Evaluating a Gene-Environment Interaction in Amyotrophic Lateral Sclerosis: Methylmercury Exposure and Mutated SOD1.

PURPOSE OF REVIEW: Gene-environment (GxE) interactions likely contribute to numerous diseases, but are often difficult to model in the laboratory. Such interactions have been widely hypothesized for amyotrophic lateral sclerosis (ALS); recent controlled laboratory studies are discussed here and hypotheses related to possible mechanisms of action are offered. Using methylmercury exposure and mutated SOD1 to model the impacts of such an interaction, we interpret evidence about their respective mechanisms of toxicity to interrogate the possibility of additive (or synergistic) effects when combined. RECENT FINDINGS: Recent work has converged on mechanisms of calcium-mediated glutamate excitotoxicity as a likely contributor in one model of a gene-environment interaction affecting the onset and progression of ALS-like phenotype. The current experimental literature on mechanisms of metal-induced neuronal injury and their relevant interactions with genetic contributions in ALS is sparse, but we describe those studies here and offer several integrative hypotheses about the likely mechanisms involved.

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis.

Since the linking of mutations in the Cu,Zn superoxide dismutase gene (sod1) to amyotrophic lateral sclerosis (ALS) in 1993, researchers have sought the connection between SOD1 and motor neuron death. Disease-linked mutations tend to destabilize the native dimeric structure of SOD1, and plaques containing misfolded and aggregated SOD1 have been found in the motor neurons of patients with ALS. Despite advances in understanding of ALS disease progression and SOD1 folding and stability, cytotoxic species and mechanisms remain unknown, greatly impeding the search for and design of therapeutic interventions. Here, we definitively link cytotoxicity associated with SOD1 aggregation in ALS to a nonnative trimeric SOD1 species. We develop methodology for the incorporation of low-resolution experimental data into simulations toward the structural modeling of metastable, multidomain aggregation intermediates. We apply this methodology to derive the structure of a SOD1 trimer, which we validate in vitro and in hybridized motor neurons. We show that SOD1 mutants designed to promote trimerization increase cell death. Further, we demonstrate that the cytotoxicity of the designed mutants correlates with trimer stability, providing a direct link between the presence of misfolded oligomers and neuron death. Identification of cytotoxic species is the first and critical step in elucidating the molecular etiology of ALS, and the ability to manipulate formation of these species will provide an avenue for the development of future therapeutic strategies.

Parkin Protects Against Misfolded SOD1 Toxicity by Promoting Its Aggresome Formation and Autophagic Clearance.

Mutations in Cu/Zn superoxide dismutase (SOD1) cause autosomal dominant amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease with no effective treatment. Despite ample evidence indicating involvement of mutation-induced SOD1 protein misfolding and aggregation in ALS pathogenesis, the molecular mechanisms that control cellular management of misfolded, aggregation-prone SOD1 mutant proteins remain unclear. Here, we report that parkin, an E3 ubiquitin-protein ligase which is linked to Parkinson's disease, is a novel regulator of cellular defense against toxicity induced by ALS-associated SOD1 mutant proteins. We find that parkin mediates K63-linked polyubiquitination of SOD1 mutants in cooperation with the UbcH13/Uev1a E2 enzyme and promotes degradation of these misfolded SOD1 proteins by the autophagy-lysosome system. In response to strong proteotoxic stress associated with proteasome impairment, parkin promotes sequestration of misfolded and aggregated SOD1 proteins to form perinuclear aggresomes, regulates positioning of lysosomes around misfolded SOD1 aggresomes, and facilitates aggresome clearance by autophagy. Our findings reveal parkin-mediated cytoprotective mechanisms against misfolded SOD1 toxicity and suggest that enhancing parkin-mediated cytoprotection may provide a novel therapeutic strategy for treating ALS.

A novel SOD1-ALS mutation separates central and peripheral effects of mutant SOD1 toxicity.

Transgenic mouse models expressing mutant superoxide dismutase 1 (SOD1) have been critical in furthering our understanding of amyotrophic lateral sclerosis (ALS). However, such models generally overexpress the mutant protein, which may give rise to phenotypes not directly relevant to the disorder. Here, we have analysed a novel mouse model that has a point mutation in the endogenous mouse Sod1 gene; this mutation is identical to a pathological change in human familial ALS (fALS) which results in a D83G change in SOD1 protein. Homozgous Sod1(D83G/D83G) mice develop progressive degeneration of lower (LMN) and upper motor neurons, likely due to the same unknown toxic gain of function as occurs in human fALS cases, but intriguingly LMN cell death appears to stop in early adulthood and the mice do not become paralyzed. The D83 residue coordinates zinc binding, and the D83G mutation results in loss of dismutase activity and SOD1 protein instability. As a result, Sod1(D83G/D83G) mice also phenocopy the distal axonopathy and hepatocellular carcinoma found in Sod1 null mice (Sod1(-/-)). These unique mice allow us to further our understanding of ALS by separating the central motor neuron body degeneration and the peripheral effects from a fALS mutation expressed at endogenous levels.

Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective and progressive loss of motor neurons. Cystatin C (CysC), an endogenous cysteine protease inhibitor, is a major protein component of Bunina bodies observed in the spinal motor neurons of sporadic ALS and is decreased in the cerebrospinal fluid of ALS patients. Despite prominent deposition of CysC in ALS, the roles of CysC in the central nervous system remain unknown. Here, we identified the neuroprotective activity of CysC against ALS-linked mutant Cu/Zn-superoxide dismutase (SOD1)-mediated toxicity. We found that exogenously added CysC protected neuronal cells including primary cultured motor neurons. Moreover, the neuroprotective property of CysC was dependent on the coordinated activation of two distinct pathways: autophagy induction through AMPK-mTOR pathway and inhibition of cathepsin B. Furthermore, exogenously added CysC was transduced into the cells and aggregated in the cytosol under oxidative stress conditions, implying a relationship between the neuroprotective activity of CysC and Bunina body formation. These data suggest CysC is an endogenous neuroprotective agent and targeting CysC in motor neurons may provide a novel therapeutic strategy for ALS.

Identification of a misfolded region in superoxide dismutase 1 that is exposed in amyotrophic lateral sclerosis.

Mutations and aberrant post-translational modifications within Cu,Zn-superoxide dismutase (SOD1) cause this otherwise protective enzyme to misfold, leading to amyotrophic lateral sclerosis (ALS). The C4F6 antibody selectively binds misfolded SOD1 in spinal cord tissues from postmortem human ALS cases, as well as from an ALS-SOD1 mouse model, suggesting that the C4F6 epitope reports on a pathogenic conformation that is common to misfolded SOD1 variants. To date, the residues and structural elements that comprise this epitope have not been elucidated. Using a chemical cross-linking and mass spectrometry approach, we identified the C4F6 epitope within several ALS-linked SOD1 variants, as well as an oxidized form of WT SOD1, supporting the notion that a similar misfolded conformation is shared among pathological SOD1 proteins. Exposure of the C4F6 epitope was modulated by the SOD1 electrostatic (loop VII) and zinc binding (loop IV) loops and correlated with SOD1-induced toxicity in a primary microglia activation assay. Site-directed mutagenesis revealed Asp(92) and Asp(96) as key residues within the C4F6 epitope required for the SOD1-C4F6 binding interaction. We propose that stabilizing the functional loops within SOD1 and/or obscuring the C4F6 epitope are viable therapeutic strategies for treating SOD1-mediated ALS.

Abnormal intracellular calcium signaling and SNARE-dependent exocytosis contributes to SOD1G93A astrocyte-mediated toxicity in amyotrophic lateral sclerosis.

Motor neurons are progressively and predominantly degenerated in ALS, which is not only induced by multiple intrinsic pathways but also significantly influenced by the neighboring glial cells. In particular, astrocytes derived from the SOD1 mutant mouse model of ALS or from human familial or sporadic ALS patient brain tissue directly induce motor neuron death in culture; however, the mechanisms of pathological astroglial secretion remain unclear. Here we investigated abnormal calcium homeostasis and altered exocytosis in SOD1G93A astrocytes. We found that purinergic stimulation induces excess calcium release from the ER stores in SOD1G93A astrocytes, which results from the abnormal ER calcium accumulation and is independent of clearance mechanisms. Furthermore, pharmacological studies suggested that store-operated calcium entry (SOCE), a calcium refilling mechanism responsive to ER calcium depletion, is enhanced in SOD1G93A astrocytes. We found that oxidant-induced increased S-glutathionylation and calcium-independent puncta formation of the ER calcium sensor STIM1 underlies the abnormal SOCE response in SOD1G93A astrocytes. Enhanced SOCE contributes to ER calcium overload in SOD1G93A astrocytes and excess calcium release from the ER during ATP stimulation. In addition, ER calcium release induces elevated ATP release from SOD1G93A astrocytes, which can be inhibited by the overexpression of dominant-negative SNARE. Selective inhibition of exocytosis in SOD1G93A astrocytes significantly prevents astrocyte-mediated toxicity to motor neurons and delays disease onset in SOD1G93A mice. Our results characterize a novel mechanism responsible for calcium dysregulation in SOD1G93A astrocytes and provide the first in vivo evidence that astrocyte exocytosis contributes to the pathogenesis of ALS.

Human superoxide dismutase 1 overexpression in motor neurons of Caenorhabditis elegans causes axon guidance defect and neurodegeneration.

Strong evidence indicates that mutant Cu, Zn-superoxide dismutase 1 (SOD1) exerts toxic effect on motor neurons in amyotrophic lateral sclerosis (ALS). However, the nature of mutant SOD1-mediated motor neuron degeneration is poorly understood. To provide new insight into the mechanism by which mutant SOD1 induces motor neuron injury, we developed novel Caenorhabditis elegans models of ALS. Expression of human wild type or G93A SOD1 specifically in motor neurons of C. elegans caused progressive locomotion defect and paralytic phenotype, which recapitulate some characteristic features of ALS including age-dependent motor dysfunction and degeneration of motor neurons associated with SOD1 aggregation. In addition, the motor neuron loss is independent of cell death protein 3 (CED-3)/cell death protein 4 (CED-4) caspase pathway. We also found that before motor neurons began to die in adulthood, axon guidance defect of motor neuron appeared during the development stages. When green fluorescent protein (GFP)-tagged proteins related to axon guidance were examined in motor neurons, a significantly decreased density and number of GFP-tagged puncta were observed in the transgenic worms. Our models mimic axon developmental defect and the adult-onset degeneration of motor neurons in ALS. Using this model, we uncovered the cell-autonomous damage caused by human SOD1 to motor neurons in vivo, and provided a new insight into the developmental defect mechanism that may contribute to motor neuron degeneration in ALS.

Disulfide scrambling in superoxide dismutase 1 reduces its cytotoxic effect in cultured cells and promotes protein aggregation.

Mutations in the gene coding for superoxide dismutase 1 (SOD1) are associated with familiar forms of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). These mutations are believed to result in a "gain of toxic function", leading to neuronal degeneration. The exact mechanism is still unknown, but misfolding/aggregation events are generally acknowledged as important pathological events in this process. Recently, we observed that demetallated apoSOD1, with cysteine 6 and 111 substituted for alanine, is toxic to cultured neuroblastoma cells. This toxicity depended on an intact, high affinity Zn(2+) site. It was therefor contradictory to discover that wild-type apoSOD1 was not toxic, despite of its high affinity for Zn(2+). This inconsistency was hypothesized to originate from erroneous disulfide formation involving C6 and C111. Using high resolution non-reducing SDS-PAGE, we have in this study demonstrated that the inability of wild-type apoSOD1 to cause cell death stems from formation of non-native intra-molecular disulfides. Moreover, monomeric apoSOD1 variants capable of such disulfide scrambling aggregated into ThT positive oligomers under physiological conditions without agitation. The oligomers were stabilized by inter-molecular disulfides and morphologically resembled what has in other neurodegenerative diseases been termed protofibrils. Disulfide scrambling thus appears to be an important event for misfolding and aggregation of SOD1, but may also be significant for protein function involving cysteines, e.g. mitochondrial import and copper loading.

Small peptides against the mutant SOD1/Bcl-2 toxic mitochondrial complex restore mitochondrial function and cell viability in mutant SOD1-mediated ALS.

Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS) in 20% of familial cases (fALS). Mitochondria are one of the targets of mutant SOD1 (mutSOD1) toxicity. We previously demonstrated that at the mitochondria, mutSOD1 forms a toxic complex with Bcl-2, which is then converted into a toxic protein via a structural rearrangement that exposes its toxic BH3 domain (Pedrini et al., 2010). Here we now show that formation of this toxic complex with Bcl-2 is the primary event in mutSOD1-induced mitochondrial dysfunction, inhibiting mitochondrial permeability to ADP and inducing mitochondrial hyperpolarization. In mutSOD1-G93A cells and mice, the newly exposed BH3 domain in Bcl-2 alters the normal interaction between Bcl-2 and VDAC1 thus reducing permeability of the outer mitochondrial membrane. In motor neuronal cells, the mutSOD1/Bcl-2 complex causes mitochondrial hyperpolarization leading to cell loss. Small SOD1-like therapeutic peptides that specifically block formation of the mutSOD1/Bcl-2 complex, recover both aspects of mitochondrial dysfunction: they prevent mitochondrial hyperpolarization and cell loss as well as restore ADP permeability in mitochondria of symptomatic mutSOD1-G93A mice.

Inhibition of fast axonal transport by pathogenic SOD1 involves activation of p38 MAP kinase.

Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS.

Expression of zinc-deficient human superoxide dismutase in Drosophila neurons produces a locomotor defect linked to mitochondrial dysfunction.

More than 130 different mutations in the Cu/Zn superoxide dismutase (SOD1) gene have been associated with amyotrophic lateral sclerosis but the mechanism of this toxicity remains controversial. To gain insight into the importance of the zinc site in the pathogenesis of SOD1 in vivo, we generated a Drosophila model with transgenic expression of a zinc-deficient human SOD1. Expression of zinc-deficient SOD1 in Drosophila resulted in a progressive movement defect with associated mitochondrial cristae vacuolization and reductions in adenosine triphosphate (ATP) levels. Furthermore, these flies are sensitized to mitochondrial toxins, paraquat, and zinc. Importantly, we show that the zinc-deficient SOD1-induced motor defect can be ameliorated by supplementing the endogenous fly respiratory chain machinery with the single-subunit NADH-ubiquinone oxidoreductase from yeast (NADH is nicotinamide adenine dinucleotide, reduced form.). These results demonstrate that zinc-deficient SOD1 is neurotoxic in vivo and suggest that mitochondrial dysfunction plays a critical role in this toxicity. The robust behavioral, pathological, and biochemical phenotypes conferred by zinc-deficient SOD1 in Drosophila have general implications for the role of the zinc ion in familial and sporadic amyotrophic lateral sclerosis.

Over-expression of N-type calcium channels in cortical neurons from a mouse model of Amyotrophic Lateral Sclerosis.

Voltage-gated Ca(2+) channels (VGCCs) mediate calcium entry into neuronal cells in response to membrane depolarisation and play an essential role in a variety of physiological processes. In Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disease caused by motor neuron degeneration in the brain and spinal cord, intracellular calcium dysregulation has been shown, while no studies have been carried out on VGCCs. Here we show that the subtype N-type Ca(2+) channels are over expressed in G93A cultured cortical neurons and in motor cortex of G93A mice compared to Controls. In fact, by western blotting, immunocytochemical and electrophysiological experiments, we observe higher membrane expression of N-type Ca(2+) channels in G93A neurons compared to Controls. G93A cortical neurons filled with calcium-sensitive dye Fura-2, show a net calcium entry during membrane depolarization that is significantly higher compared to Control. Analysis of neuronal vitality following the exposure of neurons to a high K(+) concentration (25 mM, 5h), shows a significant reduction of G93A cellular survival compared to Controls. N-type channels are involved in the G93A higher mortality because ω-conotoxin GVIA (1 µM), which selectively blocks these channels, is able to abolish the higher G93A mortality when added to the external medium. These data provide robust evidence for an excess of N-type Ca(2+) expression in G93A cortical neurons which induces a higher mortality following membrane depolarization. These results may be central to the understanding of pathogenic pathways in ALS and provide novel molecular targets for the design of rational therapies for the ALS disorder.

Substituted pyrazolones require N2 hydrogen bond donating ability to protect against cytotoxicity from protein aggregation of mutant superoxide dismutase 1.

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal neurodegenerative disease. Although the cause remains unknown, misfolded protein aggregates are seen in neurons of sporadic ALS patients, and familial ALS mutations, including mutations in superoxide dismutase 1 (SOD1), produce proteins with an increased propensity to misfold and aggregate. A structure activity relationship of a lead scaffold exhibiting neuroprotective activity in a G93A-SOD1 mouse model for ALS has been further investigated in a model PC12 cellular assay. Synthesis of biotinylated probes at the N(1) nitrogen of the pyrazolone ring gave compounds (5d-e) that retained activity within 10-fold of the proton-bearing lead compound (5a) and were equipotent with a sterically less cumbersome N(1)-methyl substituted analogue (5b). However, when methyl substitution was introduced at N(1) and N(2) of the pyrazolone ring, the compound was inactive (5c). These data led us to investigate further the pharmacophoric nature of the pyrazolone unit. A range of N(1) substitutions were tolerated, leading to the identification of an N(1)-benzyl substituted pyrazolone (5m), equipotent with 5a. Substitution at N(2) or excision of N(2), however, removed all activity. Therefore, the hydrogen bond donating ability of the N(2)-H of the pyrazolone ring appears to be a critical part of the structure, which will influence further analogue synthesis.

Cytotoxicity of superoxide dismutase 1 in cultured cells is linked to Zn2+ chelation.

Neurodegeneration in protein-misfolding disease is generally assigned to toxic function of small, soluble protein aggregates. Largely, these assignments are based on observations of cultured neural cells where the suspect protein material is titrated directly into the growth medium. In the present study, we use this approach to shed light on the cytotoxic action of the metalloenzyme Cu/Zn superoxide dismutase 1 (SOD1), associated with misfolding and aggregation in amyotrophic lateral sclerosis (ALS). The results show, somewhat unexpectedly, that the toxic species of SOD1 in this type of experimental setting is not an aggregate, as typically observed for proteins implicated in other neuro-degenerative diseases, but the folded and fully soluble apo protein. Moreover, we demonstrate that the toxic action of apoSOD1 relies on the protein's ability to chelate Zn(2+) ions from the growth medium. The decreased cell viability that accompanies this extraction is presumably based on disturbed Zn(2+) homeostasis. Consistently, mutations that cause global unfolding of the apoSOD1 molecule or otherwise reduce its Zn(2+) affinity abolish completely the cytotoxic response. So does the addition of surplus Zn(2+). Taken together, these observations point at a case where the toxic response of cultured cells might not be related to human pathology but stems from the intrinsic limitations of a simplified cell model. There are several ways proteins can kill cultured neural cells but all of these need not to be relevant for neurodegenerative disease.

An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1.

Recent studies suggest that Cu/Zn superoxide dismutase (SOD1) could be pathogenic in both familial and sporadic amyotrophic lateral sclerosis (ALS) through either inheritable or nonheritable modifications. The presence of a misfolded WT SOD1 in patients with sporadic ALS, along with the recently reported evidence that reducing SOD1 levels in astrocytes derived from sporadic patients inhibits astrocyte-mediated toxicity on motor neurons, suggest that WT SOD1 may acquire toxic properties similar to familial ALS-linked mutant SOD1, perhaps through posttranslational modifications. Using patients' lymphoblasts, we show here that indeed WT SOD1 is modified posttranslationally in sporadic ALS and is iper-oxidized (i.e., above baseline oxidation levels) in a subset of patients with bulbar onset. Derivatization analysis of oxidized carbonyl compounds performed on immunoprecipitated SOD1 identified an iper-oxidized SOD1 that recapitulates mutant SOD1-like properties and damages mitochondria by forming a toxic complex with mitochondrial Bcl-2. This study conclusively demonstrates the existence of an iper-oxidized SOD1 with toxic properties in patient-derived cells and identifies a common SOD1-dependent toxicity between mutant SOD1-linked familial ALS and a subset of sporadic ALS, providing an opportunity to develop biomarkers to subclassify ALS and devise SOD1-based therapies that go beyond the small group of patients with mutant SOD1.

Daf-2 signaling modifies mutant SOD1 toxicity in C. elegans.

The DAF-2 Insulin/IGF-1 signaling (IIS) pathway is a strong modifier of Caenorhabditis elegans longevity and healthspan. As aging is the greatest risk factor for developing neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS), we were interested in determining if DAF-2 signaling modifies disease pathology in mutant superoxide dismutase 1 (SOD1) expressing C. elegans. Worms with pan-neuronal G85R SOD1 expression demonstrate significantly impaired locomotion as compared to WT SOD1 expressing controls and they develop insoluble SOD1 aggregates. Reductions in DAF-2 signaling, either through a hypomorphic allele or neuronally targeted RNAi, decreases the abundance of aggregated SOD1 and results in improved locomotion in a DAF-16 dependant manner. These results suggest that manipulation of the DAF-2 Insulin/IGF-1 signaling pathway may have therapeutic potential for the treatment of ALS.

A novel variant of human superoxide dismutase 1 harboring amyotrophic lateral sclerosis-associated and experimental mutations in metal-binding residues and free cysteines lacks toxicity in vivo.

Mutations in superoxide dismutase 1 (SOD1) cause familial amyotrophic lateral sclerosis. The Cu-binding capacity of SOD1 has spawned hypotheses that implicate metal-mediated production of reactive species as a potential mechanism of toxicity. In past experiments, we have tested such hypotheses by mutating residues in SOD1 that normally coordinate the binding of Cu, finding that such mutants retain the capacity to induce motor neuron disease. We now describe the lack of disease in mice that express a variant of human SOD1 in which residues that coordinate the binding of Cu and Zn have been mutated (SODMD). SODMD encodes three disease-causing and four experimental mutations that ultimately eliminate all histidines involved in the binding of metals; and includes one disease-causing and one experimental mutation that eliminate secondary metal binding at C6 and C111. We show that the combined effect of these mutations produces a protein that is unstable but does not aggregate on its own, is not toxic, and does not induce disease when co-expressed with high levels of wild-type SOD1. In cell culture models, we determine that the combined mutation of C6 and C111 to G and S, respectively, dramatically reduces the aggregation propensity of SODMD and may account for the lack of toxicity for this mutant.

28-day repeated dose oral toxicity of human copper-zinc superoxide dismutase from recombinant Pichia pastori in rats.

Human copper/zinc superoxide dismutase from recombinant Pichia pastori (RH-Cu/Zn-SOD) was orally administered, via gavage, to Sprague-Dawley rats at 500, 1,000, and 2,000 mg/kg body weight/day for 28 days. During the 28-day period, animals were examined for evidence of toxicity; there were no deaths, and in-life physical signs were normal. On day 29, the animals were exsanguinated, examined for gross pathology, and tissues were preserved for histopathology. Although statistical differences were noted in some hematology and clinical chemistry, they were of questionable biological significance. The results of the 28-day oral administration demonstrated a lack of toxicity of RH-Cu/Zn-SOD in rats. There were no treatment-related, toxicologically relevant changes in clinical signs, growth, food consumption, hematology, clinical chemistry, organ weights, or pathology. The no observed adverse effect level was greater than 2,000 mg/kg/day for RH-Cu/Zn-SOD in rats.

Neuroprotective effect of Cu,Zn-superoxide dismutase fused to a TCTP-derived protein transduction domain.

Previously, we have reported that a 10-amino acid peptide (MIIYRDLISH) derived from the NH(2)-terminus of the human translationally controlled tumor protein (TCTP) functions as a protein transduction domain (PTD). In this study, we evaluated the transduction ability of SOD fused to TCTP-PTD (TCTP-SOD) into various cell lines. We also evaluated its ability to protect cells against paraquat-induced cell damage, in vitro and its neuroprotective effect in vivo against kainic acid-induced neuronal damage in an animal model. TCTP-SOD was transduced into various cell lines in a dose- and time-dependent manner without cytotoxic effect. Furthermore, TCTP-SOD showed cytoprotective activity in SH-SY5Y cells, and intraperitoneally, injected TCTP-SOD was delivered into the mouse brain and protected the cells in the hippocampal region against the damage induced by kainic acid. We propose TCTP-SOD as a potential candidate drug for treatment of brain diseases.