The small heat shock proteins αB-crystallin and Hsp27 suppress SOD1 aggregation in vitro

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease. The mechanism that underlies amyotrophic lateral sclerosis (ALS) pathology remains unclear, but protein inclusions are associated with all forms of the disease. Apart from pathogenic proteins, such as TDP-43 and SOD1, other proteins are associated with ALS inclusions including small heat shock proteins. However, whether small heat shock proteins have a direct effect on SOD1 aggregation remains unknown. In this study, we have examined the ability of small heat shock proteins αB-crystallin and Hsp27 to inhibit the aggregation of SOD1 in vitro. We show that these chaperone proteins suppress the increase in thioflavin T fluorescence associated with SOD1 aggregation, primarily through inhibiting aggregate growth, not the lag phase in which nuclei are formed. αB-crystallin forms high molecular mass complexes with SOD1 and binds directly to SOD1 aggregates. Our data are consistent with an overload of proteostasis systems being associated with pathology in ALS.     

A model for gain of function in superoxide dismutase

Studies have found that mutant, misfolded superoxide dismutase [Cu–Zn] (SOD1) can convert wild type SOD1 (wtSOD1) in a prion-like fashion, and that misfolded wtSOD1 can be propagated by release and uptake of protein aggregates. In developing a prion-like mechanism for this propagation of SOD1 misfolding we have previously shown how enervation of the SOD1 electrostatic loop (ESL), caused by the formation of transient non-obligate SOD1 oligomers, can lead to an experimentally observed gain of interaction (GOI) that results in the formation of SOD1 amyloid-like filaments. It has also been shown that freedom of ESL motion is essential to catalytic function. This work investigates the possibility that restricting ESL mobility might not only compromise superoxide catalytic activity but also serve to promote the peroxidase activity of SOD1, thus implicating the formation of SOD1 oligomers in both protein misfolding and in protein oxidation.     

Elevated cortical extracellular fluid glutamate in transgenic mice expressing human mutant (G93A) Cu/Zn superoxide dismutase

Transgenic mice expressing a mutated (G93A) human Cu/Zn superoxide dismutase (SOD1) develop motor neuron pathology and clinical symptoms similar to those seen in patients with amyotrophic lateral sclerosis. Loss of motor neurons is most prominent in lumbar, followed by cervical cord and then brainstem. No significant cell death has been reported in motor cortex. The integrity of the cortical glutamate reuptake systems was evaluated using intracerebral microdialysis and western immunoblot assays for the glutamate transporters GLT-1, GLAST, and EAAC1. The basal extracellular fluid levels of aspartate, glutamate, glutamine, 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindole-3-acetic acid were evaluated by HPLC. The extraction fraction of L-3H]glutamate, corrected with [14C]mannitol, was also evaluated. GLT-1, EAAC1, and GLAST protein levels were determined by semiquantitative chemiluminescence immunoblot of proteins from membrane-enriched fractions. The relative optical density of film was translated into relative protein level by comparison with a standard control mouse. The SOD1 mutant mice demonstrated a significant (p < 0.05) increase in basal levels of extracellular aspartate and glutamate. In addition, when the glutamate extraction fraction was challenged with exogenous unlabeled glutamate (500 microM) by reversed microdialysis, the glutamate extraction fraction in the mutant SOD1 mice was decreased significantly from control levels. The SOD1 mutant mice demonstrated no difference in the cortical protein levels of the glutamate transporter subtypes. This study demonstrates that in areas of no visible pathology and no loss of glutamate transporter proteins, SOD1 mutant mice have elevated extracellular fluid aspartate and glutamate levels and a decreased capacity to clear glutamate from the extracellular space.     

Gene Expression Profiles of APP and BACE1 in Tg SOD1G93A Cortical Cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse model (Tg SOD1G93A) shows pathological features that closely mimic those seen in ALS patients. An hypothetic link between AD and ALS was suggested by finding an higher amount of amyloid precursor protein (APP) in the spinal cord anterior horn neurons, and of Aβ peptides in ALS patients skin. In this work, we have investigated the expression of some genes involved in Alzheimer’s disease, as APP, β- and γ-secretase, in an animal model of ALS, to understand some possible common molecular mechanisms between these two pathologies. For gene expression analysis, we carried out a quantitative RT-PCR in ALS mice and in transgenic mice over-expressing human wild-type SOD1 (Tg hSOD1). We found that APP and BACE1 mRNA levels were increased 1.5-fold in cortical cells of Tg SOD1G93A mice respect to Tg hSOD1, whereas the expression of γ-secretase genes, as PSEN1, PSEN2, Nicastrin, and APH1a, showed no statistical differences between wild-type and ALS mice. Biochemical analysis carried out by immunostaining and western blotting, did not show any significant modulation of the protein expression compared to the genes, suggesting the existence of post-translational mechanisms that modify protein levels.     

Comparative study of behavioural tests in the SOD1G93A mouse model of amyotrophic lateral sclerosis

In preclinical trials, a sensitive functional test is required to detect changes in the motor behaviour of the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS). We evaluated changes in body weight and motor impairment in behavioural tests, such as the rotarod, the hanging-wire test and the treadmill, of transgenic and wild type mice. We found differences in detection of the onset of symptoms and progression of the disease between the different tests assessed. Moreover, the data showed significant gender differences in the motor behaviour of this mouse model. The rotarod and the hanging-wire test were more sensitive to detect early motor impairment. Moreover, the results suggested that the rotarod and hanging-wire became the most accurate tests rather than treadmill to characterise the ALS disease phenotype.     

Arylsulfanyl pyrazolones block mutant SOD1-G93A aggregation. Potential application for the treatment of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an orphan neurodegenerative disease currently without a cure. Mutations in copper/zinc superoxide dismutase 1 (SOD1) have been implicated in the pathophysiology of this disease. Using a high-throughput screening assay expressing mutant G93A SOD1, two bioactive chemical hit compounds (1 and 2), identified as arylsulfanyl pyrazolones, were identified. The structural optimization of this scaffold led to the generation of a more potent analogue (19) with an EC₅₀ of 170 nM. To determine the suitability of this class of compounds for further optimization, 1 was subjected to a battery of pharmacokinetic assays; most of the properties of 1 were good for a screening hit, except it had a relatively rapid clearance and short microsomal half-life stability. Compound 2 was found to be blood-brain barrier penetrating with a brain/plasma ratio = 0.19. The optimization of this class of compounds could produce novel therapeutic candidates for ALS patients.     

Structures of mouse SOD1 and human/mouse SOD1 chimeras

Mutations in human copper-zinc superoxide dismutase (SOD1) cause an inherited form of amyotrophic lateral sclerosis (ALS). Inclusions enriched in pathogenic SOD1 accumulate in the spinal cords of transgenic mice expressing these proteins, but endogenous mouse SOD1 is not found as a component of these aggregates. In the accompanying paper, Karch and colleagues analyze aggregation propensities of human/mouse SOD1 chimeras in cell culture and identify two sequence elements in the human enzyme that seem to enhance its aggregation relative to the mouse enzyme. Here, we report the first structure of mouse SOD1 along with those of SOD1 chimeras in which residues 1-80 come from human SOD1 and residues 81-153 come from mouse SOD1 and vice versa. Taken together, the structural and cell-based data suggest a model in which residues Q42 and Q123 in mouse SOD1 modulate non-native SOD1-SOD1 intermolecular interactions at edge strands in the SOD1 Greek key β-barrel.     

Mutant SOD1 linked to familial amyotrophic lateral sclerosis,but not wild-type SOD1, induces ER stress in COS7 cells and transgenic mice

Mutations in a Cu, Zn-superoxide dismutase (SOD1) cause motor neuron death in human familial amyotrophic lateral sclerosis (FALS) and its mouse model, suggesting that mutant SOD1 has a toxic effect on motor neurons. However, the question of how the toxic function is gained has not been answered. Here, we report that the mutant SOD1s linked to FALS, but not wild-type SOD1, aggregated in association with the endoplasmic reticulum (ER) and induced ER stress in the cDNA-transfected COS7 cells. These cells showed an aberrant intracellular localization of mitochondria and microtubules, which might lead to a functional disturbance of the cells. Motor neurons of the spinal cord in transgenic mice with a FALS-linked mutant SOD1 also showed a marked increase of GRP78/BiP, an ER-resident chaperone, just before the onset of motor symptoms. These data suggest that ER stress is involved in the pathogenesis of FALS with an SOD1 mutation.     

Co-chaperone CHIP associates with mutant Cu/Zn-superoxide dismutase proteins linked to familial amyotrophic lateral sclerosis and promotes their degradation by proteasomes

Although the ubiquitin-proteasome system and the molecular chaperones are implicated to play an important role in pathogenesis of familial amyotrophic lateral sclerosis (FALS) caused by mutations in Cu/Zn-superoxide dismutase (SOD1), the mechanism underlying the causes of this fatal disease is still poorly understood. Here we found that co-chaperone CHIP (carboxyl terminus of Hsc70-interacting protein), together with molecular chaperones Hsc70/Hsp70 and Hsp90, associates with FALS-linked mutant SOD1 proteins in cultured human cells. S5a subunit of 26S proteasomes, which recognizes polyubiquitylated proteins, also interacts with mutant SOD1 proteins. Over-expression of CHIP leads to the reduction in cellular levels of mutant SOD1 as well as the suppression of cytotoxicity induced by mutant SOD1. Unusually, rather than increasing the level of poly-ubiquitylated SOD1, over-expressed CHIP alters the ubiquitylation pattern of mutant SOD1 proteins. Both down-regulation and ubiquitylation of mutant SOD1 are greatly reduced by a mutant CHIP protein lacking U-box domain. Taken together, these results suggest that co-chaperone CHIP, possibly with another E3 ligase(s), modulates the ubiquitylation of mutant SOD1 and renders them more susceptible for proteasomal degradation.     

The structure of holo and metal-deficient wild-type human Cu,Zn superoxide dismutase and its relevance to familial amyotrophic lateral sclerosis

Cu, Zn superoxide dismutase (SOD1) forms a crucial component of the cellular defence against oxidative stress. Zn-deficient wild-type and mutant human SOD1 have been implicated in the disease familial amyotrophic lateral sclerosis (FALS). We present here the crystal structures of holo and metal-deficient (apo) wild-type protein at 1.8A resolution. The P21 wild-type holo enzyme structure has nine independently refined dimers and these combine to form a "trimer of dimers" packing motif in each asymmetric unit. There is no significant asymmetry between the monomers in these dimers, in contrast to the subunit structures of the FALS G37R mutant of human SOD1 and in bovine Cu,Zn SOD. Metal-deficient apo SOD1 crystallizes with two dimers in the asymmetric unit and shows changes in the metal-binding sites and disorder in the Zn binding and electrostatic loops of one dimer, which is devoid of metals. The second dimer lacks Cu but has approximately 20% occupancy of the Zn site and remains structurally similar to wild-type SOD1. The apo protein forms a continuous, extended arrangement of beta-barrels stacked up along the short crystallographic b-axis, while perpendicular to this axis, the constituent beta-strands form a zig-zag array of filaments, the overall arrangement of which has a similarity to the common structure associated with amyloid-like fibrils.     

Establishment of a cell model of ALS disease: Golgi apparatus disruption occurs independently from apoptosis

The Golgi apparatus (GA) appears disrupted in motor neurons of amyotrophic lateral sclerosis (ALS). Here, mouse motor neuron-like NSC-34 cell lines stably expressing human superoxide dismutase 1 (hSOD1)^wt and mutant hSOD1^G93A, as an ALS cell model, were constructed. The number of cells with disrupted GA increased from 14% to 34%. Furthermore, NSC-34/hSOD1^G93A cells showed lower levels of proliferation and differentiation. GA disruption was not caused by apoptosis as determined by several techniques including caspase-3 activation. Similarly, spinal cords from ALS patients did not show caspase-3 activation. Therefore, NSC-34/hSOD1^G93A cells are a suitable cell model to study GA dysfunction in ALS.     

Structural and thermodynamic effects of post-translational modifications in mutant and wild type Cu, Zn superoxide dismutase

Aggregation of Cu,Zn superoxide dismutase (SOD1) is implicated in amyotrophic lateral sclerosis. Glutathionylation and phosphorylation of SOD1 is omnipresent in the human body, even in healthy individuals, and has been shown to increase SOD1 dimer dissociation, which is the first step on the pathway toward SOD1 aggregation. We found that post-translational modification of SOD1, especially glutathionylation, promotes dimer dissociation. We discovered an intermediate state in the pathway to dissociation, a conformational change that involves a “loosening” of the β-barrels and a loss or shift of dimer interface interactions. In modified SOD1, this intermediate state is stabilized as compared to unmodified SOD1. The presence of post-translational modifications could explain the environmental factors involved in the speed of disease progression. Because post-translational modifications such as glutathionylation are often induced by oxidative stress, post-translational modification of SOD1 could be a factor in the occurrence of sporadic cases of amyotrophic lateral sclerosis, which represent 90% of all cases of the disease.     

肌萎缩性侧索硬化症相关的Cu,Zn-SOD

超氧化物歧化酶（superoxide dismutase,SOD）被称为生物体内自由基的清洁剂,其主要形式Cu,Zn-SOD称SOD1. SOD1突变体可引起致死性运动神经元疾病肌萎缩性侧索硬化症(ALS).但是,SOD1的毒性机理尚未完全清楚.本文概述了SOD1、Cu分子伴侣（copper chaperone for SOD1,CCS）的分子结构和CCS活化SOD1的机理,重点分析了突变体SOD1构象变化的原因及其在ALS中的可能致病机制的最新研究进展.     

Choline Acetyltransferase Activities in Single Spinal Motor Neurons from Patients with Amyotrophic Lateral Sclerosis

Activities of choline acetyltransferase (ChAT) were microassayed in individual cell bodies of motor neurons, isolated from freeze-dried sections after autopsy of lumbar spinal cords from four patients with sporadic amyotrophic lateral sclerosis (ALS) and four control patients with nonneurological diseases. Numerous large neurons were found in the anterior horn at the early degeneration stage of ALS, but the cell bodies atrophied and decreased in number at the late advanced stage. The small, atrophied neurons were very fragile and were easily destroyed during the isolation procedure with a microknife. The average activity, expressed on a dry weight basis, of 58 ALS neurons was lower than that of 67 control neurons. The large, well-preserved neurons at the early nonadvanced stage had markedly lower ChAT activities than control neurons. The specific activity gradually increased with the progress of atrophy but did not return to the control level.