Abstract
Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1G93A motor neurons.
Highlights
Amyotrophic lateral sclerosis (ALS)2 is a debilitating lateonset, progressive form of motor neuron disease that primarily targets motor neurons in the cortex, brainstem, and the anterior horn of the spinal cord, leading to paralysis and eventually death, typically within 5 years of diagnosis [1,2,3,4,5]
The Integrity and Sucrose Sedimentation Pattern of the Dynein Complex Are Not Compromised in SOD1G93A and SOD1G93A;Dync1h1Loa/ϩ—We have previously shown that dynein-mediated retrograde axonal transport is impaired in cultured motor neurons isolated from SOD1G93A embryos and that the Loa mutation rescues this defect [17]
These data show that the bulk of the dynein complex, represented by DHC, DIC, and dynein light intermediate chain, sediments in fractions 9, 10, and 11 and that their sedimentation patterns in wild-type (ϩ/ϩ) brain matched those of SOD1G93A brain, and likewise, the sedimentation patterns of these polypeptides from Dync1h1Loa/ϩ brain matched those from SOD1G93A; Dync1h1Loa/ϩ
Summary
Animals and Tissue Collection—Congenic heterozygous Dync1h1Loa/ϩ female mice, maintained on a C57BL/6 (Harlan UK) genetic background, were crossed with male transgenic mice expressing human SOD1G93A, maintained on an F1 (SJL x C57BL/6) genetic background, to produce four genetically distinct groups of littermates: Dync1h1ϩ/ϩ, Dync1h1Loa/ϩ heterozygote, SOD1G93A hemizygote, and SOD1G93A; Dync1h1Loa/ϩ double-heterozygote (represented in the figures as ϩ/ϩ, Loa/ϩ, SOD1G93A, and SOD1G93A/Loa, respectively) mice. Samples of the SOD1G93A and SOD1G93A;Dync1h1Loa/ϩ cerebral cortex and spinal cord buoyant density gradient (BDG) fractions containing equal amounts of proteins were subjected to treatments with salt, alkali, and digestion with proteinase K in the presence and absence of detergents and analyzed by SDS-PAGE and immunoblotting. Samples of the SOD1G93A and SOD1G93A;Dync1h1Loa/ϩ cerebral cortex and spinal cord microsomal fractions containing equal amounts of proteins were subjected to chemical insults with salt and alkali and digestion with proteinase K in the presence and absence of detergents and analyzed by SDS-PAGE and immunoblotting. Preparation of Mitochondria-enriched Fractions for Polarographic Studies and Measurement of Oxygen Consumption— Brain cortex and spinal cord tissues were dissected and washed once in ice-cold tissue wash buffer (TWB; 0.3 M mannitol, 20 mM MOPS, 2 mM EDTA (pH 7.5)) homogenized with a loose pestle in ϳ1 ml of TWB supplemented with 1% bovine serum albumin and 7 mM L-cysteine hydrochloride monohydrate.
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