Abstract
The constitutive secretion of antioxidant Cu-Zn Superoxide dismutase (SOD1) has been widely demonstrated in many cellular lines. In addition, we showed that as well as the basal SOD1 secretion, this enzyme is also exported through depolarization of excitable cells by high extracellular K concentration. Recent data showed that SOD1 was able to activate muscarinic M1 receptor producing the activation, via phospholipase C, of ERK1-2 and AKT pathways. It is also known that about 20% of familial amyotrophic lateral sclerosis (fALS) is due to mutations in the gene coding for SOD1. The aim of the present research is to evaluate whether, analogously to wild type SOD1 (SOD1wt), the mutated form of SOD1G93A is able to activate ERK1-2 and AKT through muscarinic M1 receptor in SK-N-BE as well as in motoneuron like NSC-34. Our results demonstrated that in NSC-34 and SK-N-BE cells mutated SOD1G93A carried out a more evident activation of ERK1-2 and AKT and a stronger increase of intracellular calcium levels compared to SOD1WT; we also demonstrated that these effects are mediated by the M1 receptor as shown using pirenzepine, a specific M1 inhibitor and the calcium chelator BAPTA. Of note, M1 receptor pathway activation by SOD1G93A, but not by SOD1WT, is associated with both an increase of reactive oxygen species and a cytotoxic effect.
Highlights
Cu, Zn superoxide dismutase (SOD1) is a dimeric ubiquitous enzyme mainly localized in the cytosol that catalyzes the dismutation of superoxide radical into hydrogen peroxide and molecular oxygen; in association with non-enzymatic antioxidants it plays a pivotal role in the cellular homeostasis of reactive oxygen species (ROS) (Fridovich, 1995; Russo et al, 2011)
Activating M1 muscarinic receptor, and the downstream cascade of reactions leading to the ERK1-2 and AKT phosphorylation and subsequent increase of intracellular calcium concentrations (Damiano et al, 2013)
We previously demonstrated that SOD1 interacts with the cell surface of SK-N-BE by M1 muscarinic receptor, activating the cascade of reactions leading to the ERK1-2 and AKT phosphorylation and subsequent increase of intracellular calcium concentration
Summary
Zn superoxide dismutase (SOD1) is a dimeric ubiquitous enzyme mainly localized in the cytosol that catalyzes the dismutation of superoxide radical into hydrogen peroxide and molecular oxygen; in association with non-enzymatic antioxidants it plays a pivotal role in the cellular homeostasis of reactive oxygen species (ROS) (Fridovich, 1995; Russo et al, 2011). We showed that depolarization, caused by high extracellular K+ concentrations, induces an additional inducible calcium-dependent SOD1 secretion through T and V SNARE complexes in excitable GH3 rat pituitary cells (Santillo et al, 2007). This enzyme, through the activation of a calcium dependent pathway, can participate in various biological functions (Mondola et al, 2002; Viggiano et al, 2012). The mechanism involved in the molecular secretory pathway of SOD1 is not yet clear (Mellman and Warren, 2000; Bosco et al, 2010; Forsberg et al, 2010)
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