In vivo kinetic approach reveals slow SOD1 turnover in the CNS
Matthew J. Crisp, … , Randall J. Bateman, Timothy M. Miller
Matthew J. Crisp, … , Randall J. Bateman, Timothy M. Miller
Published June 15, 2015
Citation Information: J Clin Invest. 2015;125(7):2772-2780. https://doi.org/10.1172/JCI80705.
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Research Article Neuroscience

In vivo kinetic approach reveals slow SOD1 turnover in the CNS

Abstract

Therapeutic strategies that target disease-associated transcripts are being developed for a variety of neurodegenerative syndromes. Protein levels change as a function of their half-life, a property that critically influences the timing and application of therapeutics. In addition, both protein kinetics and concentration may play important roles in neurodegeneration; therefore, it is essential to understand in vivo protein kinetics, including half-life. Here, we applied a stable isotope-labeling technique in combination with mass spectrometric detection and determined the in vivo kinetics of superoxide dismutase 1 (SOD1), mutation of which causes amyotrophic lateral sclerosis. Application of this method to human SOD1-expressing rats demonstrated that SOD1 is a long-lived protein, with a similar half-life in both the cerebral spinal fluid (CSF) and the CNS. Additionally, in these animals, the half-life of SOD1 was longest in the CNS when compared with other tissues. Evaluation of this method in human subjects demonstrated successful incorporation of the isotope label in the CSF and confirmed that SOD1 is a long-lived protein in the CSF of healthy individuals. Together, the results of this study provide important insight into SOD1 kinetics and support application of this technique to the design and implementation of clinical trials that target long-lived CNS proteins.

Authors

Matthew J. Crisp, Kwasi G. Mawuenyega, Bruce W. Patterson, Naveen C. Reddy, Robert Chott, Wade K. Self, Conrad C. Weihl, Jennifer Jockel-Balsarotti, Arun S. Varadhachary, Robert C. Bucelli, Kevin E. Yarasheski, Randall J. Bateman, Timothy M. Miller

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Figure 3

Kinetic data and model from SOD1 G93A rats.

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Kinetic data and model from SOD1 G93A rats. (A) Schematic of the oral la...
(A) Schematic of the oral labeling paradigm. SOD1 G93A rats were fed 13C6-leucine for 7 days and then chased with unlabeled leucine for an additional 56 days. Tissues were collected at the indicated time points, and detergent soluble SOD1 was immunoprecipitated, digested, and analyzed by LC/tandem MS. (B) Mole fraction labeled plasma-free 13C6-leucine and liver, kidney, cortex, CSF, and spinal cord SOD1 G93A were plotted over time (individual points) and modeled as previously described (solid lines). The slower kinetics of SOD1 G93A in spinal cord, cortex, and CSF are reflected in the gradual rise and fall of the curves compared with the steep slopes seen in liver and kidney. (C) Misfolded SOD1 G93A was immunoprecipitated from previously labeled SOD1 G93A rat tissue with the α-misfolded SOD1 B8H10 antibody, digested, analyzed by LC/tandem MS, and modeled as previously described (solid lines represent total soluble SOD1; dashed lines represent misfolded SOD1). Labeled misfolded pools in both spinal cord and liver reveal accelerated turnover rates when compared with total soluble SOD1 within each tissue. For both graphs, light red shading between 0 and 7 days represents the 13C6-leucine pulse interval. n = 3 for all time points with the exception of day 14 (n = 2).