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Altered axonal architecture by removal of the heavily phosphorylated neurofilament tail domains strongly slows superoxide dismutase 1 mutant-mediated ALS.

ABSTRACT: Eliminating assembled neurofilaments (NFs) from axons or misaccumulating NFs in motor neuron cell bodies strongly slows disease in mouse models of mutant superoxide dismutase 1 (SOD1)-induced amyotrophic lateral sclerosis. One proposal for how reducing axonal NFs can increase survival is that the multiphosphorylated tail domains of the two larger NF subunits act in motor neuron cell bodies as phosphorylation sinks where they mitigate cyclin-dependent kinase 5 dysregulation induced by mutant SOD1. Elimination by gene targeting in mice of the NF medium and NF heavy tail domains and their 58 known phosphorylation sites accelerates aberrant phosphorylation of other neuronal substrates while leaving overall NF content unaltered. However, disease onset is significantly delayed and survival is extended, inconsistent with the ameliorative property of altered NF content protecting by serving as substrates for dysregulation of any NF kinase. Moreover, at comparable disease stages significantly more surviving motor neurons and axons were found in SOD1 mutant mice deleted in the NF tails than in similar mice with wild-type NFs. This finding supports noncell autonomous toxicity in SOD1 mutant-mediated amyotrophic lateral sclerosis: removal of the NF tails slows damage developed directly within motor neurons, but SOD1 mutant damage within nonneuronal supporting cells reduces motor neuron functionality.

SUBMITTER: Lobsiger CS

PROVIDER: S-EPMC1177385 | biostudies-literature | 2005 Jul

REPOSITORIES: biostudies-literature

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Altered axonal architecture by removal of the heavily phosphorylated neurofilament tail domains strongly slows superoxide dismutase 1 mutant-mediated ALS.

Lobsiger Christian S CS Garcia Michael L ML Ward Christopher M CM Cleveland Don W DW

Proceedings of the National Academy of Sciences of the United States of America 20050707 29

Eliminating assembled neurofilaments (NFs) from axons or misaccumulating NFs in motor neuron cell bodies strongly slows disease in mouse models of mutant superoxide dismutase 1 (SOD1)-induced amyotrophic lateral sclerosis. One proposal for how reducing axonal NFs can increase survival is that the multiphosphorylated tail domains of the two larger NF subunits act in motor neuron cell bodies as phosphorylation sinks where they mitigate cyclin-dependent kinase 5 dysregulation induced by mutant SOD1 ...[more]

PMID: 16002469

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Project description:Over 130 mutations to copper, zinc superoxide dismutase (SOD) are implicated in the selective death of motor neurons found in 25% of patients with familial amyotrophic lateral sclerosis (ALS). Despite their widespread distribution, ALS mutations appear positioned to cause structural and misfolding defects. Such defects decrease SOD's affinity for zinc, and loss of zinc from SOD is sufficient to induce apoptosis in motor neurons in vitro. To examine the importance of the zinc site in the structure and pathogenesis of human SOD, we determined the 2.0-A-resolution crystal structure of a designed zinc-deficient human SOD, in which two zinc-binding ligands have been mutated to hydrogen-bonding serine residues. This structure revealed a 9 degrees twist of the subunits, which opens the SOD dimer interface and represents the largest intersubunit rotational shift observed for a human SOD variant. Furthermore, the electrostatic loop and zinc-binding subloop were partly disordered, the catalytically important Arg143 was rotated away from the active site, and the normally rigid intramolecular Cys57-Cys146 disulfide bridge assumed two conformations. Together, these changes allow small molecules greater access to the catalytic copper, consistent with the observed increased redox activity of zinc-deficient SOD. Moreover, the dimer interface is weakened and the Cys57-Cys146 disulfide is more labile, as demonstrated by the increased aggregation of zinc-deficient SOD in the presence of a thiol reductant. However, equimolar Cu,Zn SOD rapidly forms heterodimers with zinc-deficient SOD (t1/2 approximately 15 min) and prevents aggregation. The stabilization of zinc-deficient SOD as a heterodimer with Cu,Zn SOD may contribute to the dominant inheritance of ALS mutations. These results have general implications for the importance of framework stability on normal metalloenzyme function and specific implications for the role of zinc ion in the fatal neuropathology associated with SOD mutations.

Project description:BackgroundMutations in superoxide dismutase 1 (SOD1), which are one cause of familial amyotrophic lateral sclerosis (fALS), induce misfolding and aggregation of the protein. Misfolding can be detected by the binding of antibodies raised against peptide epitopes that are normally buried in the native conformation, shifts in solubility in non-ionic detergents, and the formation of macromolecular inclusions. In the present study, we investigate the relationship between detergent-insoluble and sedimentable forms of mutant SOD1, forms of mutant SOD1 with aberrantly accessible epitopes, and mutant protein in inclusions with the goal of defining the spectrum of misfolded states that mutant SOD1 can adopt.ResultsUsing combined approaches in cultured cell models, we demonstrate that a substantial fraction of mutant SOD1 adopts a non-native conformation that remains soluble and freely mobile. We also show that mutant SOD1 can produce multimeric assemblies of which some are insoluble in detergent and large enough to sediment by ultracentrifugation and some are large enough to detect visually. Three conformationally restricted antibodies were found to be useful in discriminating mal-folded forms of mutant SOD1. An antibody termed C4F6 displays properties consistent with recognition of soluble, freely mobile, mal-folded mutant SOD1. An antibody termed SEDI, which recognizes C-terminal residues, detects larger inclusion structures as well as soluble misfolded entities. An antibody termed hSOD1, which recognizes aa 24-36, detects an epitope shared by soluble non-natively folded WT and mutant SOD1. This epitope becomes inaccessible in aggregates of mutant SOD1.ConclusionsOur studies demonstrate how different methods of detecting misfolding and aggregation of mutant SOD1 reveal different forms of aberrantly folded protein. Immunological and biochemical methods can be used in combination to detect soluble and insoluble misfolded forms of mutant SOD1. Our findings support the view that mutant SOD1 can adopt multiple misfolded conformations with the potential that different structural variants mediate different aspects of fALS.

Dataset Information

Altered axonal architecture by removal of the heavily phosphorylated neurofilament tail domains strongly slows superoxide dismutase 1 mutant-mediated ALS.

Publications

Altered axonal architecture by removal of the heavily phosphorylated neurofilament tail domains strongly slows superoxide dismutase 1 mutant-mediated ALS.

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