Investigation of mitochondrial calcium uptake in G93AhSOD1 model of ALS

Abstract

Mitochondrial calcium overload and excitotoxicity represent hallmarks of selective motor neuron degeneration in amyotrophic lateral sclerosis (ALS) (1,2). Here, we specifically investigated the role of the mitochondrial Ca2+ uniporter (MCU) in cultured embryonic motor neurons from non-transgenic and G93AhSOD1 mice, a widely accepted ALS model. Immunocytochemistry and qPCR were used to examine expression of MCU. For survival assay we challenged our cultures with excitotoxicity inducer kainate (100 µM, 12h) and applied KN-62 (10 µM) and kaempferol (25 µM). Functional effects of these drugs were examined by single cell calcium imaging using fura 2-AM. Reduced mitochondrial Ca2+ buffering (p<0.05) and MCU overexpression (p<0.05) were confirmed in G93AhSOD1 motor neurons. Ca2+/calmodulin-dependent protein kinase inhibitor KN-62 reduced MCU expression (p<0.01) and protected G93AhSOD1 motor neurons against kainate (p<0.01). MCU activator kaempferol did not influence MCU expression or neuronal viability. Still, its acute application enhanced spontaneous Ca2+ activity, especially in G93AhSOD1 neurons. We provide evidence that mitochondrial Ca2+ uptake plays a major role in G93AhSOD1 motor neurons and that pharmacological manipulation of MCU may be a highly valuable neuroprotective strategy in ALS.