ORIGINAL CONTRIBUTION Mitochondrial Respiratory Chain Dysfunction in Muscle From Patients With Amyotrophic Lateral Sclerosis Veronica Crugnola, MD; Costanza Lamperti, MD, PhD; Valeria Lucchini, MD; Dario Ronchi, PhD; Lorenzo Peverelli, MD; Alessandro Prelle, MD; Monica Sciacco, MD, PhD; Andreina Bordoni, BS; Elisa Fassone, PhD; Francesco Fortunato, BS; Stefania Corti, MD; Vincenzo Silani, MD; Nereo Bresolin, MD; Salvatore Di Mauro, MD; Giacomo Pietro Comi, MD; Maurizio Moggio, MD Background: Amyotrophic lateral sclerosis (ALS) is a of 100 fibers), 8 had moderate (5-10 COX-negative fibers major cause of neurological disability and its pathogen- of 100), and 7 had severe (Ͼ10 COX-negative fibers of 100) esis remains elusive despite a multitude of studies. Al- COX deficiency. Spectrophotometric measurement of res- though defects of the mitochondrial respiratory chain have piratory chain activities showed that 3 patients with severe been described in several ALS patients, their pathogenic histochemical COX deficiency also showed combined en- significance is unclear. zyme defects. In 1 patient, COX deficiency worsened in a second biopsy taken 9 months after the first. Among the pa- Objective: To review systematically the muscle biopsy tients with severe COX deficiency, one had a new mutation specimens from patients with typical sporadic ALS to search in the SOD1 gene, another a mutation in the TARDBP gene, for possible mitochondrial oxidative impairment. and a third patient with biochemically confirmed COX de- ficiency had multiple mitochondrial DNA deletions detect- Design: Retrospective histochemical, biochemical, and able by Southern blot analysis. molecular studies of muscle specimens. Conclusions: Our data confirm that the histochemical Setting: Tertiary care university. finding of COX-negative fibers is common in skeletal Subjects: Fifty patients with typical sporadic ALS (mean muscle from patients with sporadic ALS. We did not find age, 55 years). a correlation between severity of the oxidative defect and age of the patients or duration of the disease. However, Main Outcome Measure: Number of patients show- the only patient who underwent a second muscle bi- ing a clear muscle mitochondrial dysfunction assessed opsy did show a correlation between severity of symp- through histochemical and biochemical muscle analysis. toms and worsening of the respiratory chain defect. In 7 patients, the oxidative defect was severe enough to sup- Results: Histochemical data showed cytochrome c oxidase port the hypothesis that mitochondrial dysfunction must (COX)–negative fibers in 46% patients. Based on COX his- play a role in the pathogenesis of the disease. tochemical activity, patients fell into 4 groups: 27 had nor- mal COX activity; and 8 had mild (2-4 COX-negative fibers Arch Neurol. 2010;67(7):849-854. MYOTROPHIC LATERAL SCLE- copper/zinc superoxide dismutase (SOD1), rosis (ALS) is a progres- which is encoded by a gene on chromo- sive neurodegenerative dis- some 21. Cases of SOD1-associated ALS order characterized by loss are due to a toxic gain of function of the of motor neurons in the an- enzyme that leads to increased oxidative Aterior horns of the spinal cord, brainstem stress.3 Mice expressing mutant Sod1 de- motor nuclei, and cerebral cortex. The dis- velop motor neuron degeneration de- ease usually starts in the fourth or fifth de- spite increased SOD1 activity.4 cade of life. Presentation is heterogeneous, Other genes that are mutated in rarer and weakness may initially be limited to 1 forms of ALS are involved in DNA re- limb or even to 1 muscle group. As a rule, pair,5-7 axonal transport,8-10 mitochondrial the disease progresses rapidly and causes respiration,11 or gene expression and splic- widespread paralysis and spasticity.1,2 ing.12,13 Mutations in the TARDBP gene have The prevalence of ALS is approxi- been detected in approximately 1% to 5% mately 1 to 2 in 100 000 individuals. About of patients with familial ALS and approxi- Author Affiliations are listed at 10% of ALS cases are familial, of which mately 1% of patients with sporadic ALS.14 the end of this article. 20% are caused by dominant mutations in Recently, mutations in another RNA- (REPRINTED) ARCH NEUROL / VOL 67 (NO. 7), JULY 2010 WWW.ARCHNEUROL.COM 849 ©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 binding protein, fused in sarcoma/translated in liposar- tionswereprocessedaccordingtostandardhistologicaltechniques.36 coma (FUS/TLS), have been described in familial ALS pa- Histochemistry for COX, succinic dehydrogenase (SDH), and com- tients.15,16 It is likely that additional genetic factors predispose bined COX/SDH stains were performed as previously described.37 individuals to the disease17 or modify its onset and pro- gression.18,19 Oxidative stress results from mitochondrial BIOCHEMISTRY dysfunction and may play a role in the pathogenesis of ALS by worsening or even initiating the motor neuron in- Mitochondrial respiratory chain enzyme and citrate synthase jury.20-22 In fact, several reports describe mitochondrial al- activities were measured spectrophotometrically in all pa- terations in ALS.23 Transgenic mice with muscular over- tients with histochemical evidence of COX deficiency by de- 38 expression of uncoupling protein 1, a potent mitochondrial scribed assays. The specific activity of each complex was nor- uncoupler, displayed age-dependent deterioration of the malized to that of citrate synthase. neuromuscular junction correlated with progressive signs of denervation and a mild late-onset motor neuron pathol- MOLECULAR GENETICS ogy.24 On the other hand, some authors are still question- ing the presence of mitochondrial dysfunction.25,26 In the 7 patients with severe COX deficiency, we sequenced both the SOD1 gene (OMIM 147450) and mtDNA. We also per- Motor neuron death might also be caused by calcium- 39 mediated excitotoxicity or by activation of the intrinsic apop- formed Southern blot analysis of muscle mtDNA. Molecular 27 examination for TARDBP (OMIM 605078) was also per- totic pathway. Paradoxically, both decreased mitochon- formed.40 Quantitation of mtDNA was performed using South- drial number and increased mitochondrial mass (with ern blot analysis as described. increased intramitochondrial calcium concentrations) have Genomic DNA was extracted from peripheral blood of all pa- been reported in intramuscular nerves, spinal cord, and skel- tients and used as a template for polymerase chain reaction (PCR) etal muscles of patients with sporadic ALS.28-30 Abnormal amplification of each of the 5 exons of SOD1. Mitochondrial DNA mitochondria were also seen by electron microscopy in was PCR-amplified using the VariantSEQr Resequencing Sys- muscle31 and in the anterior horn cells of the spinal cord.32 tem (Applied Biosystem, Foster City, California). All PCR prod- One patient with motor neuron degeneration had se- ucts were purified and directly sequenced using the BigDye Ter- vere muscle cytochrome c oxidase (COX) deficiency due minator protocol on an automated 3100 ABI Prism Genetic to a mutation in the mitochondrial DNA (mtDNA)– Analyzer (Applied Biosystem). The following nuclear genes were analyzed in patient 7: PEO1, POLG1, POLG2, ANT1, and OPA1. encoded subunit I of COX.33 Hirano et al34 have recently reviewed several articles documenting how respiratory chain defects can mimic ALS or spinal muscular atro- RESULTS phy. To assess the importance of mitochondria respira- tory chain defects in ALS, we have studied muscle biop- CLINICAL AND MUSCLE BIOPSY FINDINGS sies from a cohort of 50 typical patients. In all patients, histopathologic examination showed a METHODS chronic neurogenic pattern, with small groups of atro- phic fibers and fiber-type grouping. Histochemically, COX- negative fibers were observed in 23 patients (46%). The oxi- SUBJECTS dative defect was mild in 8 patients (2-4 COX-negative fibers per 100 fibers), moderate in 8 (5-10 COX-negative fibers We studied biceps brachii muscle biopsy specimens from 50 pa- per 100), and severe in 7 (Ͼ10 COX-negative fibers per tients (14 women and 36 men ranging in age from 24 to 78 years; 100) (Figure 1). In patient 6, a second muscle biopsy per- mean age, 55 years) with sporadic ALS defined according to El Escorial35 criteria who were admitted to our clinic from 2000 to formed 9 months after the first one documented further 2008. Of these specimens, 29 were biopsied within 1 year and decreasing of the COX activity (almost all muscle fibers 20 within 2 years from onset of the disease. In 1 patient, the first lacked COX activity) (Figure 2). The clinical, morpho- symptoms had appeared 4 years before the muscle biopsy. logical and biochemical features of the 7 patients with se- For controls, we used biceps brachii muscle biopsy speci- vere oxidative defects are presented in the Table. mens from 8 normal, healthy controls, 10 biopsies from patients All patients were men; age at onset ranged from 31 to with peripheral neuropathy, and 10 biopsies from patients with 75 years; and two-thirds of patients were younger than 50 nonmitochondrial metabolic diseases (5 carnitine palmitoyltrans- years. At onset, all patients had predominantly lower mo- ferase II deficiencies, 2 myoadenylate deaminase deficiencies, and tor neuron involvement and variable disease severity. 3 glycogenoses). Control muscle samples came from individuals of similar ages as the ALS patients. All specimens were obtained from the DNA, Muscle and Nerve Tissue Bank