Overexpression of human mutated G93A SOD1 changes dynamics of the ER mitochondria calcium cycle specifically in mouse embryonic motor neurons

Abstract

Motor neurons vulnerable to the rapidly progressive deadly neurodegenerative disease amyotrophic lateral sclerosis (ALS) inherently express low amounts of calcium binding proteins (CaBP), likely to allow physiological motor neuron firing frequency modulation. At the same time motor neurons are susceptible to AMPA receptor mediated excitotoxicity and internal calcium deregulation which is not fully understood. We analysed ER mitochondria calcium cycle (ERMCC) dynamics with subsecond resolution in G93A hSOD1 overexpressing motor neurons as a model of ALS using fluorescent calcium imaging. When comparing vulnerable motor neurons and non-motor neurons from G93A hSOD1 mice and their non-transgenic littermates, we found a decelerated cytosolic calcium clearance in the presence of G93A hSOD1. While both non-transgenic as well as G93A hSOD1 motor neurons displayed large mitochondrial calcium uptake by the mitochondrial uniporter (mUP), the mitochondrial calcium extrusion system was altered in the presence of G93A hSOD1. In addition, ER calcium uptake by the sarco-/endoplasmic reticulum ATPase (SERCA) was increased in G93A hSOD1 motor neurons. In survival assays, blocking the mitochondrial sodium calcium exchanger (mNCE) by CGP37157 as well as inhibiting SERCA by cyclopiazonic acid showed protective effects against kainate induced excitotoxicity. Thus, our study shows for the first time that the functional consequence of G93A hSOD1 overexpression in intact motor neurons is indeed a disturbance of the ER mitochondria calcium cycle, and identified two promising targets for therapeutic intervention in the pathology of ALS.

Keywords: ALS; AMPA; Amyotrophic lateral sclerosis; CPA; CaBP; Calcium; CsA; EMD; ER; ER mitochondria calcium cycle; ERMCC; FUS/TLS; Mitochondria; Motor neuron; SERCA; TDP-43; TTX; UPR; VAPB; VCP; VGCC; X-box binding protein 1; XBP1; amyotrophic lateral sclerosis; calcium binding proteins; cyclopiazonic acid; cyclosporine A; eIF2α; earth mover's distance; eukaryotic translation initiation factor 2 α; fused in sarcoma/translated in liposarcoma; hSOD1; human superoxide dismutase 1; mNCE; mPTP; mUP; mitochondrial permeability transition pore; mitochondrial sodium calcium exchanger; mitochondrial uniporter; sarco-/endoplasmic reticulum ATPase; tetrodotoxin; transactive response (TAR) DNA-binding protein-43; unfolded protein response; valosin containing protein; vesicle-associated membrane protein (VAMP)-associated protein B; voltage gated calcium channels; α-amino-5-methyl-3-hydroxyisoxazolone-4-propionat.