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...

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 ...

We observed that 14 biologically metallated mutants of copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis all exhibited aberrantly accelerated mobility during partially denaturing PAGE and increased sensitivity to proteolytic digestion compared with wild type SOD1. Decreased metal binding site occupancy and exposure to the disulfide-reducing agents dithiothreitol, Tris(2-carboxyethyl)phosphine (TCEP), or reduced glutathione increased the fraction of anomalously migrating mutant SOD1 proteins. Furthermore, the incubation of mutant SOD1s with TCEP increased the accessibility to iodoacetamide of cysteine residues that normally participate in the formation of the intrasubunit disulfide bond (Cys-57 to Cys-146) or are buried within the core of the ␤-barrel (Cys-6). SOD1 enzymes in spinal cord lysates from G85R and G93A mutant but not wild type SOD1 transgenic mice also exhibited abnormal vulnerability to TCEP, which exposed normally inaccessible cysteine residues to modification by maleimide conjugated to polyethylene glycol. These results implicate SOD1 destabilization under cellular disulfide-reducing conditions at physiological pH and temperature as a shared property that may be relevant to amyotrophic lateral sclerosis mutant neurotoxicity.Amyotrophic lateral sclerosis (ALS) 1 is an age-dependent degenerative disorder of motor neurons in the spinal cord, brain stem, and brain (1). Approximately 10% of ALS cases are familial, and ϳ20% of these individuals inherit one of Ͼ90 autosomal dominant mutations in the gene encoding copper/ zinc superoxide dismutase 1 (SOD1) (2). 2 SOD1 is a 32-kDa homodimeric enzyme expressed predominantly in the cytosol that decreases the intracellular concentration of superoxide radicals (O 2 . ) by catalyzing their dismutation to O 2 and H 2 O 2 . ALS-associated mutations of conserved residues throughout the protein impart a toxic property to the enzyme that appears unrelated to its normal dismutase activity (reviewed in Ref.3). Whereas transgenic mice that overexpress mutant SOD1s consistently develop lethal motor neuron degeneration (4 -8), mice that overexpress the wild type (WT) enzyme exhibit only subtle motor abnormalities (9). In addition, SOD1 knock-out mice are not susceptible to motor neuron loss unless following axonal injury (10). Mutant SOD1 enzymes have been proposed to facilitate aberrant copper-mediated chemistry, disrupt protein recycling or chaperone function, form toxic aggregates, or induce organelle dysfunction or apoptosis (3,11,12), but the precise mechanism of specific motor neuron toxicity has not been elucidated. The observation that some mutant SOD1s exhibit accelerated turnover in vivo or increased proteolytic susceptibility compared with the WT enzyme (13-15) suggests that biologically significant perturbations of mutant SOD1 conformation occur. The induction of chaperone proteins that can protect cultured motor neurons from mutant SOD1 toxicity (16) and appear to associate with SOD1 mutants (17) provides further...