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J. Biol. Chem., Vol. 283, Issue 13, 8340-8350, March 28, 2008

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Detergent-insoluble Aggregates Associated with Amyotrophic Lateral Sclerosis in Transgenic Mice Contain Primarily Full-length, Unmodified Superoxide Dismutase-1^*

Bryan F. Shaw¹², Herman L. Lelie¹, Armando Durazo¹, Aram M. Nersissian, Guillan Xu^¶, Pik K. Chan, Edith B. Gralla, Ashutosh Tiwari^||3, Lawrence J. Hayward^||4, David R. Borchelt^¶5, Joan S. Valentine⁵, and Julian P. Whitelegge^**56

From the Departments of Chemistry and Biochemistry and the ^**NPI-Semel Institute, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, the Department of Chemistry, Occidental College, Los Angeles, California 90041, the ^¶Department of Neuroscience, Santa Fe Health Alzheimer's Disease Research Center, McKnight Brain Institute, University of Florida, Gainesville, Florida 32611, and the ^||Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

Determining the composition of aggregated superoxide dismutase 1 (SOD1) species associated with amyotrophic lateral sclerosis (ALS), especially with respect to co-aggregated proteins and post-translational modifications, could identify cellular or biochemical factors involved in the formation of these aggregates and explain their apparent neurotoxicity. The results of mass spectrometric and shotgun-proteomic analyses of SOD1-containing aggregates isolated from spinal cords of symptomatic transgenic ALS mice using two different isolation strategies are presented, including 1) resistance to detergent extraction and 2) size exclusion-coupled anti-SOD1 immunoaffinity chromatography. Forty-eight spinal cords from three different ALS-SOD1 mutant mice were analyzed, namely G93A, G37R, and the unnatural double mutant H46R/H48Q. The analysis consistently revealed that the most abundant proteins recovered from aggregate species were full-length unmodified SOD1 polypeptides. Although aggregates from some spinal cord samples contained trace levels of highly abundant proteins, such as vimentin and neurofilament-3, no proteins were consistently found to co-purify with mutant SOD1 in stoichiometric quantities. The results demonstrate that the principal protein in the high molecular mass aggregates whose appearance correlates with symptoms of the disease is the unmodified, full-length SOD1 polypeptide.

Received for publication, September 14, 2007 , and in revised form, December 18, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental "Experimental Procedures," references, Figs. S1–S3, and Tables S1 and S2.

¹ These authors contributed equally to this work.

² Supported by a TSR&TP fellowship for predoctoral support. Present address: Dept. of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138.

³ Supported by the ALS Association and the ALS Therapy Alliance.

⁴ Supported by Grant R01-NS44170 from the NINDS, National Institutes of Health (NIH).

⁵ Supported by NINDS, NIH Grant P01 NS049134-01.

⁶ To whom correspondence should be addressed: NPI-Semel Institute, David Geffen School of Medicine, UCLA, 405 Hilgard Ave., Los Angeles, CA 90095. Tel.: 310-206-7886; Fax: 310-206-2161; E-mail: jpw{at}chem.ucla.edu.

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