Mutations in the RNA-binding protein FUS (fused in sarcoma) are linked to amyotrophic lateral sclerosis (ALS), but the mechanism by which these mutants cause motor neuron degeneration is not known. We report a novel ALS truncation mutant (R495X) that leads to a relatively severe ALS clinical phenotype compared with FUS missense mutations. Expression of R495X FUS, which abrogates a putative nuclear localization signal at the C-terminus of FUS, in HEK-293 cells and in the zebrafish spinal cord caused a striking cytoplasmic accumulation of the protein to a greater extent than that observed for recessive (H517Q) and dominant (R521G) missense mutants. Furthermore, in response to oxidative stress or heat shock conditions in cultures and in vivo, the ALS-linked FUS mutants, but not wild-type FUS, assembled into perinuclear stress granules in proportion to their cytoplasmic expression levels. These findings demonstrate a potential link between FUS mutations and cellular pathways involved in stress responses that may be relevant to altered motor neuron homeostasis in ALS.

Adult-onset neurodegenerative diseases (AONDs) comprise a heterogeneous group of neurological disorders characterized by a progressive, age-dependent decline in neuronal function and loss of selected neuronal populations. Alterations in synaptic function and axonal connectivity represent early and critical pathogenic events in AONDs, but molecular mechanisms underlying these defects remain elusive. The large size and complex subcellular architecture of neurons render them uniquely vulnerable to alterations in axonal transport (AT). Accordingly, deficits in AT have been documented in most AONDs, suggesting a common defect acquired through different pathogenic pathways. These observations suggest that many AONDs can be categorized as dysferopathies, diseases in which alterations in AT represent a critical component in pathogenesis. Topics here address various molecular mechanisms underlying alterations in AT in several AONDs. Illumination of such mechanisms provides a framework for the development of novel therapeutic strategies aimed to prevent axonal and synaptic dysfunction in several major AONDs.

Over 90 different mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) cause ϳ2% of amyotrophic lateral sclerosis (ALS) cases by an unknown mechanism. We engineered 14 different human ALSrelated SOD1 mutants and obtained high yields of biologically metallated proteins from an Sf21 insect cell expression system. Both the wild type and mutant "as isolated" SOD1 variants were deficient in copper and were heterogeneous by native gel electrophoresis. By contrast, although three mutant SOD1s with substitutions near the metal binding sites (H46R, G85R, and D124V) were severely deficient in both copper and zinc ions, zinc deficiency was not a consistent feature shared by the as isolated mutants. Eight mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133⌬) exhibited normal SOD activity over pH 5.5-10.5, per equivalent of copper, consistent with the presumption that bound copper was in the proper metal-binding site and was fully active. The H48Q variant contained a high copper content yet was 100-fold less active than the wild type enzyme and exhibited a blue shift in the visible absorbance peak of bound Cu(II), indicating rearrangement of the Cu(II) coordination geometry. Further characterization of these as-isolated SOD1 proteins may provide new insights regarding mutant SOD1 enzyme toxicity in ALS.Amyotrophic lateral sclerosis (ALS, 1 Lou Gehrig's disease) is an age-dependent, degenerative disorder of motor neurons in the spinal cord and brain. Progressive dysfunction of both upper and lower motor neurons causes death from respiratory paralysis, usually within 5 years. Investigation of the causes of familial ALS, which comprises ϳ10% of cases, may contribute insights relevant to the pathophysiology of sporadic ALS and of other motor neuron diseases (1, 2).A subset of autosomal dominant ALS is caused by over 90 mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) (3, 4) (see an updated list of all mutations on the World Wide Web at www.alsod.org). SOD1 is a 32-kDa homodimeric enzyme that functions as an antioxidant, converting two molecules of superoxide anion (O 2 . ) to O 2 and H 2 O 2 .This redox cycle involves alternate reduction (reaction 1) and reoxidation (reaction 2) of the catalytic copper ion by O 2 . .REACTIONS 1 and 2 SOD1 contains an eight-stranded ␤-barrel motif, an intrasubunit disulfide bond, and a zinc binding site that contribute to its extreme thermochemical stability (Fig. 1). The mutant residues are scattered throughout the protein, including some residues important for copper or zinc coordination, others located near the dimer interface or at either pole of the ␤-barrel, and several in the charged loop near the C terminus that may guide O 2 . to the active site. Although most are missense substitutions, some are predicted to truncate the C terminus of the protein, including the charged loop. No null mutations have been described. Mutant SOD1 most likely causes motor neuron death by gain of an unknown toxic property rather than by deficiency of dismutase a...

Mutations in the SOD1 gene cause the autosomal dominant, neurodegenerative disorder familial amyotrophic lateral sclerosis (FALS). In spinal cord neurons of human FALS patients and in transgenic mice expressing these mutant proteins, aggregates containing FALS SOD1 are observed. Accumulation of SOD1 aggregates is believed to interfere with axonal transport, protein degradation and anti-apoptotic functions of the neuronal cellular machinery. Here we show that metal-deficient, pathogenic SOD1 mutant proteins crystallize in three different crystal forms, all of which reveal higher-order assemblies of aligned beta-sheets. Amyloid-like filaments and water-filled nanotubes arise through extensive interactions between loop and beta-barrel elements of neighboring mutant SOD1 molecules. In all cases, non-native conformational changes permit a gain of interaction between dimers that leads to higher-order arrays. Normal beta-sheet-containing proteins avoid such self-association by preventing their edge strands from making intermolecular interactions. Loss of this protection through conformational rearrangement in the metal-deficient enzyme could be a toxic property common to mutants of SOD1 linked to FALS.

FUS/TLS is a nucleic acid binding protein that, when mutated, can cause a subset of familial amyotrophic lateral sclerosis (fALS). Although FUS/TLS is normally located predominantly in the nucleus, the pathogenic mutant forms of FUS/TLS traffic to, and form inclusions in, the cytoplasm of affected spinal motor neurons or glia. Here we report a yeast model of human FUS/TLS expression that recapitulates multiple salient features of the pathology of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, inclusion formation, and cytotoxicity. Protein domain analysis indicates that the carboxyl-terminus of FUS/TLS, where most of the ALS-associated mutations are clustered, is required but not sufficient for the toxicity of the protein. A genome-wide genetic screen using a yeast over-expression library identified five yeast DNA/RNA binding proteins, encoded by the yeast genes ECM32, NAM8, SBP1, SKO1, and VHR1, that rescue the toxicity of human FUS/TLS without changing its expression level, cytoplasmic translocation, or inclusion formation. Furthermore, hUPF1, a human homologue of ECM32, also rescues the toxicity of FUS/TLS in this model, validating the yeast model and implicating a possible insufficiency in RNA processing or the RNA quality control machinery in the mechanism of FUS/TLS mediated toxicity. Examination of the effect of FUS/TLS expression on the decay of selected mRNAs in yeast indicates that the nonsense-mediated decay pathway is probably not the major determinant of either toxicity or suppression.

We report the thermal stability of wild type (WT) and 14 different variants of human copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis (FALS). Multiple endothermic unfolding transitions were observed by differential scanning calorimetry for partially metallated SOD1 enzymes isolated from a baculovirus system. We correlated the metal ion contents of SOD1 variants with the occurrence of distinct melting transitions. Altered thermal stability upon reduction of copper with dithionite identified transitions resulting from the unfolding of copper-containing SOD1 species. We demonstrated that copper or zinc binding to a subset of "WT-like" FALS mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133⌬) conferred a similar degree of incremental stabilization as did metal ion binding to WT SOD1. However, these mutants were all destabilized by ϳ1-6°C compared with the corresponding WT SOD1 species. Most of the "metal binding region" FALS mutants (H46R, G85R, D124V, D125H, and S134N) exhibited transitions that probably resulted from unfolding of metal-free species at ϳ4 -12°C below the observed melting of the least stable WT species. We conclude that decreased conformational stability shared by all of these mutant SOD1s may contribute to SOD1 toxicity in FALS.Copper/zinc superoxide dismutase (SOD1) 1 catalyzes the disproportionation of two molecules of superoxide anion (O 2 . )into O 2 and H 2 O 2 (1, 2) in all eukaryotic cells. Many specific, highly conserved structural interactions confer upon SOD1 a remarkable thermal stability (3-6) and resistance to chemical denaturation (7-9).Each subunit of homodimeric SOD1 is built upon a flattened ␤-barrel motif with additional loop regions that contribute to metal ion binding and formation of the active site (10). One catalytic copper ion and one buried zinc ion per subunit are bound at the active site on the external surface of the ␤-barrel. Occupancy of the metal ion binding sites confers greater thermal stabilization to the bovine SOD1 apoenzyme (3, 4). The copper and zinc ions are linked directly via the imidazolate side chain of the shared His-63 residue 2 and indirectly via extended interactions between their respective ligands. SOD1 dimerization is stabilized by optimized hydrophobic interactions at the contact interface between complementary patches on each subunit (10 -12). A conserved intrasubunit disulfide bond involving Cys-57 also stabilizes the enzyme by anchoring a loop that forms part of the dimer interface to the ␤-barrel at Cys-146.A subset of SOD1 mutations in familial amyotrophic lateral sclerosis (FALS) have been proposed to destabilize the ␤-barrel or disrupt dimerization of SOD1 monomers (13,14). A crystal structure obtained for the G37R SOD1 mutant shows minimal perturbation of the averaged backbone conformation but exhibits unusually high atomic displacement parameters, suggestive of increased molecular flexibility in some regions of the molecule (15). Consistent with this, some mutant SOD1s exhibit acceler...

The relative stabilities and structural properties of a representative set of 20 ALS-mutant Cu,Zn-superoxide dismutase apoproteins were examined by using differential scanning calorimetry and hydrogen-deuterium (H͞D) exchange followed by MS. Contrary to recent reports from other laboratories, we found that ALS-mutant apoproteins are not universally destabilized by the disease-causing mutations. For example, several of the apoproteins with substitutions at or near the metal binding region (MBR) (MBR mutants) exhibited melting temperatures (Tm) in the range 51.6°C to 56.2°C, i.e., similar to or higher than that of the WT apoprotein (Tm ‫؍‬ 52.5°C). The apoproteins with substitutions remote from the MBR (WT-like mutants) showed a wide range of Tms, 40.0°C to 52.4°C. The H͞D exchange properties of the mutants were also wideranging: the MBR mutant apoproteins exhibited H͞D exchange kinetics similar to the WT apoprotein, as did some of the more stable WT-like mutant apoproteins, whereas the less stable apoproteins exhibited significantly less protection from H͞D exchange than the WT apoprotein. Most striking were the three mutant apoproteins, D101N, E100K, and N139K, which have apparently normal metallation properties, and differ little from the WT apoprotein in either thermal stability or H͞D exchange kinetics. Thus, the ALS mutant Cu,Zn-superoxide dismutase apoproteins do not all share reduced global stability, and additional properties must be identified and understood to explain the toxicity of all of the mutant proteins.differential scanning calorimetry ͉ hydrogen-deuterium exchange ͉ protein stability ͉ protein aggregation ͉ neurodegenerative disease P rotein misfolding and aggregation have been linked to many diseases, including Alzheimer's disease, cystic fibrosis, transmissible spongiform encephalopathies, and ALS, but the pathways followed by pathogenic proteins from translation to disease-causing states are not completely understood (1-3). In some cases, partial or complete unfolding from the native state precedes protein aggregation, and thus the stability of a protein's native state may provide one measure of its propensity to aggregate. However, many familial protein misfolding diseases are caused by proteins that are not destabilized relative to their WT counterparts (4-6), implying that additional intrinsic or extrinsic factors may be required for protein aggregation.Our recent studies of a large number of ALS-mutant Cu,Znsuperoxide dismutase (SOD1) proteins have revealed that there is great diversity in the biophysical properties of these proteins (7-12). In contrast, Lindberg et al. (13) reported in 2002 that instability of the apoproteins of ALS-mutant SOD1 proteins is a ''common denominator'' among the nearly 100 known ALSlinked SOD1 mutations. More recently, Furukawa and O'Halloran (14) have reported that some of the destabilized mutant apoproteins studied by Lindberg et al. are further destabilized when the intrasubunit disulfide bond is reduced, again suggesting that protein destabilization is ...

More than 90 point mutations in human CuZn superoxide dismutase lead to the development of familial amyotrophic lateral sclerosis, known also as motor neuron disease. A growing body of evidence suggests that a subset of mutations located close to the dimeric interface can lead to a major destabilization of the mutant enzymes. We have determined the crystal structures of the Ala4Val (A4V) and Ile113Thr (I113T) mutants to 1.9 and 1.6 Å, respectively. In the A4V structure, small changes at the dimer interface result in a substantial reorientation of the two monomers. This effect is also seen in the case of the I113T crystal structure, but to a smaller extent. X-ray solution scattering data show that in the solution state, both of the mutants undergo a more pronounced conformational change compared with wild-type superoxide dismutase (SOD) than that observed in the A4V crystal structure. Shape reconstructions from the x-ray scattering data illustrate the nature of this destabilization. Comparison of these scattering data with those for bovine CuZn SOD measured at different temperatures shows that an analogous change in the scattering profile occurs for the bovine enzyme in the temperature range of 70 -80°C. These results demonstrate that the A4V and I113T mutants are substantially destabilized in comparison with wild-type SOD1, and it is possible that the pathogenic properties of this subset of familial amyotrophic lateral sclerosis mutants are at least in part due to this destabilization.human superoxide dismutase ͉ crystal structure ͉ x-ray solution scattering ͉ neurodegenerative disease