprecipitated from the brain lysate (apoE is shown as an example). The precipitated proteins were then eluted from the antibody beads and subjected to trypsin digestion. The resulting peptide mixture was separated and analyzed via ultra performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) (yellow = apoE, blue = sepharose bead, red = trypsin). (B) To observe the bioavailability of the 13 C6-leucine, plasma samples were collected at sequential time points following the bolus injection and subjected to GC-MS analysis. The tracer-to-tracee ratio (TTR, shown as labeled/unlabeled leucine) was then measured by quantifying the relative amounts of 13 C6-leucine and dividing by the amount of unlabeled leucine in each sample. Each time point in the graph represents the average value from 5–6 individual mice

Mentions: The outline of our experimental design for the labeling of mice and tissue processing is shown in Figure 1A. Mice were intraperitoneally (IP) injected with a bolus of 13 C6-leucine, a non-radioactive stable isotope form of the amino acid leucine. We chose 13 C6-labeled leucine because it is one of the essential amino acids that rapidly crosses the blood brain barrier via facilitative neutral amino acid transport [34]. Intraperitoneal administration of the label was chosen because it is straightforward and quick, and it allows for high bioavailability upon absorption into the bloodstream. Following the injection, we observed a rapid increase in the amount of 13 C6-leucine in the plasma of the mice over the first hour, as measured by the ratio of labeled to unlabeled free leucine quantified by GC-MS (Figure 1B). After predetermined time points, the mice were euthanized and the brains were quickly removed and frozen. Upon collection of all of the brain samples for each time course, the tissue was then lysed in a 1% Triton X-100 lysis buffer and the protein of interest (apoE and Aβ for this study) was immunoprecipitated with protein-specific antibodies covalently coupled to protein G sepharose beads. Only the cortex of the brain was used in this study; however this technique could easily be applied to measure turnover rates in other regions of the brain. The isolated proteins were eluted off the beads using formic acid, and the concentrated samples were digested with trypsin to generate protein-specific peptides for each protein. These peptide mixtures were then subjected to LC-MS analysis for identification and characterization as described below.


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

Basak JM, Kim J, Pyatkivskyy Y, Wildsmith KR, Jiang H, Parsadanian M, Patterson BW, Bateman RJ, Holtzman DM - Mol Neurodegener (2012)

Experimental schematic for stable isotope labeling and isolation of mouse brain proteins. (A) Cohorts of mice were pulse-labeled with 13 C6-leucine via a bolus intraperitoneal injection (200 mg/kg of body weight). After a pre-determined time following the 13 C6-leucine administration, the mice were euthanized and the brains were removed. The brain tissue was then lysed using a 1% Triton X-100 lysis buffer, and the protein of interest was immunoprecipitated from the brain lysate (apoE is shown as an example). The precipitated proteins were then eluted from the antibody beads and subjected to trypsin digestion. The resulting peptide mixture was separated and analyzed via ultra performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) (yellow = apoE, blue = sepharose bead, red = trypsin). (B) To observe the bioavailability of the 13 C6-leucine, plasma samples were collected at sequential time points following the bolus injection and subjected to GC-MS analysis. The tracer-to-tracee ratio (TTR, shown as labeled/unlabeled leucine) was then measured by quantifying the relative amounts of 13 C6-leucine and dividing by the amount of unlabeled leucine in each sample. Each time point in the graph represents the average value from 5–6 individual mice
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3405485&req=5

Figure 1: Experimental schematic for stable isotope labeling and isolation of mouse brain proteins. (A) Cohorts of mice were pulse-labeled with 13 C6-leucine via a bolus intraperitoneal injection (200 mg/kg of body weight). After a pre-determined time following the 13 C6-leucine administration, the mice were euthanized and the brains were removed. The brain tissue was then lysed using a 1% Triton X-100 lysis buffer, and the protein of interest was immunoprecipitated from the brain lysate (apoE is shown as an example). The precipitated proteins were then eluted from the antibody beads and subjected to trypsin digestion. The resulting peptide mixture was separated and analyzed via ultra performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) (yellow = apoE, blue = sepharose bead, red = trypsin). (B) To observe the bioavailability of the 13 C6-leucine, plasma samples were collected at sequential time points following the bolus injection and subjected to GC-MS analysis. The tracer-to-tracee ratio (TTR, shown as labeled/unlabeled leucine) was then measured by quantifying the relative amounts of 13 C6-leucine and dividing by the amount of unlabeled leucine in each sample. Each time point in the graph represents the average value from 5–6 individual mice
Mentions: The outline of our experimental design for the labeling of mice and tissue processing is shown in Figure 1A. Mice were intraperitoneally (IP) injected with a bolus of 13 C6-leucine, a non-radioactive stable isotope form of the amino acid leucine. We chose 13 C6-labeled leucine because it is one of the essential amino acids that rapidly crosses the blood brain barrier via facilitative neutral amino acid transport [34]. Intraperitoneal administration of the label was chosen because it is straightforward and quick, and it allows for high bioavailability upon absorption into the bloodstream. Following the injection, we observed a rapid increase in the amount of 13 C6-leucine in the plasma of the mice over the first hour, as measured by the ratio of labeled to unlabeled free leucine quantified by GC-MS (Figure 1B). After predetermined time points, the mice were euthanized and the brains were quickly removed and frozen. Upon collection of all of the brain samples for each time course, the tissue was then lysed in a 1% Triton X-100 lysis buffer and the protein of interest (apoE and Aβ for this study) was immunoprecipitated with protein-specific antibodies covalently coupled to protein G sepharose beads. Only the cortex of the brain was used in this study; however this technique could easily be applied to measure turnover rates in other regions of the brain. The isolated proteins were eluted off the beads using formic acid, and the concentrated samples were digested with trypsin to generate protein-specific peptides for each protein. These peptide mixtures were then subjected to LC-MS analysis for identification and characterization as described below.

Bottom Line: 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.However, ABCA1 had no effect on the FCR of Aβ, suggesting that ABCA1 does not regulate Aβ metabolism in the brain.Our SILK strategy represents a straightforward, cost-effective, and efficient method to measure the clearance of proteins in the mouse brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Saint Louis, Missouri 63110, USA.

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 13C6-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.

Show MeSH
Related in: MedlinePlus