Heat shock protein 70 protects motor neuronal cells expressing mutant Cu/Zn superoxide dismutase (SOD1) against altered calcium homeostasis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive loss of motor neurons leading to paralysis and death. Mutations of the human Cu/Zn superoxide dismutase (SOD1) are found in some cases of familial ALS (fALS). Recent evidences suggest the accumulation of intracellular calcium is one of the primary mechanisms of motor neuronal degeneration. In this study, we attempted to test the protective effect of Hsp 70 on mutant SOD1 against altered calcium homeostasis. To test the effect of Hsp70 on constitutively expressing mutant SOD1, motoneuron-neuroblastoma hybrid cells (VSC4.1) co-expressing HSP70 and human mutant SOD1 (G93A, A4V) was established. Calcium mobilizer through cell membrane (4-bromo-calcium ionophore A23187) or endogenous calcium releasers (ryanodine, thapsigargin, 8-bromo-cyclic ADP) were treated and the viability determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. To determine the evidence of apoptotic cell death, treated cells were stained with Hoechst 33342 or assayed for caspase 3 activity. To confirm the suppression of SOD1 aggregates by Hsp70, VSC 4.1 cells expressing Hsp70 were transfected with mutant SOD1 gene (G93A), and then, A23187 or thapsigargin were treated for 1 day. Mutant SOD1 aggregates were quantified under the fluorescence microscope. The effect of exogenous NO was also tested with S-nitrosoglutathione (GSNO). Calcium ionophore A23187, thapsigargin and GSNO decreased viability of cells expressing mutant SOD-1 (A4V, G93A) in a dose-dependent manner. These cells showed dispersed chromatin or nuclear fragmentation by Hoechst 33342 staining. In addition, increased intracellular calcium levels by A23187 or thapsigargin increased caspase 3 activity and mutant SOD1 aggregates. Our data showed the protective effect of Hsp 70 against altered calcium homeostasis by inhibiting the caspase 3 activation. These findings suggest Hsp 70 may have beneficial effects on the SOD1-mediated motor neuronal degeneration in some forms of ALS.