ABSTRACT
The combination of Ca(2+) influx during neurotransmission and low cytosolic Ca(2+) buffering contributes to the preferential vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS). This study investigated the relationship among Ca(2+) accumulation in intracellular compartments, mitochondrial abnormalities, and protein aggregation in a model of familial ALS (fALS1). Human SOD1, wild type (SOD1(WT)) or with the ALS-causing mutation G93A (SOD1(G93A)), was expressed in motor neurons of dissociated murine spinal cord-dorsal root ganglia (DRG) cultures. Elevation of mitochondrial Ca(2+) ([Ca(2+)](m)), decreased mitochondrial membrane potential (Δψ) and rounding of mitochondria occurred early, followed by increased endoplasmic reticular Ca(2+) ([Ca(2+)](ER)), elevated cytosolic Ca(2+) ([Ca(2+)](c)), and subsequent appearance of SOD1(G93A) inclusions (a consequence of protein aggregation). [Ca(2+)](c) was elevated to a greater extent in neurons with inclusions than in those with diffusely distributed SOD1(G93A) and promoted aggregation of mutant protein, not vice versa: both [Ca(2+)](c) and the percentage of neurons with SOD1(G93A) inclusions were reduced by co-expressing the cytosolic Ca(2+)-buffering protein, calbindin D-28K; treatment with the heat shock protein inducer, geldanamycin, prevented inclusions but not the increase in [Ca(2+)](c), [Ca(2+)](m) or loss of Δψ, and inhibiting proteasome activity with epoxomicin, known to promote aggregation of disease-causing mutant proteins including SOD1(G93A), had no effect on Ca(2+) levels. Both expression of SOD1(G93A) and epoxomicin-induced inhibition of proteasome activity caused mitochondrial rounding, independent of Ca(2+) dysregulation and reduced Δψ. That geldanamycin prevented inclusions and mitochondrial rounding, but not Ca(2+) 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.
