Mice that transgenically express mutated forms of human superoxide dismutase I (SOD1) that produce familial amyotrophic lateral sclerosis (fALS) develop progressive loss of motor nerve terminals that often precedes the eventual death of motor neuron cell bodies in the spinal cord. In addition to this early vulnerabiltiy to fALS-induced degeneration, we found that motor terminals of pre- symptomatic SOD1-G93A mice (high-copy number) are also more susceptible than wild-type terminals to degeneration caused by brief ischemia/reperfusion stress. In healthy motor terminals, mitochondria are essential for handling large, stimulation-induced Ca2+ loads. The proposed studies will test the hypothesis that motor terminals of different fALS mouse models become increasingly vulnerable to stresses due to deficient mitochondrial Ca2+ handling. Stresses to be tested include high frequency stimulation of motor nerves, energy stresses (ischemia/reperfusion, oxygen/glucose deprivation) and hydrogen peroxide. We hypothesize that mitochondria of fALS mouse terminals eventually lose their capacity to increase respiration sufficiently to sequester the Ca2+ overload associated with these stresses, resulting in mitochondrial depolarization, toxic increases in cytosolic [Ca2+] and calpain-mediated degeneration. We will use SOD1-fALS models that possess (G93A) or lack (G85R) dismutase activity, and that differ in their level of SOD1 expression level (high and low copy number G93A), to test if these fALS mutants will exhibit a lower threshold to these stresses than mice expressing wild-type human SOD1. Imaging of fluorescent indicator dyes in living terminals will be used to assay changes in motor terminal cytosolic and mitochondrial [Ca2+] and mitochondrial membrane potential. Inhibitors will be used to block hypothesized routes of damage, including excessive accumulation of cytosolic Ca2+, opening of the mitochondrial permeability transition pore, calpain activation and generation of reactive oxygen species. The extent of stress-induced muscle denervation and any protective effect of applied treatments will be assayed using motor terminals in which yellow fluorescent protein is transgenically expressed and muscle endplates are identified with fluorescently-labelled 1- bungarotoxin. The proposed experiments are important because they will test whether defective mitochondrial Ca2+ handling is a major upstream mechanism mediating motor terminal damage, and will identify agents that can protect motor terminals against this damage.PROJECT NARRATIVE Mouse models of familial amyotrophic lateral sclerosis (fALS) exhibit early degeneration of motor nerve terminals. The proposed experiments will test the hypothesis that motor terminals in multiple strains of fALS mice become especially vulnerable to energy stresses, and that this vulnerability involves defective mitochondrial handling of calcium loads. We will test multiple strategies for protecting motor terminals against these stresses. Treatments to preserve remaining motor nerve terminals should slow the progression of paralysis, and thus may become an important new component of therapies for treating ALS. ? ?
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|Zhang, Zhongsheng; Nguyen, Khanh T; Barrett, Ellen F et al. (2010) Vesicular ATPase inserted into the plasma membrane of motor terminals by exocytosis alkalinizes cytosolic pH and facilitates endocytosis. Neuron 68:1097-108|
|Nguyen, Khanh T; García-Chacón, Luis E; Barrett, John N et al. (2009) The Psi(m) depolarization that accompanies mitochondrial Ca2+ uptake is greater in mutant SOD1 than in wild-type mouse motor terminals. Proc Natl Acad Sci U S A 106:2007-11|