Ryanodine receptor-mediated Ca2+ release underlies iron-induced mitochondrial fission and stimulates mitochondrial Ca2+ uptake in primary hippocampal neurons

Cecilia Hidalgo,Alejandra Garcã­A,Pablo Barattini,Steffen Hartel,Carol D Sanmartã­N,Andrea C Paula-Lima,Marco T Nãºã±Ez

doi:10.3389/fnmol.2014.00013

Abstract

Mounting evidence indicates that iron accumulation impairs brain function. We have reported previously that addition of sub-lethal concentrations of iron to primary hippocampal neurons produces Ca2+ signals and promotes cytoplasmic generation of reactive oxygen species. These Ca2+ signals, which emerge within seconds after iron addition, arise mostly from Ca2+ release through the redox-sensitive ryanodine receptor (RyR) channels present in the endoplasmic reticulum. We have reported also that addition of synaptotoxic amyloid-β oligomers to primary hippocampal neurons stimulates RyR-mediated Ca2+ release, generating long-lasting Ca2+ signals that activate Ca2+-sensitive cellular effectors and promote the disruption of the mitochondrial network. Here, we describe that 24 h incubation of primary hippocampal neurons with iron enhanced agonist-induced RyR-mediated Ca2+ release and promoted mitochondrial network fragmentation in 43% of neurons, a response significantly prevented by RyR inhibition and by the antioxidant agent N-acetyl-L-cysteine. Stimulation of RyR-mediated Ca2+ release by a RyR agonist promoted mitochondrial Ca2+ uptake in control neurons and in iron-treated neurons that displayed non-fragmented mitochondria, but not in neurons with fragmented mitochondria. Yet, the global cytoplasmic Ca2+ increase induced by the Ca2+ ionophore ionomycin prompted significant mitochondrial Ca2+ uptake in neurons with fragmented mitochondria, indicating that fragmentation did not prevent mitochondrial Ca2+ uptake but presumably decreased the functional coupling between RyR-mediated Ca2+ release and the mitochondrial Ca2+ uniporter. Taken together, our results indicate that stimulation of redox-sensitive RyR-mediated Ca2+ release by iron causes significant neuronal mitochondrial fragmentation, which presumably contributes to the impairment of neuronal function produced by iron accumulation.

Highlights

Under physiological conditions, mitochondria undergo constant structural changes, forming interconnected networks or punctiform organelles depending on cell type (Kuznetsov et al, 2009)
These combined effects promote a sustained increase in calcium with time, which is much faster in cells pre-incubated with Fe-NTA
These results indicate that prolonged exposure to Fe-NTA favored agonist-induced ryanodine receptor (RyR)-mediated Ca2+ release, presumably via RyR redox modifications produced by iron-generated reactive oxygen species (ROS)

Summary

Introduction

Mitochondria undergo constant structural changes, forming interconnected networks or punctiform organelles depending on cell type (Kuznetsov et al, 2009). The hFis protein and the large GTPase Dynamin-related protein 1 (Drp1) are key molecules for the regulation of mitochondrial fission (Smirnova et al, 2001). Neurons in primary culture (Zampese et al., 2011) and neuronal cells lines (Spat et al, 2008) exhibit a physical and functional association between the ER and mitochondria. This association allows mitochondrial Ca2+ uptake through the uniporter following Ca2+ release mediated by the ER resident Ca2+ channels, the ryanodine receptor (RyR; Szalai et al, 2000)

Methods

Results

Conclusion