Background and aims. Amyotrophic lateral sclerosis (ALS) is caused by the selective degeneration of motoneurons in spinal cord, brain stem and cerebral cortex. The degeneration of motoneurons leads to skeletal muscle atrophy, paralysis and death. About 10 % of ALS cases are familial and mutation of Cu, Zn superoxide dismutase (SOD1) has been demonstrated to be a culprit among 20 % of the familial cases. Although contraversial, mitochondrial abnormalities have been reported in ALS-patients and in mouse model such as mutated human SOD1 transgenic mice and mnd mouse. To address this issue we assess mitochondrial function in transgenic rats overexpressed a human mutated SOD1 associated with ALS. Material and methods. Spinal cords and cerebellum from G93A and non-transgenic rats were homogenized in isolation buffer. The homogenate was centrifuged at 1,000 g for 10 min, and the resulting supernatant was layered onto 7.5% Ficoll medium on top of the 10% Ficoll medium and centrifuged at 79,000 g for 30 min. The mitochondiral pellet was resuspended in isolation buffer. 10 μg isolated mitochondria were separated on 15% SDS-PAGE and transferred to a nitorocellulose membrane. The membrane was immunostained rabbit anti-human SOD1 antibody or mouse anti-cytochrome c oxidase antibody. Secondary antibodies conjugated with HRP were used. Mitochondria were suspended in respiration buffer at 28 oC, and oxygen-consumption rates were measured in a closed-chamber cuvette with a mini-stirring bar using Clark-type electrode. To assess mitochondrial respiration mediated through complex I, mitochondria were preincubated with 10 mM glutamate and 5 mM for 2.5 min before 500 μM ADP was added to induce state 3 respiration. For complex II mediated respiration, mitochondria were preincubated for 2.5 min with 10 mM succinate before 500 μM ADP was added. For complex IV mediated respiration, mitochondria were preincubated for 2.5 min before 10 mM ascorbate and 0.2 mM N,N,N′, N′-tetramethyl-benzendiamine were added to induce oxygen consumption. Results. Significant level of human mutated SOD1 was detected in purified mitochondria of G93A rat, but not endogeneous rat SOD1. SOD1 aggregates were detected in G93A spinal cord mitochondria. Significant reduction of O2 consumption using complex I substrates were observed in the spinal cord mitochondria from G93A rats. On the other hand, enzyme activities, which are normalized against citrate synthase activities showed no significant changes in complex I. Polarographical study of mitochondrial respiration via complex II or complex IV showed no significant difference between nontransgenic and G93A rat mitochondria. difference between non-transgenic and G93A rat mitochondria. Conclusion. In transgenic G93A rat model of ALS, we showed a specific reduction of mitochondria respiration mediated through complex I, but not complex II and IV. This functional abnormality was only detected in intact mitochondria of G93A spinal cord, but not in freeze-thawed lysed mitochondria.This study supports the role of mitochondial defects in the pathogenesis of ALS.
|Journal||Journal of Cerebral Blood Flow and Metabolism|
|Issue number||SUPPL. 1|
|Publication status||Published - Nov 13 2007|
ASJC Scopus subject areas
- Clinical Neurology
- Cardiology and Cardiovascular Medicine