Mitochondrial-shaping proteins are altered in NSC-34 and Neuro-2a cells bearing the fALS mutant G93A-hSOD1

ALAIMO A.; GOROJOD R.; UCHITEL O.D; KOTLER M.L

Praga

Congreso; Fens Featured Regional Meeting; 2013

FENS (Federation of European Neurosciences Societies)

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease that affects both lower and upper motor neurons. The loss of these neurons leads to muscle atrophy and weakness, fasciculations and spasticity. ALS is traditionally classified into two categories: familial and sporadic (fALS and sALS, respectively), both sharing several clinical manifestations. Mutations in Cu-Zn superoxide dismutase (SOD1) underlie about 20% of fALS. However, recent studies suggest that SOD1 could be pathogenic in both fALS and sALS through either inheritable or non heritable modifications. Otherwise, the molecular mechanisms of SOD1-mediated motor neuron death are still unclear. Mitochondria exist in a dynamic state and their proper function relies on a critical balance of fission and fusion events, both regulated by mitochondrial-shaping proteins (e.g. Opa-1 and Drp-1, among others). Impairment of this delicate balance leads to mitochondrial dysfunction which has several physiological consequences on cellular survival, especially in neurons, due to its high energy demand. In the present work we studied the mitochondrial dynamics in an in vitro model of fALS. For this purpose, we transiently transfected mouse motor neuron-like (NSC-34) and neuroblastoma (Neuro-2a) cell lines with both normal and mutant (WT or G93A) hSOD1 cDNAs. Employing MTT and Neutral Red assays we demonstrated that cells expressing mutant G93A hSOD1 were more prone to cell death. In addition, using MitoTracker Red CMX Ros probe and fluorescence microscopy we observed a mitochondrial network derangement induced by the mutant hSOD1. These results were in accordance with alterations in mitochondrial-shaping proteins expressions levels evaluated by western blot analysis. Moreover, to mimic the occurrence of age- or environmental-induced oxidative stress (OS) we challenged cells with 250µM H202 during 90 min. Viability assays indicated that G93A hSOD1-bearing NSC-34 and Neuro-2a cells resulted to be more susceptible to exogenous OS than both controls and cells over-expressing WT SOD1. The scenario resulted of a higher complexity when the mitochondrial-shaping proteins expression was analyzed. In this case we found that both cell lines exhibited different vulnerabilities to OS. Furthermore, we studied the effects of Mdivi-1 (1µM), a newly pharmacological fission inhibitor, determining that this molecule protects cells carrying the G93A hSOD1 mutant against cell death. In conclusion, we focused our attention in the mitochondrial dynamics as a new paradigm for ALS research. Compounds that modulate mitochondrial fission-fusion could have therapeutic value in the intervention of this heterogeneous disorder.