Interaction of amyotrophic lateral sclerosis (ALS)-related mutant copper-zinc superoxide dismutase with the dynein-dynactin complex contributes to inclusion formation

An important consequence of protein misfolding related to neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), is the formation of proteinaceous inclusions or aggregates within the central nervous system. We have previously shown that several familial ALS-linked copper-zinc superoxide dismutase (SOD1) mutants (A4V, G85R, and G93A) interact and co-localize with the dynein-dynactin complex in cultured cells and affected tissues of ALS mice. In this study, we report that the interaction between mutant SOD1 and the dynein motor plays a critical role in the formation of large inclusions containing mutant SOD1. Disruption of the motor by overexpression of the p50 subunit of dynactin in neuronal and non-neuronal cell cultures abolished the association between aggregation-prone SOD1 mutants and the dynein-dynactin complex. The p50 overexpression also prevented mutant SOD1 inclusion formation and improved the survival of cells expressing A4V SOD1. Furthermore, we observed that two ALS-linked SOD1 mutants, H46R and H48Q, which showed a lower propensity to interact with the dynein motor, also produced less aggregation and fewer large inclusions. Overall, these data suggest that formation of large inclusions depends upon association of the abnormal SOD1s with the dynein motor. Whether the misfolded SOD1s directly perturb axonal transport or impair other functional properties of the dynein motor, this interaction could propagate a toxic effect that ultimately causes motor neuron death in ALS.     

Alterations in local stability and dynamics of A4V SOD1 in the presence of trifluoroethanol

Alterations in the local dynamics of Cu/Zn Superoxide dismutase (SOD1) due to mutations affect the protein folding, stability, and function leading to misfolding and aggregation seen in amyotrophic lateral sclerosis (ALS). Here, we study the structure and dynamics of the most devastating ALS mutation, A4V SOD1 in aqueous trifluoroethanol (TFE) through experiments and simulation. Far‐UV circular dichroism (CD) studies shows that TFE at intermediate concentrations (～15% ‐ 30%) induce partially unfolded β‐sheet‐rich extended conformations in A4V SOD1 which subsequently aggregates. Molecular dynamics (MD) simulation results shows that A4V SOD1 increases local dynamics in the active site loops that leads to the destabilization of the β‐barrel and loss of hydrophobic contacts, thus stipulating a basis for aggregation. Free energy landscape (FEL) and essential dynamics (ED) analysis demonstrates the conformational heterogeneity in A4V SOD1. Our results thus shed light on the role of local unfolding and conformational dynamics in aggregation of SOD1.     

Cysteine to Serine Conversion at 111th Position Renders the Disaggregation and Retains the Stabilization of Detrimental SOD1 A4V Mutant Against Amyotrophic Lateral Sclerosis in Human—A Discrete Molecular Dynamics Study

Protein aggregation is a hallmark of various neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) in humans. Mutations in Cu/Zn superoxide dismutase (SOD1) protein were found to be a prominent cause behind the majority of the familial ALS cases with abnormal protein aggregates. Herein, we report the biophysical characterization of the beneficial mutation C111S that stabilizes the SOD1 harboring A4V mutation, one of the most lethal diseases causing mutant that leads to protein destabilization and aggregation. In this study, we utilized discrete molecular dynamics (DMD) simulations, which stipulated an outlook over the systematic action of C111S mutation in the A4V mutant that stabilizes the protein and impedes the formation of protein aggregation. Herewith, the findings from our study manifested that the mutation of C111S in SOD1 could aid in regaining the protein structural conformations that protect against the formation of toxic aggregates, thereby hindering the disease pathogenicity subtly. Hence, our study provides a feasible pharmaceutical strategy in developing the treatment for incurable ALS affecting the mankind.     

Toxic SOD1 trimers are off-pathway in the formation of amyloid-like fibrils in ALS

Accumulation of insoluble amyloid fibrils is widely studied as a critical factor in the pathology of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. Misfolded Cu, Zn superoxide dismutase (SOD1) was the first protein linked to ALS, and non-native SOD1 trimeric oligomers were recently linked to cytotoxicity, while larger oligomers were protective to cells. The balance between trimers and larger aggregates in the process of SOD1 aggregation is, thus, a critical determinant of potential therapeutic approaches to treat ALS. However, it is unknown whether these trimeric oligomers are a necessary intermediate for larger aggregate formation or a distinct off-pathway species competing with fibril formation. Depending on the on- or off-pathway scenario of trimer formation, we expect drastically different therapeutic approaches. Here, we show that the toxic SOD1 trimer is an off-pathway intermediate competing with protective fibril formation. We design mutant SOD1 constructs that remain in a trimeric state (super-stable trimers) and show that stabilizing the trimeric SOD1 prevents formation of fibrils in vitro and in a motor neuron-like cell model (NSC-34). Using size exclusion chromatography, we track the aggregation kinetics of purified SOD1 and show direct competition of trimeric SOD1 with larger oligomer and fibril formation. Finally, we show the trimer is structurally independent of both larger soluble oligomers and insoluble fibrils using circular dichroism spectroscopy and limited proteolysis.     

DNA is a template for accelerating the aggregation of copper, zinc superoxide dismutase

The proteinaceous aggregates rich in copper, zinc superoxide dismutase (SOD1) have been shown to be involved in pathogenesis of amyotrophic lateral sclerosis (ALS). Since negatively charged species such as nucleic acids have frequently been found associated with the proteinaceous deposits in the tissues of patients with amyloid diseases, we examined here the aggregation behavior of SOD1 in the presence of DNA under acidic conditions that facilitate protein aggregation. Several forms of double-stranded DNA were tested to trigger SOD1 aggregation by light scattering, single- and double-fluorescence imaging with dyes, atomic force microscopy, and direct observations under visible light. The results reveal that DNA acts as a template for accelerating the formation of SOD1 aggregates and is incorporated into SOD1 aggregates. The spherical and ellipsoidal SOD1 aggregates were characterized in both hydrated and dried states and have morphology similar to those identified in the diseased neurons. Light scattering experiments indicate that the aggregation first undergoes a rapid phase where the aggregates with average diameters of 40-80 nm rapidly form in <2 min, and then passes through a slow phase where the average diameters of aggregates were increased to at least 200-260 nm in 2 h. All forms of DNAs tested can lead to the aggregation of SOD1 at nanomolar levels. The association of SOD1 with DNA, driven by electrostatic interactions between both, can restrict the orientation of SOD1 molecules and increase a SOD1 population along DNA strands. This facilitates the hydrophobic interactions between SOD1 molecules, as indicated by hydrophobic probe binding and chemical denaturant treatment experiments. Demonstration of the DNA-accelerated aggregation of SOD1 might establish a possible role of DNA in the pathogenesis of some diseases because of the ubiquitous expression of SOD1 and the coexistence of SOD1 and DNA in the crowded molecular environment of a cell.     

A yeast model of optineurin proteinopathy reveals a unique aggregation pattern associated with cellular toxicity

Many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) are linked to the accumulation of specific protein aggregates in affected regions of the nervous system. SOD1, TDP‐43, FUS and optineurin (OPTN) proteins were identified to form intraneuronal inclusions in ALS patients. In addition, mutations in OPTN are associated with both ALS and glaucoma. As the pathological role of OPTN in neuronal degeneration remains unresolved, we created a yeast model to study its potential for aggregation and toxicity. We observed that both wild type and disease‐associated mutants of OPTN form toxic non‐amyloid aggregates in yeast. Similar to reported cell culture and mouse models, the OPTN E50K mutant shows enhanced toxicity in yeast, implying a conserved gain‐of‐function mechanism. Furthermore, OPTN shows a unique aggregation pattern compared to other disease‐related proteins in yeast. OPTN aggregates colocalize only partially with the insoluble protein deposit (IPOD) site markers, but coincide perfectly with the prion seed‐reducing protein Btn2 and several other aggregation‐prone proteins, suggesting that protein aggregates are not limited to a single IPOD site. Importantly, changes in the Btn2p level modify OPTN toxicity and aggregation. This study generates a mechanistic framework for investigating how OPTN may trigger pathological changes in ALS and other OPTN‐linked neurodegenerative disorders.     

Amyotrophic lateral sclerosis mutations have the greatest destabilizing effect on the apo- and reduced form of SOD1, leading to unfolding and oxidative aggregation

Mutant forms of Cu,Zn-superoxide dismutase (SOD1) that cause familial amyotrophic lateral sclerosis (ALS) exhibit toxicity that promotes the death of motor neurons. Proposals for the toxic properties typically involve aberrant catalytic activities or protein aggregation. The striking thermodynamic stability of mature forms of the ALS mutant SOD1 (Tm>70 degrees C) is not typical of protein aggregation models that involve unfolding. Over 44 states of the polypeptide are possible, depending upon metal occupancy, disulfide status, and oligomeric state; however, it is not clear which forms might be responsible for toxicity. Recently the intramolecular disulfide has been shown to be required for SOD1 activity, leading us to examine these states of several disease-causing SOD1 mutants. We find that ALS mutations have the greatest effect on the most immature form of SOD1, destabilizing the metal-free and disulfide-reduced polypeptide to the point that it is unfolded at physiological temperatures (Tm<37 degrees C). We also find that immature states of ALS mutant (but not wild type) proteins readily form oligomers at physiological concentrations. Furthermore, these oligomers are more susceptible to mild oxidative stress, which promotes incorrect disulfide cross-links between conserved cysteines and drives aggregation. Thus it is the earliest disulfide-reduced polypeptides in the SOD1 assembly pathway that are most destabilized with respect to unfolding and oxidative aggregation by ALS-causing mutations.     

Elucidation of insulin assembly at acidic and neutral pH: Characterization of low molecular weight oligomers

Deficiency in insulin secretion and function that characterize type 2 diabetes often requires administration of extraneous insulin, leading to injection‐site amyloidosis. Insulin aggregation at neutral pH is not well understood. Although oligomer formation is believed to play an important role, insulin oligomers have not been fully characterized yet. Here, we elucidate similarities and differences between in vitro insulin aggregation at acidic and neutral pH for a range of insulin concentrations (2.5–100 μM) by using kinetic thioflavin T fluorescence, circular dichroism, atomic force and electron microscopy imaging. Importantly, we characterize the size distribution of insulin oligomers at different assembly stages by the application of covalent cross‐linking and gel electrophoresis. Our results show that at the earliest assembly stage, oligomers comprise up to 40% and 70% of soluble insulin at acidic and neutral pH, respectively. While the highest oligomer order increases with insulin concentration at acidic pH, the opposite tendency is observed at neutral pH, where oligomers up to heptamers are formed in 10 μM insulin. These findings suggest that oligomers may be on‐ and off‐pathway assemblies for insulin at acidic and neutral pH, respectively. Agitation, which is required to induce insulin aggregation at neutral pH, is shown to increase fibril formation rate and fibrillar mass both by an order of magnitude. Insulin incubated under agitated conditions at neutral pH rapidly aggregates into large micrometer‐sized aggregates, which may be of physiological relevance and provides insight into injection‐site amyloidosis and toxic pulmonary aggregates induced by administration of extraneous insulin.     

Nonamyloid aggregates arising from mature copper/zinc superoxide dismutases resemble those observed in amyotrophic lateral sclerosis

Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS) where aggregation of copper/zinc superoxide dismutase (SOD1) is implicated in pathogenesis. We report here that fully metallated (holo) SOD1 under physiologically relevant solution conditions can undergo changes in metallation and/or dimerization over time and form aggregates that do not exhibit classical characteristics of amyloid. The relevance of the observed aggregation to disease is demonstrated by structural and tinctorial analyses, including the novel observation of binding of an anti-SOD1 antibody that specifically recognizes aggregates in ALS patients and mice models. ALS-associated SOD1 mutations can promote aggregation but are not essential. The SOD1 aggregation is characterized by a lag phase, which is diminished by self- or cross-seeding and by heterogeneous nucleation. We interpret these findings in terms of an expanded aggregation mechanism consistent with other in vitro and in vivo findings that point to multiple pathways for the formation of toxic aggregates by different forms of SOD1.     

Novel Mutations that Enhance or Repress the Aggregation Potential of SOD1

Mutations in SOD1 cause FALS by a gain of function likely related to protein misfolding and aggregation. SOD1 mutations encompass virtually every domain of the molecule, making it difficult to identify motifs important in SOD1 aggregation. Zinc binding to SOD1 is important for structural integrity, and is hypothesized to play a role in mutant SOD1 aggregation. To address this question, we mutated the unique zinc binding sites of SOD1 and examined whether these changes would influence SOD1 aggregation. We generated single and multiple mutations in SOD1 zinc binding residues (H71, H80 and D83) either alone or in combination with an aggregate forming mutation (A4V) known to cause disease. These SOD1 mutants were assayed for their ability to form aggregates.Using an in vitro cellular SOD1 aggregation assay, we show that combining A4V with mutations in non-zinc binding domains (G37R or G85R) increases SOD1 aggregation potential. Mutations at two zinc binding residues (H71G and D83G) also increase SOD1 aggregation potential. However, an H80G mutation at the third zinc binding residue decreases SOD1 aggregation potential even in the context of other aggregate forming SOD1 mutations. These results demonstrate that various mutations have different effects on SOD1 aggregation potential and that the H80G mutation appears to uniquely act as a dominant inhibitor of SOD1 aggregation.     

An intersubunit disulfide bond prevents in vitro aggregation of a superoxide dismutase-1 mutant linked to familial amytrophic lateral sclerosis

Familial amyotrophic lateral sclerosis (FALS) is linked to over 90 point mutations in superoxide dismutase-1 (SOD1), a dimeric metalloenzyme. The postmortem FALS brain is characterized by SOD1 inclusions in the motor neurons of regions in which neuronal loss is most significant. These findings, together with animal modeling studies, suggest that aggregation of mutant SOD1 produces a pathogenic species. We demonstrate here that a mutant form of SOD1 (A4V) that is linked to a particularly aggressive form of FALS aggregates in vitro, while wild-type SOD1 (WT) is stable. Some A4V aggregates resemble amyloid pores formed by other disease-associated proteins. The WT dimer is significantly more stable than the A4V dimer, suggesting that dimer dissociation may be the required first step of aggregation. To test this hypothesis, an intersubunit disulfide bond between symmetry-related residues at the A4V dimer interface was introduced. The resultant disulfide bond (V148C-V148C') eliminated the concentration-dependent loss of enzymatic activity of A4V, stabilized the A4V dimer, and completely abolished aggregation. A drug-like molecule that could stabilize the A4V dimer could slow the onset and progression of FALS.     

Intracellular conformational alterations of mutant SOD1 and the implications for fALS-associated SOD1 mutant induced motor neuron cell death

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the selective death of motor neurons. Approximately 10% of ALS cases are familial (fALS) and about 25% of fALS patients inherit autosomal dominant mutations in the gene encoding copper-zinc superoxide dismutase (SOD1). Over 90 different SOD1 mutations have been identified in fALS patients. It has been established that the ALS-linked SOD1 mutations provoke a new toxic function, the nature of which remains unclear. In vitro studies using various biophysical techniques have demonstrated that the SOD1 mutants share a reduced conformational stability. However, conformational alterations of the ALS mutants have not been directly demonstrated in vivo. We employed an SOD1-GFP fusion protein system in this study to monitor the intracellular protein conformation. We demonstrate that the ALS-linked SOD1 mutants adopt different conformations from the wild-type (WT) protein in living cells. Moreover, the conformational alterations of mutant SOD1 render the mutants susceptible to the formation of high-molecular-weight complexes prior to the appearance of detergent-resistant aggregates. Finally, we show that the motor neuron-like cells expressing mutant SOD1 are more susceptible to H2O2 induced cell death compared to the cells expressing WT SOD1. This study provides direct evidence of in vivo conformational differences between WT and mutant SOD1. In addition, the SOD1-GFP system can be exploited in future studies to investigate how conformational alterations of mutant SOD1 lead to protein aggregation and to study the potential toxicity of such aggregates in familial ALS.     

Unsaturated fatty acids induce cytotoxic aggregate formation of amyotrophic lateral sclerosis-linked superoxide dismutase 1 mutants

Formation of misfolded protein aggregates is a remarkable hallmark of various neurodegenerative diseases including Alzheimer disease, Parkinson disease, Huntington disease, prion encephalopathies, and amyotrophic lateral sclerosis (ALS). Superoxide dismutase 1 (SOD1) immunoreactive inclusions have been found in the spinal cord of ALS animal models and patients, implicating the close involvement of SOD1 aggregates in ALS pathogenesis. Here we examined the molecular mechanism of aggregate formation of ALS-related SOD1 mutants in vitro. We found that long-chain unsaturated fatty acids (FAs) promoted aggregate formation of SOD1 mutants in both dose- and time-dependent manners. Metal-deficient SOD1s, wild-type, and mutants were highly oligomerized compared with holo-SOD1s by incubation in the presence of unsaturated FAs. Oligomerization of SOD1 is closely associated with its structural instability. Heat-treated holo-SOD1 mutants were readily oligomerized by the addition of unsaturated FAs, whereas wild-type SOD1 was not. The monounsaturated FA, oleic acid, directly bound to SOD1 and was characterized by a solid-phase FA binding assay using oleate-Sepharose. The FA binding characteristics were closely correlated with the oligomerization propensity of SOD1 proteins, which indicates that FA binding may change SOD1 conformation in a way that favors the formation of aggregates. High molecular mass aggregates of SOD1 induced by FAs have a granular morphology and show significant cytotoxicity. These findings suggest that SOD1 mutants gain FA binding abilities based on their structural instability and form cytotoxic granular aggregates.     

Oxidation-induced misfolding and aggregation of superoxide dismutase and its implications for amyotrophic lateral sclerosis

The presence of intracellular aggregates that contain Cu/Zn superoxide dismutase (SOD1) in spinal cord motor neurons is a pathological hallmark of amyotrophic lateral sclerosis (ALS). Although SOD1 is abundant in all cells, its half-life in motor neurons far exceeds that in any other cell type. On the basis of the premise that the long half-life of the protein increases the potential for oxidative damage, we investigated the effects of oxidation on misfolding/aggregation of SOD1 and ALS-associated SOD1 mutants. Zinc-deficient wild-type SOD1 and SOD1 mutants were extremely prone to form visible aggregates upon oxidation as compared with wild-type holo-protein. Oxidation of select histidine residues that bind metals in the active site mediates SOD1 aggregation. Our results provide a plausible model to explain the accumulation of SOD1 aggregates in motor neurons affected in ALS.     

SOD1 and amyotrophic lateral sclerosis: mutations and oligomerization

There are about 100 single point mutations of copper, zinc superoxide dismutase 1 (SOD1) which are reported (http://alsod.iop.kcl.ac.uk/Als/index.aspx) to be related to the familial form (fALS) of amyotrophic lateral sclerosis (ALS). These mutations are spread all over the protein. It is well documented that fALS produces protein aggregates in the motor neurons of fALS patients, which have been found to be associated to mitochondria. We selected eleven SOD1 mutants, most of them reported as pathological, and characterized them investigating their propensity to aggregation using different techniques, from circular dichroism spectra to ThT-binding fluorescence, size-exclusion chromatography and light scattering spectroscopy. We show here that these eleven SOD1 mutants, only when they are in the metal-free form, undergo the same general mechanism of oligomerization as found for the WT metal-free protein. The rates of oligomerization are different but eventually they give rise to the same type of soluble oligomeric species. These oligomers are formed through oxidation of the two free cysteines of SOD1 (6 and 111) and stabilized by hydrogen bonds, between beta strands, thus forming amyloid-like structures. SOD1 enters the mitochondria as demetallated and mitochondria are loci where oxidative stress may easily occur. The soluble oligomeric species, formed by the apo form of both WT SOD1 and its mutants through an oxidative process, might represent the precursor toxic species, whose existence would also suggest a common mechanism for ALS and fALS. The mechanism here proposed for SOD1 mutant oligomerization is absolutely general and it provides a common unique picture for the behaviors of the many SOD1 mutants, of different nature and distributed all over the protein.     

Cu/Zn superoxide dismutase can form pore-like structures

Mutations in Cu/Zn superoxide dismutase (SOD) are associated with familial amyotrophic lateral sclerosis (FALS), a neurodegenerative disease that is characterized by the selective death of motor neurons. Despite the genetic association made between the protein and the disease, the mechanism by which the mutant SOD proteins become toxic is still a mystery. Using wild-type SOD and three pathogenic mutants (A4V, G37R, and G85R), we show that the copper-induced oxidation of metal-depleted SOD causes its in vitro aggregation into pore-like structures, as determined by atomic force microscopy. Because toxic pores have been recently implicated in the pathogenic mechanism of other neurodegenerative diseases, these results raise the possibility that the aberrant self-assembly of oxidatively damaged SOD mutants into toxic oligomers or pores may have a pathological role in FALS.     

Cupric Ions Induce the Oxidation and Trigger the Aggregation of Human Superoxide Dismutase 1

Background

Amyotrophic lateral sclerosis (ALS), partly caused by the mutations and aggregation of human copper, zinc superoxide dismutase (SOD1), is a fatal degenerative disease of motor neurons. Because SOD1 is a major copper-binding protein present at relatively high concentration in motor neurons and copper can be a harmful pro-oxidant, we want to know whether aberrant copper biochemistry could underlie ALS pathogenesis. In this study, we have investigated and compared the effects of cupric ions on the aggregation of ALS-associated SOD1 mutant A4V and oxidized wild-type SOD1.

Methodology/Principal Findings

As revealed by 90° light scattering, dynamic light scattering, SDS-PAGE, and atomic force microscopy, free cupric ions in solution not only induce the oxidation of either apo A4V or Zn₂-A4V and trigger the oligomerization and aggregation of oxidized A4V under copper-mediated oxidative conditions, but also trigger the aggregation of non-oxidized form of such a pathogenic mutant. As evidenced by mass spectrometry and SDS-PAGE, Cys-111 is a primary target for oxidative modification of pathological human SOD1 mutant A4V by either excess Cu²⁺ or hydrogen peroxide. The results from isothermal titration calorimetry show that A4V possesses two sets of independent binding sites for Cu²⁺: a moderate-affinity site (10⁶ M^-1) and a high-affinity site (10⁸ M^-1). Furthermore, Cu²⁺ binds to wild-type SOD1 oxidized by hydrogen peroxide in a way similar to A4V, triggering the aggregation of such an oxidized form.

Conclusions/Significance

We demonstrate that excess cupric ions induce the oxidation and trigger the aggregation of A4V SOD1, and suggest that Cu²⁺ plays a key role in the mechanism of aggregation of both A4V and oxidized wild-type SOD1. A plausible model for how pathological SOD1 mutants aggregate in ALS-affected motor neurons with the disruption of copper homeostasis has been provided.     

Selective interactions of polyanions with basic surfaces on human immunodeficiency virus type 1 gp120

It is well established that the gp120 V3 loop of T-cell-line-adapted human immunodeficiency virus type 1 (HIV-1) binds both cell-associated and soluble polyanions. Virus infectivity is increased by interactions between HIV-1 and heparan sulfate proteoglycans on some cell types, and soluble polyanions such as heparin and dextran sulfate neutralize HIV-1 in vitro. However, the analysis of gp120-polyanion interactions has been limited to T-cell-line-adapted, CXCR4-using virus and virus-derived gp120, and the polyanion binding ability of gp120 regions other than the V3 loop has not been addressed. Here we demonstrate by monoclonal-antibody inhibition, labeled heparin binding, and surface plasmon resonance studies that a second site, most probably corresponding to the newly defined, highly conserved coreceptor binding region on gp120, forms part of the polyanion binding surface. Consistent with the binding of polyanions to the coreceptor binding surface, dextran sulfate interfered with the gp120-CXCR4 association while having no detectable effect on the gp120-CD4 interaction. The interaction between polyanions and X4 or R5X4 gp120 was readily detectable, whereas weak or undetectable binding was observed with R5 gp120. Analysis of mutated forms of X4 gp120 demonstrated that the V3 loop is the major determinant for polyanion binding whereas other regions, including the V1/V2 loop structure and the NH(2) and COOH termini, exert a more subtle influence. A molecular model of the electrostatic potential of the conserved coreceptor binding region confirmed that it is basic but that the overall charge on this surface is dominated by the V3 loop. These results demonstrate a selective interaction of gp120 with polyanions and suggest that the conserved coreceptor binding surface may present a novel and conserved target for therapeutic intervention.     

p62 accumulates and enhances aggregate formation in model systems of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurode-generative disease characterized by motor neuron death. A hallmark of the disease is the appearance of protein aggregates in the affected motor neurons. We have found that p62, a protein implicated in protein aggregate formation, accumulated progressively in the G93A mouse spinal cord. The accumulation of p62 was in parallel to the increase of polyubiquitinated proteins and mutant SOD1 aggregates. Immunostaining studies showed that p62, ubiquitin, and mutant SOD1 co-localized in the protein aggregates in affected cells in G93A mouse spinal cord. The p62 protein selectively interacted with familial ALS mutants, but not WT SOD1. When p62 was co-expressed with SOD1 in NSC34 cells, it greatly enhanced the formation of aggregates of the ALS-linked SOD1 mutants, but not wild-type SOD1. Cell viability was measured in the presence and absence of overexpressed p62, and the results suggest that the large aggregates facilitated by p62 were not directly toxic to cells under the conditions in this study. Deletion of the ubiquitin-association (UBA) domain of p62 significantly decreased the p62-facilitated aggregate formation, but did not completely inhibit it. Further protein interaction experiments also showed that the truncated p62 with the UBA domain deletion remained capable of interacting with mutant SOD1. The findings of this study show that p62 plays a critical role in forming protein aggregates in familial ALS, likely by linking misfolded mutant SOD1 molecules and other cellular proteins together.     

Elevation of the Hsp70 chaperone does not effect toxicity in mouse models of familial amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) account for 10-20% of a familial form of amyotrophic lateral sclerosis (ALS). A common feature of SOD1 mutants is abnormal aggregation of the aberrant SOD1 in neurons and glia. We now report that in ALS transgenic mouse models the constitutively expressed heat shock protein 70 (Hsp70) is mislocalized into aggregates together with mutant SOD1 and ubiquitin. Forcing increased synthesis of Hsp70 ameliorates both aggregate formation and toxicity in primary motor neurons in culture. However, chronic increase in an inducible form of Hsp70 to about 10-fold its normal level is shown here not to affect disease course or pathology developed in mice from accumulation of any of three familial ALS causing SOD1 mutants with different underlying biochemical characteristics. Therefore, increasing Hsp70 to a level that is protective in mouse models of acute ischemic insult and selected neurodegenerative disorders is not sufficient to ameliorate mutant SOD1-mediated toxicity.