This Program Project brings together five researchers with very different expertise and experimental capabilities to work together on elucidating the underlying mechanisms of S0D1-linked familial amyotrophic lateral sclerosis (fALS) pathogenesis. It is well established that S0D1 multimers and larger aggregates are associated with disease but the toxic species and in vivo mechanism remain unknown. The overall goals of this proposal are to gain an extensive understanding of the role of aggregation in disease, to characterize further the biochemical properties associated with mutant S0D1 and its aggregation, to uncover clues about the initiation and progression of disease, to exploit this understanding to develop targeted blockers of multimerization. The PPG collaboration will encompass five primary investigators with four projects and a technical Core. Projects 1 (Dr. Joan Valentine) and 4 (Dr. David Eisenberg) will take an in vitro structural and biophysical approach to studying the mechanism of S0D1 multimerization and the structures of the multimers, with the goals of understanding the mechanism(s) of multimerization and designing inhibitors of aggregation. Project 2 (Dr. Martina Wiedau-Pazos) will use stem cell-derived motor neurons and glia and project 3 (Dr. David Borchelt) will use a mouse,and cell culture models to probe the toxicity of multimers and to characterize the changes in mutant S0D1 that lead toward disease. A particular emphasis on the latter project will be toward in vivo metal loading as it pertains to S0D1 stability, and metal homeostasis as it pertains toward cellular toxicity. Core A (Dr. Julian Whitelegge) will serve as the backbone of these investigations by providing and maintaining the necessary instrumentation and data delivery. Disease models from projects 2 and 3 will be used to test the efficacy of inhibitors from projects 1 and 4. Finally, ALS tissue will be used as a source to validate the findings and test new hypotheses.

Public Health Relevance

A critical unsolved question in understanding amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases is the role of various aggregated forms of proteins in causing disease. This proposal addresses this question for ALS in particular using approaches that combine some of the best cell culture and animal model systems available with advanced biophysical and biochemical methods. PROJECT 1 Principal Investigator: Joan Valentine Title: Not provided. Description (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by the selective death of motor neurons. While the most common form of ALS is sporadic and has no known cause, a subset of cases caused by genetic mutations are familial, of which those caused by mutations in the protein copper-zinc superoxide dismutase (SODl) represent the most extensively studied model of ALS. The formation of SODl-rich fibrillar inclusions in the spinal cord is a prominent feature of SODI-linked familial ALS in human patients and animal models of this disease. In animal models, the inclusions are preceded by the formation of high-molecular-weight oligomeric forms of SODl that appear even before the onset of symptoms, suggesting that oligomerization and aggregation of SODl is an essential component of the disease etiology. Understanding how multimeric S0D1 contributes to motor neuron death is the overarching goal of the Program Project. In this project, we will address the biophysical aspects of SODl multimerization. Specifically, the goals include (1) examining the structure of multimeric SODl generated in vitro or isolated from human and animal tissue sources, (2) applying defined multimeric preparations of tagged SODl to cultured motor neurons to study if and how they are toxic (in collaboration with project 2), (3) examining the mechanism of S0D1 multimerization into fibrils to understand how structural factors that destabilize SODl contribute to this process and, (4) elucidating the role of familial ALS-causing mutations in modulating the rate of these processes. Our studies will make extensive use of an assay we developed in the prior award period for converting SODl into soluble, oligomeric species and amyloid fibrils under mild, physiologically relevant conditions. We will also make extensive use of a variety of highly sensitive biophysical methods to study a variety of structural properties such as folding,.metal content, and disulfide status of soluble and insoluble forms of SODl isolated from animal tissues. Public Health Relevance: A critical unsolved question in understanding amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases is the role of various aggregated forms of proteins in causing disease. This project addresses this question for ALS in particular using some of the best advanced biophysical and biochemical methods available.

National Institute of Health (NIH)
Research Program Projects (P01)
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National Institute of Neurological Disorders and Stroke Initial Review Group (NSD)
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Gubitz, Amelie
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University of California Los Angeles
Schools of Arts and Sciences
Los Angeles
United States
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Ming, Li-June; Valentine, Joan Selverstone (2014) Insights into SOD1-linked amyotrophic lateral sclerosis from NMR studies of Ni(2+)- and other metal-ion-substituted wild-type copper-zinc superoxide dismutases. J Biol Inorg Chem 19:647-57
Brown, Hilda H; Borchelt, David R (2014) Analysis of mutant SOD1 electrophoretic mobility by Blue Native gel electrophoresis; evidence for soluble multimeric assemblies. PLoS One 9:e104583
Ayers, Jacob; Lelie, Herman; Workman, Aron et al. (2014) Distinctive features of the D101N and D101G variants of superoxide dismutase 1; two mutations that produce rapidly progressing motor neuron disease. J Neurochem 128:305-14
Ivanova, Magdalena I; Sievers, Stuart A; Guenther, Elizabeth L et al. (2014) Aggregation-triggering segments of SOD1 fibril formation support a common pathway for familial and sporadic ALS. Proc Natl Acad Sci U S A 111:197-201
Sheng, Yuewei; Abreu, Isabel A; Cabelli, Diane E et al. (2014) Superoxide dismutases and superoxide reductases. Chem Rev 114:3854-918
Ayers, Jacob I; Xu, Guilian; Pletnikova, Olga et al. (2014) Conformational specificity of the C4F6 SOD1 antibody; low frequency of reactivity in sporadic ALS cases. Acta Neuropathol Commun 2:55
Chan, Pik K; Chattopadhyay, Madhuri; Sharma, Shivani et al. (2013) Structural similarity of wild-type and ALS-mutant superoxide dismutase-1 fibrils using limited proteolysis and atomic force microscopy. Proc Natl Acad Sci U S A 110:10934-9
Rutherford, Nicola J; Lewis, Jada; Clippinger, Amy K et al. (2013) Unbiased screen reveals ubiquilin-1 and -2 highly associated with huntingtin inclusions. Brain Res 1524:62-73
Prudencio, Mercedes; Lelie, Herman; Brown, Hilda H et al. (2012) A novel variant of human superoxide dismutase 1 harboring amyotrophic lateral sclerosis-associated and experimental mutations in metal-binding residues and free cysteines lacks toxicity in vivo. J Neurochem 121:475-85
Lelie, Herman L; Liba, Amir; Bourassa, Megan W et al. (2011) Copper and zinc metallation status of copper-zinc superoxide dismutase from amyotrophic lateral sclerosis transgenic mice. J Biol Chem 286:2795-806

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