Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease

Manoj Jaiswal, Wolf Dieter Zech, Miriam Goos, Christine Leutbecher, Alberto Ferri, Annette Zippelius, Maria Teresa Carrì, Roland Nau, Bernhard U. Keller

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Abstract

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons (MN) in the brain stem and spinal cord. Intracellular disruptions of cytosolic and mitochondrial calcium have been associated with selective MN degeneration, but the underlying mechanisms are not well understood. The present evidence supports a hypothesis that mitochondria are a target of mutant SOD1-mediated toxicity in familial amyotrophic lateral sclerosis (fALS) and intracellular alterations of cytosolic and mitochondrial calcium might aggravate the course of this neurodegenerative disease. In this study, we used a fluorescence charged cool device (CCD) imaging system to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells in an established cellular model of ALS. Results: To gain insights into the molecular mechanisms of SOD1G93A associated motor neuron disease, we simultaneously monitored cytosolic and mitochondrial calcium concentrations in individual cells. Voltage - dependent cytosolic Ca2+ elevations and mitochondria - controlled calcium release mechanisms were monitored after loading cells with fluorescent dyes fura-2 and rhod-2. Interestingly, comparable voltage-dependent cytosolic Ca2+ elevations in WT (SH-SY5YWT) and G93A (SH-SY5YG93A) expressing cells were observed. In contrast, mitochondrial intracellular Ca2+ release responses evoked by bath application of the mitochondrial toxin FCCP were significantly smaller in G93A expressing cells, suggesting impaired calcium stores. Pharmacological experiments further supported the concept that the presence of G93A severely disrupts mitochondrial Ca2+ regulation. Conclusion: In this study, by fluorescence measurement of cytosolic calcium and using simultaneous [Ca2+]i and [Ca2+]mito measurements, we are able to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells an established cellular model of ALS. The primary goals of this paper are (1) method development, and (2) screening for deficits in mutant cells on the single cell level. On the technological level, our method promises to serve as a valuable tool to identify mitochondrial and Ca2+-related defects during G93A-mediated MN degeneration. In addition, our experiments support a model where a specialized interplay between cytosolic calcium profiles and mitochondrial mechanisms contribute to the selective degeneration of neurons in ALS.

Original languageEnglish
Article number64
JournalBMC Neuroscience
Volume10
DOIs
Publication statusPublished - Jun 22 2009

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Motor Neurons
Cell Culture Techniques
Calcium
Amyotrophic Lateral Sclerosis
Nerve Degeneration
Neurodegenerative Diseases
Mitochondria
Fluorescence
Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone
Motor Neuron Disease
Fura-2
Baths
Fluorescent Dyes
Brain Stem
Spinal Cord
Pharmacology
Equipment and Supplies

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience
  • Neuroscience(all)

Cite this

Jaiswal, M., Zech, W. D., Goos, M., Leutbecher, C., Ferri, A., Zippelius, A., ... Keller, B. U. (2009). Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease. BMC Neuroscience, 10, [64]. https://doi.org/10.1186/1471-2202-10-64

Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease. / Jaiswal, Manoj; Zech, Wolf Dieter; Goos, Miriam; Leutbecher, Christine; Ferri, Alberto; Zippelius, Annette; Carrì, Maria Teresa; Nau, Roland; Keller, Bernhard U.

In: BMC Neuroscience, Vol. 10, 64, 22.06.2009.

Research output: Contribution to journalArticle

Jaiswal, M, Zech, WD, Goos, M, Leutbecher, C, Ferri, A, Zippelius, A, Carrì, MT, Nau, R & Keller, BU 2009, 'Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease', BMC Neuroscience, vol. 10, 64. https://doi.org/10.1186/1471-2202-10-64
Jaiswal, Manoj ; Zech, Wolf Dieter ; Goos, Miriam ; Leutbecher, Christine ; Ferri, Alberto ; Zippelius, Annette ; Carrì, Maria Teresa ; Nau, Roland ; Keller, Bernhard U. / Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease. In: BMC Neuroscience. 2009 ; Vol. 10.
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