Calcium dysregulation, mitochondrial pathology and protein aggregation in a culture model of amyotrophic lateral sclerosis: Mechanistic relationship and differential sensitivity to intervention

Abstract

The combination of Ca2+ influx during neurotransmission and low cytosolic Ca2+ buffering contributes to the preferential vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS). This study investigated the relationship among Ca2+ accumulation in intracellular compartments, mitochondrial abnormalities, and protein aggregation in a model of familial ALS (fALS1). Human SOD1, wild type (SOD1WT) or with the ALS-causing mutation G93A (SOD1G93A), was expressed in motor neurons of dissociated murine spinal cord–dorsal root ganglia (DRG) cultures. Elevation of mitochondrial Ca2+ ([Ca2+]m), decreased mitochondrial membrane potential (Δψ) and rounding of mitochondria occurred early, followed by increased endoplasmic reticular Ca2+ ([Ca2+]ER), elevated cytosolic Ca2+ ([Ca2+]c), and subsequent appearance of SOD1G93A inclusions (a consequence of protein aggregation). [Ca2+]c was elevated to a greater extent in neurons with inclusions than in those with diffusely distributed SOD1G93A and promoted aggregation of mutant protein, not vice versa: both [Ca2+]c and the percentage of neurons with SOD1G93A inclusions were reduced by co-expressing the cytosolic Ca2+-buffering protein, calbindin D-28K; treatment with the heat shock protein inducer, geldanamycin, prevented inclusions but not the increase in [Ca2+]c, [Ca2+]m or loss of Δψ, and inhibiting proteasome activity with epoxomicin, known to promote aggregation of disease-causing mutant proteins including SOD1G93A, had no effect on Ca2+ levels. Both expression of SOD1G93A and epoxomicin-induced inhibition of proteasome activity caused mitochondrial rounding, independent of Ca2+ dysregulation and reduced Δψ. That geldanamycin prevented inclusions and mitochondrial rounding, but not Ca2+ dysregulation or loss of Δψ indicates that chaperone-based therapies to prevent protein aggregation may require co-therapy to address these other underlying mechanisms of toxicity.