Overexpression of mutated Cu,Zn-SOD in neuroblastoma cells results in cytoskeletal change

Abstract

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and the motor cortex. It has been shown that 15–20% of patients with familial ALS (FALS) have defects in the Sod1 gene, which encodes Cu,Zn-superoxide dismutase (SOD). To elucidate the pathological role of mutated Cu,Zn-SOD, we examined the issue of whether mutated Cu,Zn-SOD affects the cell cycle. Mouse neuroblastoma Neuro-2a cells were transfected with human wild-type or mutated (G37R, G93A) Cu,Zn-SOD. Mutated, Cu,Zn-SOD-transfected cells exhibited marked retardation in cell growth and G2/M arrest. They also displayed lower reactivity to phalloidin, indicating that the cytoskeleton was disrupted. Immunoprecipitation, two-dimensional gel electrophoresis, and Western blot analysis indicated that mutated Cu,Zn-SOD associates with actin. Similar results were obtained by in vitro incubation experiments with purified actin and mutated Cu,Zn-SOD (G93A). These results suggest that mutated Cu,Zn-SOD in FALS causes cytoskeletal changes by associating with actin, which subsequently causes G2/M arrest and growth retardation.

Proteins That Bind to Misfolded Mutant Superoxide Dismutase-1 in Spinal Cords from Transgenic Amyotrophic Lateral Sclerosis (ALS) Model Mice

“…In the study with immobilized apo G93A, many more protein spots were indeed visible and identified ( Fig. 3 and Table 2), some of which have already been shown to bind SOD1: actin, (16,29), tubulin (16,30), CCS (31), ␣␤-crystallin (32), Hsp27 (24), ATPA, ATPB, GAPDH (16), and Hsp70, the inducible homolog of Hsc70 (16,24,(33)(34). RINI has apparently not been described to bind hSOD1 before.…”

Biochemical and molecular analyses of copper–zinc superoxide dismutase from a C4 plant Pennisetum glaucum reveals an adaptive role in response to oxidative stress

“…Acclimatization of plants to oxidative stress generated during abiotic stress conditions is primarily and significantly by SODs that form the first line of defense against ROS. Mutational studies in mouse and human CuZnSOD have shown to cause premature neuron death, cytoskeletal and cell cycle changes (Jaarsma et al, 2000;Takamiya et al, 2005). Numerous stress inducible isoforms of CuZnSODs have been identified and characterized from various monocot and dicot plants (Dong et al, 2011;Hernández-Nistal et al, 2002;Kernodle and Scandalios, 2001;Qu et al, 2010;Sunkar et al, 2006;Wu et al, 2011;Zhang et al, 2008).…”

Nodularin Exposure Induces SOD1 Phosphorylation and Disrupts SOD1 Co-localization with Actin Filaments

“…Both proteins are high abundant proteins which always increase the risk of unspecific binding [22] and immunoprecipitation of F-actin is usually not very efficient. It should be noted that previous studies have only been able to immunoprecipitate minor amounts of wild type SOD1 together with actin [14]. Only a minor amount of SOD1 was pulled down together with actin also in our untreated cells (Figure 11A,B).…”

“…Interestingly, expression of SOD1 has been shown to be essential for the preservation of the cytoskeleton [15], and disease-related SOD1 mutants interact directly with the actin cytoskeleton [14,21]. We therefore compared the localization of SOD1 and actin filaments in nodularin-exposed hepatocytes.…”

“…SOD1 is a prime defender against oxidative stress [12], but also a regulatory protein with possible implications for a range of sub-cellular localized processes, including cytoskeletal rearrangement [13]. SOD1 is essential for the preservation of the cytoskeleton, thus dysregulation in SOD1 results in cytoskeletal rearrangements [14,15]. We found that the initial co-localization of SOD1 and actin in hepatocytes was disrupted during nodularin exposure and that a high content of SOD1 was found in the apoptotic buds.…”