title: AMPA receptor activation causes preferential mitochondrial Ca²⁺ load and oxidative stress in motor neurons.
abstract: It is well established that motor neurons are highly vulnerable to glutamate induced excitotoxicity. The selective vulnerability of these neurons has been attributed to AMPA receptor mediated excessive rise in cytosolic calcium and consequent mitochondrial Ca(2+) loading. Earlier we have reported that in motor neurons a generic rise in [Ca(2+)]i does not always lead to mitochondrial Ca(2+) loading and membrane depolarization but it occurs upon AMPA receptor activation. The mechanism of such specific mitochondrial involvement upon AMPA receptor activation is not known. The present study examines the mitochondrial Ca(2+) regulation and oxidative stress in spinal cord neurons upon AMPA subtype of glutamate receptor activation. Stimulating the spinal neurons with AMPA exhibited a sharp rise in [Ca(2+)]m in both motor and other spinal neurons that was sustained up to the end of recording time of 30min. The rise in [Ca(2+)]m was substantially higher in motor neurons than in other spinal neurons which could be due to the differential mitochondrial homeostasis in two types of neurons. To examine this possibility, we measured AMPA induced [Ca(2+)]m loading in the presence of mitochondrial inhibitors. In both cell types the AMPA induced [Ca(2+)]m loading was blocked by mitochondrial calcium uniporter blocker ruthenium red. In motor neurons it was also inhibited substantially by CGP37157 and cyclosporine-A, the blockers of Na(+)/Ca(2+) exchanger and mitochondrial permeability transition pore (MPTP) respectively, whereas no effect of these agents was observed in other spinal neurons. Thus in motor neurons the Ca(2+) sequestration by mitochondria occurs through mitochondrial calcium uniporter as well as due to reversal of Na(+)/Ca(2+) exchanger, in contrast the latter pathway does not contribute in other spinal neurons. The ROS formation was inhibited by nitric oxide synthase (NOS) inhibitor L-NAME in both types of neurons, however the mitochondrial complex-I inhibitor rotenone suppressed the ROS formation only in motor neurons. It appears that activation of cytoplasmic nNOS leads to ROS formation in both types of spinal neurons but mitochondria is the major source of ROS in motor neurons. Spinal neurons exhibited a significant time dependent fall in glutathione (GSH) level. The GSH level in motor neurons did not recover even at 24h after AMPA exposure, whereas the other spinal neurons exhibited a tendency to maintain the GSH after a certain level suggesting that the oxidative stress is arrested in other spinal neurons but it continues to increase in motor neurons. Thus our results demonstrate that upon AMPA receptor stimulation the motor neurons employ some additional pathways for regulation of mitochondrial calcium and oxidative stress as compared to other spinal neurons. It is suggested that such differential signaling mechanisms in motor neurons could be crucial for their selective vulnerability to excitotoxicity.
authors.affiliation: Department of Biophysics National Institute of Mental Health and Neuro Sciences, Bangalore, 560 029, India.
authors.forename: Dinesh C
authors.initials: DC
authors.lastname: Joshi
authors.affiliation: Department of Biophysics National Institute of Mental Health and Neuro Sciences, Bangalore, 560 029, India.
authors.forename: Bhanu P
authors.initials: BP
authors.lastname: Tewari
authors.affiliation: Department of Biophysics National Institute of Mental Health and Neuro Sciences, Bangalore, 560 029, India.
authors.forename: Mahendra
authors.initials: M
authors.lastname: Singh
authors.affiliation: Department of Biophysics National Institute of Mental Health and Neuro Sciences, Bangalore, 560 029, India.
authors.forename: Preeti G
authors.initials: PG
authors.lastname: Joshi
authors.affiliation: Department of Biophysics National Institute of Mental Health and Neuro Sciences, Bangalore, 560 029, India. Electronic address: joshi.nanda@gmail.com.
authors.forename: Nanda B
authors.initials: NB
authors.lastname: Joshi
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009046:Motor Neurons / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D018091:Receptors, AMPA / Q000378:metabolism*; D013116:Spinal Cord / Q000166:cytology; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
chemical_list: D018690:Excitatory Amino Acid Agonists; D017382:Reactive Oxygen Species; D018091:Receptors, AMPA; D011189:Potassium Chloride; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D005978:Glutathione; D002118:Calcium
keywords: AMPA receptors; Amyotrophic lateral sclerosis; Calcium signaling; Excitotoxicity; Mitochondrial calcium; Motor neurons; Oxidative stress; Voltage gated calcium channels