Overexpression of human mutated G93A SOD1 changes dynamics of the ER mitochondria calcium cycle specifically in mouse embryonic motor neurons Janin Lautenschläger ⁎, Tino Prell, Julia Ruhmer, Lisa Weidemann, Otto W. Witte, Julian Grosskreutz Hans Berger Department of Neurology, Jena University Hospital, Erlanger Allee 101, 07747 Jena, Germany abstract article info Article history: Received 5 January 2013 Revised 14 March 2013 Accepted 28 March 2013 Available online 8 April 2013 Keywords: Amyotrophic lateral sclerosis Motor neuron Calcium Mitochondria ER 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 physiolog- ical motor neuron firing frequency modulation. At the same time motor neurons are susceptible to AMPA re- ceptor 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 neu- rons and non-motor neurons from G93A hSOD1 mice and their non-transgenic littermates, we found a decel- erated 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 tar- gets for therapeutic intervention in the pathology of ALS. © 2013 Elsevier Inc. All rights reserved. Introduction Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegener- ative disease characterised by the selective death of motor neurones. The majority of ALS cases are sporadic, while 10% of the patients show a positive familial history (Siddique and Lalani, 2002). Mutations in the human superoxide dismutase 1 (hSOD1) gene are responsible for about 20% of familial ALS (Rosen et al., 1993). In the last ten years, mu- tations in genes for TDP-43 (Neumann et al., 2006), FUS/TLS (Vance et al., 2009), optineurin (Maruyama et al., 2010), ubiquilin 2 (Deng et al., 2011), VAPB (Nishimura et al., 2004), VCP (DeJesus-Hernandez et al., 2011a; Johnson et al., 2010) and hexanucleotide repeat expansions of C9ORF72 (DeJesus-Hernandez et al., 2011b; Renton et al., 2011) have been found. Most recently, mutations in the profiling 1 gene have been reported (Wu et al., 2012). Glutamate excitotoxicity which is mediated through calcium perme- able α-amino-5-methyl-3-hydroxyisoxazolone-4-propionat (AMPA) re- ceptors represents an important disease mechanism (Carriedo et al., 1996; Rothstein and Kuncl, 1995; Rothstein et al., 1993). Glutamate tox- icity clearly depends on the presence of extracellular calcium (Carriedo et al., 1996; Van Den Bosch et al., 2000), but calcium influx through volt- age gated calcium channels (VGCC) is not able to induce motor neuron death (Van Den Bosch et al., 2002). Motor neurons appear to be more vulnerable to AMPA receptor mediated excitotoxicity (Carriedo et al., 2000; Ikonomidou et al., 1996; Saroff et al., 2000), possibly due to lower expression of calcium binding proteins (CaBP) (Alexianu et al., 1994; Ince et al., 1993; Siklos et al., 1998). Indeed, neuroprotection was attained by overexpression of CaBP (Beers et al., 2001; Roy et al., 1998), but motor neurons primarily vulnerable to ALS possibly require a relative absence of CaBP for their high frequency modulation (Lips and Keller, 1999). Intracellular calcium shuttles between the ER and mitochondria (Berridge, 2002; Verkhratsky, 2005) and has been recognised taking a major part in controlling apoptosis and other forms of cell death (Pinton et al., 2008). Mitochondria seem to exclusively buffer calcium in motor neurons (Grosskreutz et al., 2007) while the ER is chronically stressed in ALS models (Prell et al., 2012). The ER mitochondria calcium Experimental Neurology 247 (2013) 91–100 Abbreviations: ALS, amyotrophic lateral sclerosis; AMPA, α-amino-5-methyl-3- hydroxyisoxazolone-4-propionat; CaBP, calcium binding proteins; CPA, cyclopiazonic acid; CsA, cyclosporine A; eIF2α, eukaryotic translation initiation factor 2 α; EMD, earth mover's distance; ERMCC, ER mitochondria calcium cycle; FUS/TLS, fused in sarcoma/translated in liposarcoma; hSOD1, human superoxide dismutase 1; mNCE, mitochondrial sodium calcium exchanger; mPTP, mitochondrial permeability transi- tion pore; mUP, mitochondrial uniporter; SERCA, sarco-/endoplasmic reticulum ATPase; TDP-43, transactive response (TAR) DNA-binding protein-43; TTX, tetrodotoxin; UPR, un- folded protein response; VAPB, vesicle-associated membrane protein (VAMP)-associated protein B; VCP, valosin containing protein; VGCC, voltage gated calcium channels; XBP1, X-box binding protein 1. ⁎ Corresponding author. E-mail address: janin.lautenschlaeger@med.uni-jena.de (J. Lautenschläger). 0014-4886/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2013.03.027 Contents lists available at SciVerse ScienceDirect Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr