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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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13C6-leucine brain apoE labeling in the presence of ABCA1 overexpression and deletion. (A) Cohorts of wildtype and ABCA1 transgenic mice and (C) wildtype and ABCA1−/− mice were labeled with 13 C6-leucine and the brains isolated after predetermined time points. Note that separate groups of animals were used as the wildtype controls for the ABCA1 Tg and ABCA1−/− mice. The apoE labeled/unlabeled ratios were then calculated and the data plotted as in Figure 3. For FCR measurements, the monoexponential slopes were measured for (B) ABCA1 Tg and (D) ABCA1−/− mice and their respective Wt controls (n = 5-6 mice per time point, error bars represent SEM, dotted lines represent 95% confidence band).
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Figure 4: 13C6-leucine brain apoE labeling in the presence of ABCA1 overexpression and deletion. (A) Cohorts of wildtype and ABCA1 transgenic mice and (C) wildtype and ABCA1−/− mice were labeled with 13 C6-leucine and the brains isolated after predetermined time points. Note that separate groups of animals were used as the wildtype controls for the ABCA1 Tg and ABCA1−/− mice. The apoE labeled/unlabeled ratios were then calculated and the data plotted as in Figure 3. For FCR measurements, the monoexponential slopes were measured for (B) ABCA1 Tg and (D) ABCA1−/− mice and their respective Wt controls (n = 5-6 mice per time point, error bars represent SEM, dotted lines represent 95% confidence band).

Mentions: To generate APP transgenic mice that either overexpressed or were deficient in ABCA1 levels, we crossed PDAPP mice with ABCA1 Tg and ABCA1−/− mice. These animals were then injected with 13 C6-leucine and the FCRs of both apoE and Aβ were measured as described above for the LDLR Tg animals. To limit complications due to Aβ extraction from tissue with amyloid plaques, all experiments were performed on young animals (3.5 months old) prior to the onset of detectable plaque deposition. Plots of the labeled/unlabeled protein values over time along with the monoexponential slopes of these curves are shown in Figures 4 and 5 for apoE and Aβ, respectively. The PS, FCR, and PR values for apoE and Aβ are given in Table 2 and Table 3, respectively. The apoE FCR was 1.5-fold faster in ABCA1 Tg mice and 1.9-fold faster in ABCA1−/− mice compared to Wt mice; however the difference was only significant for the ABCA1−/− mice. The apoE PS decreased by 20% in ABCA1 Tg mice and by 51% in ABCA1−/− mice compared to Wt mice. No differences were observed in the PR of apoE. For Aβ, no differences were observed in the FCR, PS, or PR values (Table 3).


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)

13C6-leucine brain apoE labeling in the presence of ABCA1 overexpression and deletion. (A) Cohorts of wildtype and ABCA1 transgenic mice and (C) wildtype and ABCA1−/− mice were labeled with 13 C6-leucine and the brains isolated after predetermined time points. Note that separate groups of animals were used as the wildtype controls for the ABCA1 Tg and ABCA1−/− mice. The apoE labeled/unlabeled ratios were then calculated and the data plotted as in Figure 3. For FCR measurements, the monoexponential slopes were measured for (B) ABCA1 Tg and (D) ABCA1−/− mice and their respective Wt controls (n = 5-6 mice per time point, error bars represent SEM, dotted lines represent 95% confidence band).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: 13C6-leucine brain apoE labeling in the presence of ABCA1 overexpression and deletion. (A) Cohorts of wildtype and ABCA1 transgenic mice and (C) wildtype and ABCA1−/− mice were labeled with 13 C6-leucine and the brains isolated after predetermined time points. Note that separate groups of animals were used as the wildtype controls for the ABCA1 Tg and ABCA1−/− mice. The apoE labeled/unlabeled ratios were then calculated and the data plotted as in Figure 3. For FCR measurements, the monoexponential slopes were measured for (B) ABCA1 Tg and (D) ABCA1−/− mice and their respective Wt controls (n = 5-6 mice per time point, error bars represent SEM, dotted lines represent 95% confidence band).
Mentions: To generate APP transgenic mice that either overexpressed or were deficient in ABCA1 levels, we crossed PDAPP mice with ABCA1 Tg and ABCA1−/− mice. These animals were then injected with 13 C6-leucine and the FCRs of both apoE and Aβ were measured as described above for the LDLR Tg animals. To limit complications due to Aβ extraction from tissue with amyloid plaques, all experiments were performed on young animals (3.5 months old) prior to the onset of detectable plaque deposition. Plots of the labeled/unlabeled protein values over time along with the monoexponential slopes of these curves are shown in Figures 4 and 5 for apoE and Aβ, respectively. The PS, FCR, and PR values for apoE and Aβ are given in Table 2 and Table 3, respectively. The apoE FCR was 1.5-fold faster in ABCA1 Tg mice and 1.9-fold faster in ABCA1−/− mice compared to Wt mice; however the difference was only significant for the ABCA1−/− mice. The apoE PS decreased by 20% in ABCA1 Tg mice and by 51% in ABCA1−/− mice compared to Wt mice. No differences were observed in the PR of apoE. For Aβ, no differences were observed in the FCR, PS, or PR values (Table 3).

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