Superoxide Dismutase 1 Subunits with Mutations Linked to Familial Amyotrophic Lateral Sclerosis Do Not Affect Wild-type Subunit Function

Borchelt, David R.; Guarnieri, Michael; Wong, Philip C.; Lee, Michael K.; Slunt, Hilda S.; Xu, Zuoshang; Sisodia, Sangram S.; Price, Donald L.; Cleveland, Don W.

doi:10.1074/jbc.270.7.3234

Cited by 146 publications

(87 citation statements)

References 25 publications

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“…Several studies have shown that expression of mutant SOD1 exerts toxic effects on cultured cells (11,12). We investigated the biological effects of RNAi-mediated silencing of SOD1 in a mouse neuroblastoma (N2a) cell line that constitutively expresses high levels of FALS-linked mutant human SOD1 (SOD1 G37R ) (12,32). In this system, overexpression of SOD1 G37R does not affect cell viability under normal culture conditions but renders cells more sensitive to cytotoxic stimuli (12).…”

Section: Functional Analysis Of Rnai-mediated Silencing Of Mutant Sod1mentioning

confidence: 99%

“…HeLa cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 units͞ml penicillin, and 100 g͞ml streptomycin. Neuro-2a (N2a) murine neuroblastoma cell lines stably expressing human mutant SOD1 G37R (12,32) were maintained in DMEM supplemented with 7% FBS, 2 mM Lglutamine, and 400 g͞ml G418. DNA and siRNA transfections were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations and published procedures (33).…”

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confidence: 99%

“…N2a cells that stably express human SOD1 G37R (12,32) were transfected with siRNA as described above. After a 24-hour incubation, to permit recovery and activation of the RNAi pathway, the growth medium was removed and replaced with serum-free medium.…”

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RNA interference-mediated silencing of mutant superoxide dismutase rescues cyclosporin A-induced death in cultured neuroblastoma cells

Maxwell

Pasinelli

Kazantsev

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder resulting from selective death of motor neurons in the brain and spinal cord. In Ϸ25% of familial ALS cases, the disease is caused by dominantly acting point mutations in the gene encoding cytosolic Cu,Zn superoxide dismutase (SOD1). In cell culture and in rodent models of ALS, mutant SOD1 proteins exhibit dose-dependent toxicity; thus, agents that reduce mutant protein expression would be powerful therapeutic tools. A wealth of recent evidence has demonstrated that the mechanism of RNA-mediated interference (RNAi) can be exploited to achieve potent and specific gene silencing in vitro and in vivo. We have evaluated the utility of RNAi for selective silencing of mutant SOD1 expression in cultured cells and have identified small interfering RNAs capable of specifically inhibiting expression of ALS-linked mutant, but not wild-type, SOD1. We have investigated the functional effects of RNAi-mediated silencing of mutant SOD1 in cultured murine neuroblastoma cells. In this model, stable expression of mutant, but not wild-type, human SOD1 sensitizes cells to cytotoxic stimuli. We find that silencing of mutant SOD1 protects these cells against cyclosporin A-induced cell death. These results demonstrate a positive physiological effect caused by RNAi-mediated silencing of a dominant disease allele. The present study further supports the therapeutic potential of RNAi-based methods for the treatment of inherited human diseases, including ALS.A myotrophic lateral sclerosis (ALS) is an age-dependent, paralytic disorder resulting from selective death of motor neurons in the brain and spinal cord. ALS is characterized by progressive muscle weakness and atrophy and is usually fatal within 5 years of clinical presentation (1). Although the majority of ALS cases are sporadic, Ϸ10% of cases are familial (FALS) and display autosomal dominant inheritance (2). Approximately 25% of FALS cases are caused by mutations in the gene encoding the cytosolic Cu,Zn superoxide dismutase (SOD1) (3), an abundant cellular homodimeric enzyme whose normal function is the scavenging of superoxide radicals. To date, Ͼ100 FALS-linked mutations in human SOD1 have been identified (4), and the vast majority of these are missense mutations resulting in single amino acid substitutions.Both the association of SOD1 mutations with dominantly inherited human disease and compelling data gleaned from expression of FALS-linked mutant SOD1 in experimental model systems suggest that mutant SOD1 causes ALS through an acquired toxic property (or properties) of the mutant proteins. Transgenic mice (5-7) and rats (8) that express clinically relevant forms of mutant human SOD1 quite remarkably recapitulate the major features of the disease; in contrast, mice in which endogenous SOD1 expression has been eliminated by means of targeted deletion develop normally and do not exhibit ALS-like symptoms (9). Further, in vitro studies demonstrate that forced expression of mutant, but not wild...

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Section: Functional Analysis Of Rnai-mediated Silencing Of Mutant Sod1mentioning

confidence: 99%

mentioning

confidence: 99%

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RNA interference-mediated silencing of mutant superoxide dismutase rescues cyclosporin A-induced death in cultured neuroblastoma cells

Maxwell

Pasinelli

Kazantsev

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…Approximately 10% of ALS cases are familial [1], and about 20% of the familial amyotrophic lateral sclerosis (FALS) cases have mutations in SOD1, the gene that encodes the human Cu,Zn-superoxide dismutase (SOD) [2]. Several studies reported that Cu,Zn-SOD mutants retained high levels of dismutation activity [3][4][5][6], which suggests that the FALS mutations in SOD1 may act through a dominant cytotoxic gain-of-function [3][4][5][6][7]. The mouse models of FALS that overexpressed the mutant human Cu, Zn-SOD suggested that the gain-of-function of the Cu,Zn-SOD contributed to the pathophysiology of FALS [4,6].…”

Section: Introductionmentioning

confidence: 99%

The free radical‐generating function of a familial amyotrophic lateral sclerosis‐associated D90A Cu,Zn‐superoxide dismutase mutant

Kim¹,

Eum²,

Kwon³

et al. 1998

IUBMB Life

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SUMMARYThe free radical-generating functions of the D90A Cu,Zn-superoxide dismutase (SOD) associated with Swedish familial amyotrophic lateral sclerosis (FALS) patients are investigated. The results show that both the wild-type and mutant enzymes have identical dismutase activity, while the free radical-generating activity of the D90A mutant is enhanced relative to that of the wild-type enzyme. The studies suggest that the active channel of the D90A mutant is larger than that of the wild-type enzyme. A higher free radical-generating activity of the mutant enzyme led to the release of copper ions from the damaged protein. The generation of strand breaks in plasmid DNA was enhanced more effectively by the D90A mutant Cu,Zn-SOD than by the wildtype enzyme. The results suggest that the pathology of FALS may be attributed to oxidative damage caused by the gain-of-function of FALS Cu,Zn-SOD mutant.Key words: copper,zin-superoxide dismutase, mutant, hydroxyl radical, copper. 1191All rights of reproduction in any form reserved, Vol. 46, No. 6, 1998 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONALAndersen et al. [7] reported a Cu,Zn-SOD exon 4 mutation (D90A) with novel features in Swedish FALS patients and the disease has only been observed in patients homozygous for this mutation, whereas heterozygotes remained unaffected. Furthermore, there was essentially no reduction in erythrocyte Cu,Zn-SOD dismutation activity. Thus, [7] suggested that this Cu,Zn-SOD mutation caused ALS by a gain-of-function rather than by the loss of dismutation activity.In view of these considerations, the present work represents an investigation of the free radical-generating functions of the D90A mutant and the oxidative damage to DNA, as a first attempt to establish the extent of any correlation between the gain-of-function and the pathology of FALS. MATERIALS AND METHODS Expression and purification of wild-type and mutated human Cu, Zn-SOD

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“…However, several studies with transgenic mice (8,21), transfected cells (22,23), and lymphoblasts of patients (24) revealed that levels of total Cu,Zn-SOD dismutation activities remain high or higher than normal, which suggests that the FALS mutations in SOD1. may act through a dominant cytotoxic gain-of-function (8,(21)(22)(23)(24). We investigated whether there are any differences in the free radical-generating function between the wild-type and FALS mutant Cu,Zn-SOD.…”

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Pro-oxidant activity of Cu,Zn-superoxide dismutase

Yim

Chock

et al. 1998

AGE

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Whereas Cu,Zn-superoxide dismutase (Cu,Zn-SOD) is an essential antioxidant enzyme for superoxide dismutation in vivo(1 ), many reports have shown that an elevated level of Cu,Zn-SOD induces cell killing (2, 3), increases lipid peroxidation (4,5), and interferes with the transport of neurotransmitters (4, 6). All of these observations are archetypical results inducible by oxygen radicals. Cu,Zn-SOD (7) and FALS mutants (8) are known to develop neurodegeneration. These findings suggest that under certain conditions Cu,Zn-SOD may behave directly or indirectly as a pro-oxidant.In addition to the usual superoxide dismutation activity, Cu,Zn-SOD is known to exhibit anion binding capacity (9, 10), inactivation by its own reaction product H202 (11-13), and the purported peroxidase activity (14). Here we summarize our findings that Cu,Zn-SOD has free radical ('OH and scavenger-derived radicals)-generating activity (15,16). TheactiveCu,Zn-SOD can catalyze the generation of free 9 radicals from H202. In the presence of H202 and radical scavengers, especially with anionic character, both free 9 radicals and scavenger-derived radicals are produced (15, 16). We also found that the free radical-generating function of several FALS-associated Cu,Zn-SOD mutants is enhanced relative to that of the wild-type enzyme, while the dismutation activities remain unchanged (17,18).To monitor the production of free radicals, two spin traps, 5,5-dimethyl-l-pyrroline N-oxide (DMPO) and Ntert-butyl-c~-phenylnitrone (PBN), were used in our studies (15) to convert transient free radicals to stable free radicals adducts according to the following reactions:The nature of the trapped free radical was identified by EPR spectroscopy. When O2-" (dissolved KO 2 in dried dimethylsulfoxide) was added to the NaHCOJCO 2 buffer at pH 7.6 containing 100 mM DMPO, EPR OH adducts. DMPO-OH formation also required active Cu,Zn-SOD, because heat-inactivated Cu,Zn-SOD or Mn-SOD failed to produce these radical adducts. The direct addition of H202 in place of O2-" produced similar results. In the presence of .OH radical scavengers with anionic character such as HCO 2 or N3-, DMPO adducts of the scavenger-derived radicals, DMPO-CO2-" or DMPO-N3", were observed in addition to DMPO-OH. The concentration ratio between DMPO-OH and DMPO-scavenger-derived radical adducts observed in these experiments reflects competition reactions between DMPO and scavengers for free "OH radicals. Surprisingly, with neutral'OH radical scavengers, such as ethanol, the formation of DMPO-hydroxyethyl radical adducts was insignificant and the concentration of DMPO-OH formed was unaffected. In contrast, with a different spin trap, PBN, in place of DMPO, ethanol yields PBNhydroxyethyl radical adducts as expected in the competition reaction for free "OH radicals. These seemingly contradicting results may be caused by the different affinities of these spin traps for the positively charged active channel leading to the active site of Cu,Zn-SOD, where the'OH radicals are generated. To prove this hypo...

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Superoxide Dismutase 1 Subunits with Mutations Linked to Familial Amyotrophic Lateral Sclerosis Do Not Affect Wild-type Subunit Function

Cited by 146 publications

References 25 publications

RNA interference-mediated silencing of mutant superoxide dismutase rescues cyclosporin A-induced death in cultured neuroblastoma cells

RNA interference-mediated silencing of mutant superoxide dismutase rescues cyclosporin A-induced death in cultured neuroblastoma cells

The free radical‐generating function of a familial amyotrophic lateral sclerosis‐associated D90A Cu,Zn‐superoxide dismutase mutant

Pro-oxidant activity of Cu,Zn-superoxide dismutase

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