Increased sensitivity of myoblasts to oxidative stress in amyotrophic lateral sclerosis peripheral tissues

Abstract

We compared mitochondrial respiratory chain function, mitochondrial DNA (mtDNA) integrity, and oxidative stress levels in muscle, myoblasts, fibroblasts and cybrids, from 12 amyotrophic lateral sclerosis (ALS) patients with 28 control samples. Mitochondrial respiratory chain enzyme activities were normal in muscle, myoblast and fibroblast cultures from ALS patients, as were levels of mtDNA in muscle. Rearranged muscle mtDNA species were not detected by Southern blot hybridization in any of the samples and no difference was found in the number of deleted mtDNA species detected by long-range PCR. Platelet-derived cybrid studies confirmed the absence of a systemic mtDNA abnormality. Aconitase activity measurements did not indicate increased oxidative damage in muscle tissue, or in myoblasts or fibroblasts from ALS patients cultured under basal conditions. We did, however, find an increased sensitivity to oxidative stress in myoblasts from ALS patients exposed to paraquat. This altered sensitivity appears to be due to a nuclear rather than a mtDNA abnormality. Motor neurons have a large relative size and metabolic activity, and would be expected to be exposed to a greater degree of oxidative stress than most tissues throughout life. In addition, neurons are postmitotic cells, with poor regenerative potential. We do not have a ready method to study this in neural tissue of living patients, but the oxidative stress identified in myoblasts would translate into oxidative damage more readily in motor neurons than in other tissues.

Introduction

Amyotrophic lateral sclerosis/motor neuron disease (ALS/MND) is a rapidly progressive, fatal neurodegenerative disorder, affecting selected populations of motor neurons in the brain and spinal cord (Mitchell and Borasio, 2007). Around 1 in 400 individuals die of the condition worldwide. Approximately 10% of all patients have a positive family history of the disease. Mutations in the SOD1 gene, which encodes copper/zinc superoxide dismutase, are found in about 20% of the familial patients and occasionally in apparently sporadic patients. Other, rarer, nuclear gene mutations have been associated with ALS (Pasinelli and Brown, 2006, Valdmanis and Rouleau, 2008, Sreedharan et al., 2008), but the importance of some of these genes is uncertain at the present time. In addition, a heteroplasmic mutation in the mitochondrial MTCO1 gene has been found in muscle tissue of a single patient with ALS (Comi et al., 1998) and a heteroplasmic mutation in the mitochondrial MTTI gene has been found in brain and spinal cord of another patient with ALS and atypical neuropathological features (Borthwick et al., 2006). Mitochondrial DNA (MtDNA) haplogroup I was found to be more common in a study of 222 Italian ALS patients compared with 151 controls (Mancuso et al., 2004), but not in a larger UK cohort of 504 ALS patients and 1016 controls (Chinnery et al., 2007).

Although the cause of ALS is unknown in the majority of cases, there is an increasing body of evidence suggesting the involvement of mitochondria in the pathogenesis of ALS (Borthwick et al., 1999, Siciliano et al., 2001, Menzies et al., 2002, Orrell and Schapira, 2002, Beal, 2005, Manfredi and Xu, 2005, Appel, 2006, Hervias et al., 2006, Mattson et al., 2008). To identify an accessible biomarker of the disease, we investigated various aspects of mitochondrial function in peripheral tissues of ALS patients. Documented evidence of mitochondrial abnormalities in peripheral tissues of patients is inconsistent. Analysis of skeletal muscle tissue has been reported as showing decreased mitochondrial respiratory chain enzyme activities, decreased levels of mitochondrial DNA (mtDNA) and increased levels of deleted mtDNA by some groups (Wiedemann et al., 1998, Vielhaber et al., 1999, Veilhaber et al., 2000, Ro et al., 2003), while others found no significant differences compared to control tissue (Krasnianski et al., 2005). Similarly, in one study, transfer of platelet mtDNA from ALS subjects to mtDNA-depleted control cell lines resulted in cybrids with decreased respiratory chain enzyme activities compared to control cybrids (Swerdlow et al., 1998), whereas two other studies found no differences between patient and control cybrids (Gluck et al., 2000, Gajewski et al., 2003).

These previous reports of the presence or absence of mitochondrial abnormalities in peripheral tissues of ALS patients require clarification and confirmation, best undertaken by a comprehensive approach to a single group of patients. Therefore, in this study we compared mitochondrial respiratory chain function, mitochondrial DNA integrity, and oxidative stress levels in muscle, myoblasts, fibroblasts and cybrids, in a group of patients with ALS and controls.