Human amyloid-beta synthesis and clearance rates as measured in cerebrospinal fluid in vivo

Abstract

Certain disease states are characterized by disturbances in production, accumulation or clearance of protein. In Alzheimer disease, accumulation of amyloid-beta (Abeta) in the brain and disease-causing mutations in amyloid precursor protein or in enzymes that produce Abeta indicate dysregulation of production or clearance of Abeta. Whether dysregulation of Abeta synthesis or clearance causes the most common form of Alzheimer disease (sporadic, >99% of cases), however, is not known. Here, we describe a method to determine the production and clearance rates of proteins within the human central nervous system (CNS). We report the first measurements of the fractional production and clearance rates of Abeta in vivo in the human CNS to be 7.6% per hour and 8.3% per hour, respectively. This method may be used to search for novel biomarkers of disease, to assess underlying differences in protein metabolism that contribute to disease and to evaluate treatments in terms of their pharmacodynamic effects on proposed disease-causing pathways.

Figure 1

The amino acid sequence of Aβ is depicted in the amyloid precursor protein (APP) in the cell with the leucines (L) labeled in red to indicate possible labeling sites. The sequence of Aβ is shown below with the trypsin digest sites indicated to demonstrate the fragments that were analyzed by mass spectrometry.

Figure 2

Human CSF was collected after intravenous infusion of ¹³C₆-leucine. Representative spectra of unlabeled (a) and labeled (b) Aβ_17–28 are shown. The spectra were obtained using MS/MS analysis of unlabeled parent ion Aβ_17–28 at m/z 663.3 or labeled parent ion Aβ_17–28 at m/z 666.3. Note the MS/MS ions containing leucine (Aβ₁₇) are mass shifted by 6 Daltons demonstrating the labeled leucine. The Aβ ions without leucine at position 17 are not labeled and are not mass shifted by 6 Daltons.

Figure 3

(a) Diagram of participant with an intravenous catheter in either antecubital vein and a lumbar catheter in the L3-4 intrathecal space. Participants were admitted to the Washington University GCRC at 7 AM and 2 IVs and one lumbar catheter were placed. In one IV, ¹³C₆-labeled leucine was infused at a rate of 1.8 to 2.5 mg/kg/hr for 9 or 12 hours, after an initial bolus of 2 mg/kg. Each hour, 12 ml of plasma and 6 ml of CSF were obtained through the other IV and the lumbar catheter respectively. Each sample was then processed as described above with immunoprecipitation of Aβ, trypsin digest, and LC-ESI-MS analysis to determine the percent labeled Aβ at each hour time point. (b) Labeled leucine in CSF and blood from a participant during a 36 hour study. Note that CSF and plasma labeled leucine reaches near steady state within an hour after the initial bolus of 2mg/kg. Also note the exponential decay in labeled leucine clearance after the infusion of leucine into the bloodstream is stopped at 9 hours. The plasma labeled leucine is ~4% higher than the CSF labeled leucine during infusion. (c) Average labeled CSF Aβ over 36 hours from 6 participants. The labeled Aβ curves were averaged and the mean for each time point shown +/− SEM. Each participant was labeled for 9 or 12 hours, while sampling occurred hourly from 0 to 12, 24, or 36 hours. There is no detectable incorporation of label in the first 4 hours, followed by an increase in percent labeled Aβ which plateaus near steady state labeled leucine levels (~10%), before decreasing over the last 12 hours of the study.

Figure 3

(a) Diagram of participant with an intravenous catheter in either antecubital vein and a lumbar catheter in the L3-4 intrathecal space. Participants were admitted to the Washington University GCRC at 7 AM and 2 IVs and one lumbar catheter were placed. In one IV, ¹³C₆-labeled leucine was infused at a rate of 1.8 to 2.5 mg/kg/hr for 9 or 12 hours, after an initial bolus of 2 mg/kg. Each hour, 12 ml of plasma and 6 ml of CSF were obtained through the other IV and the lumbar catheter respectively. Each sample was then processed as described above with immunoprecipitation of Aβ, trypsin digest, and LC-ESI-MS analysis to determine the percent labeled Aβ at each hour time point. (b) Labeled leucine in CSF and blood from a participant during a 36 hour study. Note that CSF and plasma labeled leucine reaches near steady state within an hour after the initial bolus of 2mg/kg. Also note the exponential decay in labeled leucine clearance after the infusion of leucine into the bloodstream is stopped at 9 hours. The plasma labeled leucine is ~4% higher than the CSF labeled leucine during infusion. (c) Average labeled CSF Aβ over 36 hours from 6 participants. The labeled Aβ curves were averaged and the mean for each time point shown +/− SEM. Each participant was labeled for 9 or 12 hours, while sampling occurred hourly from 0 to 12, 24, or 36 hours. There is no detectable incorporation of label in the first 4 hours, followed by an increase in percent labeled Aβ which plateaus near steady state labeled leucine levels (~10%), before decreasing over the last 12 hours of the study.

Figure 3

(a) Diagram of participant with an intravenous catheter in either antecubital vein and a lumbar catheter in the L3-4 intrathecal space. Participants were admitted to the Washington University GCRC at 7 AM and 2 IVs and one lumbar catheter were placed. In one IV, ¹³C₆-labeled leucine was infused at a rate of 1.8 to 2.5 mg/kg/hr for 9 or 12 hours, after an initial bolus of 2 mg/kg. Each hour, 12 ml of plasma and 6 ml of CSF were obtained through the other IV and the lumbar catheter respectively. Each sample was then processed as described above with immunoprecipitation of Aβ, trypsin digest, and LC-ESI-MS analysis to determine the percent labeled Aβ at each hour time point. (b) Labeled leucine in CSF and blood from a participant during a 36 hour study. Note that CSF and plasma labeled leucine reaches near steady state within an hour after the initial bolus of 2mg/kg. Also note the exponential decay in labeled leucine clearance after the infusion of leucine into the bloodstream is stopped at 9 hours. The plasma labeled leucine is ~4% higher than the CSF labeled leucine during infusion. (c) Average labeled CSF Aβ over 36 hours from 6 participants. The labeled Aβ curves were averaged and the mean for each time point shown +/− SEM. Each participant was labeled for 9 or 12 hours, while sampling occurred hourly from 0 to 12, 24, or 36 hours. There is no detectable incorporation of label in the first 4 hours, followed by an increase in percent labeled Aβ which plateaus near steady state labeled leucine levels (~10%), before decreasing over the last 12 hours of the study.

Figure 4

FSR and FCR of labeled Aβ curves from 3 participants with 9 hour label infusion and 36 hour sampling. FSR (a–c) is calculated by the slope of increasing labeled Aβ divided by the predicted steady state value. The predicted steady state value was estimated as the average CSF labeled leucine measured during labeling. The slope was defined to start after 4 to 6 hours lag time when there was no increase in labeled Aβ and ending 9 hours later (solid diamonds). The slope of FCR (d–f) is calculated by the slope of the natural logarithm of percent labeled Aβ from hours 24 to 36. (g) Average Aβ FSR & FCR: The average Aβ FSR of 6 participants and the average Aβ FCR of 3 participants is shown with standard deviation.

Figure 4

FSR and FCR of labeled Aβ curves from 3 participants with 9 hour label infusion and 36 hour sampling. FSR (a–c) is calculated by the slope of increasing labeled Aβ divided by the predicted steady state value. The predicted steady state value was estimated as the average CSF labeled leucine measured during labeling. The slope was defined to start after 4 to 6 hours lag time when there was no increase in labeled Aβ and ending 9 hours later (solid diamonds). The slope of FCR (d–f) is calculated by the slope of the natural logarithm of percent labeled Aβ from hours 24 to 36. (g) Average Aβ FSR & FCR: The average Aβ FSR of 6 participants and the average Aβ FCR of 3 participants is shown with standard deviation.