Cu,Zn-Superoxide Dismutase Increases Toxicity of Mutant and Zinc-deficient Superoxide Dismutase by Enhancing Protein Stability*

Abstract

When replete with zinc and copper, amyotrophic lateral sclerosis (ALS)-associated mutant SOD proteins can protect motor neurons in culture from trophic factor deprivation as efficiently as wild-type SOD. However, the removal of zinc from either mutant or wild-type SOD results in apoptosis of motor neurons through a copper-and peroxynitrite-dependent mechanism. It has also been shown that motor neurons isolated from transgenic mice expressing mutant SODs survive well in culture but undergo apoptosis when exposed to nitric oxide via a Fas-dependent mechanism. We combined these two parallel approaches for understanding SOD toxicity in ALS and found that zincdeficient SOD-induced motor neuron death required Fas activation, whereas the nitric oxide-dependent death of G93A SODexpressing motor neurons required copper and involved peroxynitrite formation. Surprisingly, motor neuron death doubled when Cu,Zn-SOD protein was either delivered intracellularly to G93A SOD-expressing motor neurons or co-delivered with zinc-deficient SOD to nontransgenic motor neurons. These results could be rationalized by biophysical data showing that heterodimer formation of Cu,Zn-SOD with zinc-deficient SOD prevented the monomerization and subsequent aggregation of zinc-deficient SOD under thiol-reducing conditions. ALS mutant SOD was also stabilized by mutating cysteine 111 to serine, which greatly increased the toxicity of zinc-deficient SOD. Thus, stabilization of ALS mutant SOD by two different approaches augmented its toxicity to motor neurons. Taken together, these results are consistent with copper-containing zinc-deficient SOD being the elusive "partially unfolded intermediate" responsible for the toxic gain of function conferred by ALS mutant SOD.Mutations to copper/zinc superoxide dismutase (SOD) 4 are the most common genetic cause of the familial form of amyotrophic lateral sclerosis (ALS) (1, 2). Although transgenic expression of these mutant SOD genes in mice and rats is sufficient to produce a progressive motor neuron disease that mimics human pathology (3), the toxic mechanisms remain obscure. Primary motor neuron cultures have proven to be a powerful model to elucidate the toxic gain of function conferred by ALS mutations to SOD and to determine the underlying cell death pathways. Motor neurons isolated from transgenic mice carrying ALS mutant SOD are fully viable in culture (4, 5) but undergo apoptosis after incubation with low, physiologically relevant concentrations of exogenous nitric oxide (50 -100 nM). This remains the most direct evidence that mutant SOD can become directly toxic to motor neurons but also demonstrates that expression of mutant SOD alone may not be sufficient to be toxic to motor neurons.The concentrations of nitric oxide used to induce death of ALS-SOD-expressing motor neurons were not injurious to motor neurons isolated from transgenic mice expressing wildtype SOD and are even trophic to nontransgenic motor neurons (6). Over the past decade, we have accumulated considerable evidence that the reacti...

Reactive Nitrogen Species in Motor Neuron Apoptosis

“…However, these motor neurons carrying both, mutant and Cu,Zn-containing SOD are more sensitive to nitric oxide toxicity than transgenic motor neurons without intracellular delivered Cu,Zn SOD (Sahawneh et al 2010). These in vitro results closely mimic the in vivo observations of acceleration of death when transgenic mice for mutant SOD are crossbreed with mice transgenic for wild type SOD (Jaarsma et al 2000;Fukada et al 2001;Deng et al 2006).…”

“…Nitric oxide is not toxic to non-transgenic mouse and rat motor neurons Estévez et al 2000;Raoul et al 2002). In contrast, motor neurons isolated from transgenic rats and mice carrying human ALS-linked SOD G85R and G93A die after exposure to nanomolar steady state concentrations of nitric oxide (Raoul et al 2002;Sahawneh et al 2010). Transgenic motor neurons carrying mutant SOD are also ten times more sensitive to Fas-induced apoptosis, while transgenic motor neurons carrying human wild type SOD are 100 times more resistant than non-transgenic motor neurons to Fas-mediated apoptosis (Raoul et al 2002;Raoul et al 2006).…”

“…Transgenic motor neurons carrying mutant SOD are also ten times more sensitive to Fas-induced apoptosis, while transgenic motor neurons carrying human wild type SOD are 100 times more resistant than non-transgenic motor neurons to Fas-mediated apoptosis (Raoul et al 2002;Raoul et al 2006). Nitric oxide toxicity in transgenic motor neurons can be reversed by copper chelators and scavenging of superoxide and peroxynitrite (Sahawneh et al 2010), suggesting production of superoxide in transgenic motor neurons. Fig.…”

Nitration of Hsp90 on Tyrosine 33 Regulates Mitochondrial Metabolism

“…Nitrated tyrosine residues are detected in inflammation, neurodegeneration, and aging. We previously showed that tyrosine nitration plays a key role in the activation of apoptotic signaling pathways (2)(3)(4). Tyrosine-containing peptides protect PC12 cells from peroxynitrite-induced cell death, acting as scavengers of nitrating products of peroxynitrite decomposition (2,4).…”

“…We previously showed that tyrosine nitration plays a key role in the activation of apoptotic signaling pathways (2)(3)(4). Tyrosine-containing peptides protect PC12 cells from peroxynitrite-induced cell death, acting as scavengers of nitrating products of peroxynitrite decomposition (2,4). Downstream of peroxynitrite, nitration of a single tyrosine residue on Hsp90 is sufficient to induce apoptosis in PC12 cells and motor neurons (3).…”