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

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

doi:10.1186/1471-2202-10-64

Abstract

BackgroundAmyotrophic 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.ResultsTo 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.ConclusionIn 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.

Highlights

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
Glutamate excitotoxicity may disrupt intracellular calcium homeostasis and reactive oxygen species (ROS) production [27], which may be promoted by oxidative stress as glutamate transporters are susceptible to disruption by oxidants, and oxidative modifications to the transporter have been reported in ALS and the mtSOD1G93A mouse model [24,21]
The transfected cells were treated with Carbonyl cyanide p(trifluoromethoxy) phenylhydrazone (FCCP) (3 min) and the [cytosolic calcium (Ca2+]i) measured for both WT and G93A-transfected SH-SY5Y cells (Fig. 1)

Summary

Introduction

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. 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. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by a selective loss of motor neurons (MNs) in the brain stem, the spinal cord and the motor cortex, leading to progressive weakness, muscle atrophy with eventual paralysis, and death. The etiology is likely to be multifactorial because ALS involves the interplay of several mechanisms to initiate disease and propagate the spread of motor neuron cell death [36,38,39]

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