The modulation of intracellular Ca2+ homeostasis is involved in SOD1 and ApoSOD1-induced neuroprotection of motor neurons exposed to beta-methylamino-L-alanine

Amyotrophic lateral sclerosis (ALS) is a human adult-onset neurodegenerative disease characterized by the loss of upper motor neurons in the cerebral cortex and lower motor neurons in the brainstem and spinal cord. As it occurs in cellular senescence, the impairment of antioxidant cellular machinery and intracellular calcium concentrations ([Ca2+]i) represent important mechanisms underlying cellular vulnerability in ALS. Accordingly, in about 20% of cases, familial amyotrophic lateral sclerosis as well as some sporadic cases of the disease are associated with mutations in the gene encoding Cu,Zn-superoxide dismutase (SOD1)[1,2], the enzyme that catalyzes superoxide anion dismutation. However, SOD1 mutation may cause motor neuron degeneration probably through a gain of toxic function rather than a loss of catalytic function. Indeed, mutations in metal-free apoprotein, which lacks catalytic activity, may play a more important role in ALS pathogenesis than alterations in holoprotein structure[3]. This suggests that the apo state of SOD1 (eg ApoSOD1) could exert a peculiar role in motor neuron physiology. Accordingly, in a previous study we show that the neuroprotective effect of SOD1 in motor neurons exposed to the cycad toxin L-BMAA, a model exemplifying the Guamanian form of ALS, is not dependent on its catalytic activity but rather on the activation of Ca2+/Akt/ERK1/2 signaling pathway that, in turn, prevents ER stress-dependent neuronal death[4]. On the other hand, resting SOD1 release is impaired in ALS[5]. Indeed, we hypothesized that an altered [Ca2+]i modulation by SOD1 -due to an improper release from motor neurons- could affect neuronal survival. To this aim, with the help of Fura2-single cell microfluorimetry, we compared the effect of SOD1, ApoSOD1 and recombinant SOD1G93A in the modulation of [Ca2+]i in primary motor neurons. SOD1, ApoSOD1 and recombinant SOD1wt, but not SOD1G93A, induced a rapid increase in [Ca2+]i partially dependent on the release of Ca2+ from intracellular Ca2+ stores. However, this effect was partially reduced in a Ca2+-free solution, thus suggesting a possible involvement of a such plasma membrane player. To this aim, we hypothesized the involvement of the Na+/Ca2+ exchanger (NCX), a 10-transmembrane domain protein mainly involved in the regulation of [Ca2+]i homeostasis in several neurological diseases. The isoform 1, named NCX1, was highly expressed at the plasmamembrane level of motor neurons with the highest immunosignal in the neuronal cone. However the isoform 3, NCX3, was localized intracellularly. Accordingly, Fura-2-detected NCX activity was higher in the cone than in cell body of motor neurons. Interestingly, SOD1-induced [Ca2+]i increase was partially prevented by the amiloride derivative CB-DMB, a well known inhibitor of NCXs[6]. Collectively, our data indicate that SOD1 and ApoSOD1, but not SOD1G93A, modulated [Ca2+]i via intracellular and plasmamembrane targets, thus inducing neuroprotection in ALS