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

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.

[1]  J. Haines,et al.  Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis , 1993, Nature.

[2]  J. Weiss,et al.  Motor Neurons Are Selectively Vulnerable to AMPA/Kainate Receptor-Mediated Injury In Vitro , 1996, The Journal of Neuroscience.

[3]  J. Grosskreutz,et al.  The unfolded protein response in models of human mutant G93A amyotrophic lateral sclerosis , 2012, The European journal of neuroscience.

[4]  J. Grosskreutz,et al.  Rat embryonic motoneurons in long-term co-culture with Schwann cells—a system to investigate motoneuron diseases on a cellular level in vitro , 2005, Journal of Neuroscience Methods.

[5]  David Heckerman,et al.  A Hexanucleotide Repeat Expansion in C9ORF72 Is the Cause of Chromosome 9p21-Linked ALS-FTD , 2011, Neuron.

[6]  W. Robberecht,et al.  Chloride Influx Aggravates Ca2+-Dependent AMPA Receptor-Mediated Motoneuron Death , 2003, The Journal of Neuroscience.

[7]  P. Caroni,et al.  A role for motoneuron subtype–selective ER stress in disease manifestations of FALS mice , 2009, Nature Neuroscience.

[8]  Bruce L. Miller,et al.  Expanded GGGGCC Hexanucleotide Repeat in Noncoding Region of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS , 2011, Neuron.

[9]  B. Keller,et al.  Cu/Zn Superoxide Dismutase Typical for Familial Amyotrophic Lateral Sclerosis Increases the Vulnerability of Mitochondria and Perturbs Ca2+ Homeostasis in SOD1G93A Mice , 2009, Molecular Pharmacology.

[10]  J. Grosskreutz,et al.  Calcium dysregulation in amyotrophic lateral sclerosis. , 2010, Cell calcium.

[11]  R. Dengler,et al.  Temporospatial coupling of networked synaptic activation of AMPA-type glutamate receptor channels and calcium transients in cultured motoneurons , 2006, Neuroscience.

[12]  Soumitra S Ghosh,et al.  Efficient syntheses of benzothiazepines as antagonists for the mitochondrial sodium-calcium exchanger: potential therapeutics for type II diabetes. , 2003, The Journal of organic chemistry.

[13]  Takeo Kato,et al.  Mutations of optineurin in amyotrophic lateral sclerosis , 2010, Nature.

[14]  B. Keller,et al.  Activity-related calcium dynamics in motoneurons of the nucleus hypoglossus from mouse. , 1999, Journal of neurophysiology.

[15]  T. Raju,et al.  Evidence of endoplasmic reticular stress in the spinal motor neurons exposed to CSF from sporadic amyotrophic lateral sclerosis patients , 2011, Neurobiology of Disease.

[16]  V. La Bella,et al.  The role of calcium‐binding proteins in selective motoneuron vulnerability in amyotrophic lateral sclerosis , 1994, Annals of neurology.

[17]  M. Mattson,et al.  ALS-Linked Cu/Zn–SOD Mutation Increases Vulnerability of Motor Neurons to Excitotoxicity by a Mechanism Involving Increased Oxidative Stress and Perturbed Calcium Homeostasis , 1999, Experimental Neurology.

[18]  Xun Hu,et al.  Mutations in FUS, an RNA Processing Protein, Cause Familial Amyotrophic Lateral Sclerosis Type 6 , 2009, Science.

[19]  J. Haines,et al.  Mutations in UBQLN2 cause dominant X-linked juvenile and adult onset ALS and ALS/dementia , 2011, Nature.

[20]  N. Mercuri,et al.  Altered calcium homeostasis in motor neurons following AMPA receptor but not voltage-dependent calcium channels’ activation in a genetic model of amyotrophic lateral sclerosis , 2007, Neurobiology of Disease.

[21]  T. Siddique,et al.  Genetic aspects of amyotrophic lateral sclerosis. , 2002, Advances in neurology.

[22]  R. Tsien,et al.  A new generation of Ca2+ indicators with greatly improved fluorescence properties. , 1985, The Journal of biological chemistry.

[23]  W. Robberecht,et al.  Role of mitochondria in kainate-induced fast Ca2+ transients in cultured spinal motor neurons. , 2007, Cell calcium.

[24]  Manuela G. López,et al.  Mitochondrial Na+/Ca2+ exchanger, a new target for neuroprotection in rat hippocampal slices. , 2010, Biochemical and biophysical research communications.

[25]  Haibin Ling,et al.  An Efficient Earth Mover's Distance Algorithm for Robust Histogram Comparison , 2007, IEEE Transactions on Pattern Analysis and Machine Intelligence.

[26]  W. Robberecht,et al.  Ca2+-permeable AMPA receptors and selective vulnerability of motor neurons , 2000, Journal of the Neurological Sciences.

[27]  S. C. Chafe,et al.  Mutations in the Profilin 1 Gene Cause Familial Amyotrophic Lateral Sclerosis , 2012, Nature.

[28]  E. Barrett,et al.  Stimulation‐Induced Mitochondrial [Ca2+] Elevations in Mouse Motor Terminals: Comparison of Wild‐Type with SOD1‐G93A , 2003, The Journal of physiology.

[29]  H. Drexler,et al.  The Pearson product‐moment correlation coefficient is better suited for identification of DNA fingerprint profiles than band matching algorithms , 1993, Electrophoresis.

[30]  M. Berridge,et al.  The endoplasmic reticulum: a multifunctional signaling organelle. , 2002, Cell calcium.

[31]  M. Gurney,et al.  Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. , 1994, Science.

[32]  S. Petri,et al.  Neural mitochondrial Ca2+ capacity impairment precedes the onset of motor symptoms in G93A Cu/Zn‐superoxide dismutase mutant mice , 2006, Journal of neurochemistry.

[33]  J. Grosskreutz,et al.  Endoplasmic reticulum stress and the ER mitochondria calcium cycle in amyotrophic lateral sclerosis , 2012, Amyotrophic lateral sclerosis : official publication of the World Federation of Neurology Research Group on Motor Neuron Diseases.

[34]  Alexei Verkhratsky,et al.  Physiology and pathophysiology of the calcium store in the endoplasmic reticulum of neurons. , 2005, Physiological reviews.

[35]  J. Rothstein,et al.  Chronic inhibition of glutamate uptake produces a model of slow neurotoxicity. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[36]  M. Gurney,et al.  Intracellular Calcium Parallels Motoneuron Degeneration in SOD-1 Mutant Mice , 1998, Journal of neuropathology and experimental neurology.

[37]  J. Olney,et al.  Motor Neuron Degeneration Induced by Excitotoxin Agonists Has Features in Common with those Seen in the SOD-1 Transgenic Mouse Model of Amyotrophic Lateral Sclerosis , 1996, Journal of neuropathology and experimental neurology.

[38]  S. Sensi,et al.  AMPA Exposures Induce Mitochondrial Ca2+ Overload and ROS Generation in Spinal Motor Neurons In Vitro , 2000, The Journal of Neuroscience.

[39]  P. Pinton,et al.  Calcium and apoptosis: ER-mitochondria Ca2+ transfer in the control of apoptosis , 2008, Oncogene.

[40]  C. Geula,et al.  Selective vulnerability of spinal cord motor neurons to non‐NMDA toxicity , 2000, Neuroreport.

[41]  C. Heizmann,et al.  Parvalbumin and calbindin D‐28k in the human motor system and in motor neuron disease , 1993, Neuropathology and applied neurobiology.

[42]  F. Poccia,et al.  Expression of a Cu,Zn superoxide dismutase typical of familial amyotrophic lateral sclerosis induces mitochondrial alteration and increase of cytosolic Ca2+ concentration in transfected neuroblastoma SH‐SY5Y cells , 1997, FEBS letters.

[43]  A. Zippelius,et al.  Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease , 2009, BMC Neuroscience.

[44]  J. Rothstein,et al.  Neuroprotective Strategies in a Model of Chronic Glutamate‐Mediated Motor Neuron Toxicity , 1995, Journal of neurochemistry.

[45]  Michael D. Abràmoff,et al.  Image processing with ImageJ , 2004 .

[46]  D. Figlewicz,et al.  Glutamate Potentiates the Toxicity of Mutant Cu/Zn-Superoxide Dismutase in Motor Neurons by Postsynaptic Calcium-Dependent Mechanisms , 1998, The Journal of Neuroscience.

[47]  N. Ertekin-Taner,et al.  Novel p.Ile151Val mutation in VCP in a patient of African American descent with sporadic ALS , 2011, Neurology.

[48]  Bruce L. Miller,et al.  Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis , 2006, Science.

[49]  T. Gillingwater,et al.  A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. , 2004, American journal of human genetics.

[50]  M. Haine,et al.  Van Damme A. , 1986 .

[51]  W. Robberecht,et al.  An α-mercaptoacrylic acid derivative (PD150606) inhibits selective motor neuron death via inhibition of kainate-induced Ca2+ influx and not via calpain inhibition , 2002, Neuropharmacology.

[52]  C. Pérez,et al.  Benzothiazepine CGP37157 and its isosteric 2'-methyl analogue provide neuroprotection and block cell calcium entry. , 2012, ACS chemical neuroscience.

[53]  P. van Damme,et al.  G37R SOD1 mutant alters mitochondrial complex I activity, Ca(2+) uptake and ATP production. , 2011, Cell calcium.

[54]  S. Appel,et al.  Parvalbumin overexpression alters immune‐mediated increases in intracellular calcium, and delays disease onset in a transgenic model of familial amyotrophic lateral sclerosis , 2001, Journal of neurochemistry.