Mitochondrial Respiratory Chain Dysfunction in Muscle from Patients with Amyotrophic Lateral Sclerosis

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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 deficiency 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 normal 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 teriorA 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 despite 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-

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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 progression.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 respiratory 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 atrophic 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 of the Neurologi- BIOCHEMISTRY cal Unit, Fondazione Ospedale Maggiore, University of Milan, Mi- lan, Italy, with informed consent according to a protocol ap- Respiratory chain enzyme activities were normal in all pa- proved by the institutional review board of Foundation IRCCS tients but patients 3, 6, and 7. The enzyme activities were Ospedale Maggiore Policlinico, Mangiagalli and Regina Elena, Mi- normal in all patients with scattered (Ͻ10) COX-negative lan, in compliance with Italian and European Union laws. muscle fibers. In the more severe group, as defined by the presence of more than 10 histochemically deficient COX MUSCLE BIOPSY fibers, the degree of respiratory chain deficiency paral- leled the histochemical defect. For example, patients 1, 2, Muscle biopsy specimens from the left biceps brachii were fro- 4, and 5, who had borderline histochemical COX defects, zen in isopentane-cooled liquid nitrogen, and cryostat cross sec- had either normal respiratory chain activities (patients 1,

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Figure 1. Representative light microscopy images. A, Normal cytochrome c oxidase (COX) activity. B, Moderate COX deficiency (5-10 COX-deficient fibers per 100 fibers). C, Severe COX deficiency (Ͼ10 COX-deficient fibers per 100) (original magnification ϫ25).

A B C

D E F

Figure 2. Light microscopic image of sequential muscle biopsies from patient 6. Both the first biopsy (A) and the second (D) show a neurogenic pattern with small and angular muscle fibers. However, the first biopsy (B and C) shows many cytochrome c oxidase–deficient large fibers, while the second biopsy obtained 9 months later shows generalized cytochrome c oxidase deficiency (E and F) (original magnification ϫ25).

4, and 5) or showed moderate complex III deficiency (pa- had been reported in the probands’ ethnic groups as poly- tient 2). On the other hand, patients 3, 6, and 7 showed morphisms and are not likely to explain the observed COX severe combined deficiencies involving all respiratory chain deficiency. The Table summarizes the genetic findings in complexes, but more markedly COX activity (Table). Res- the 7 patients with severe COX deficiency. piratory complex activities were normal in muscle biopsy from patients with peripheral neuropathy. COMMENT MOLECULAR GENETICS Amyotrophic lateral sclerosis is a major cause of neuro- Besides having severe muscle COX deficiency, patient 2 also logical disability and its pathogenesis remains elusive de- harbored a missense mutation (c.65AϾG, p.Q22R) in spite a multitude of studies. Because the diagnosis is based SOD1,41 and patient 4 had an A382T mutation in TAR- on clinical and neurophysiologic criteria, few patients un- DBP.13 To rule out the presence of rearrangements in dergo muscle biopsy. mtDNA, we performed a Southern blot analysis and a spe- Although defects of the mitochondrial respiratory chain cific PCR assay on muscle-derived DNA in all patients. Pa- have been described in several ALS patients, their patho- tient 7 showed multiple mtDNA deletions detectable by genic significance is unclear. Comi and colleagues33 de- Southern blot analysis. In this same patient, there were no scribed a patient with early onset and rapidly progres- mutations in PEO1, POLG, POLG2, ANT1,orOPA1. Se- sive motor neuron disease who harbored a heteroplasmic quence analysis of the entire mtDNA was performed in all microdeletion of the mtDNA-encoded subunit I of COX. patients belonging to the group with severe histochemical Rubio-Gozalbo et al42 reported on a child with spinal mus- COX deficiency. The nucleotide variations that were found cular atrophy, cardiomyopathy, and reduced COX ac-

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 Table. Characteristics of Seven Patients With Amyotrophic Lateral Sclerosis and Severe Oxidative Defects

Normal Patient No. Value, Characteristic Mean (SD) 1 2 3 4 56A6B7 Sex/age at M/62 M/40 M/27 M/59 M/32 M/32 M/33 M/75 biopsy, y Neurological Walking Fasciculations Weakness of Weakness of Fasciculations Weakness and At second Weakness of signs difficulties in the whole lower limbs; lower limbs; and bilateral atrophy of biopsy: much upper and at biopsy for 1 y; weak body for 2 y; fasciculations; cramps and foot drop for lower limbs more atrophy lower limbs; and lingual dysphagia for fasciculations 1 y; cramps for 4 y; right and dysphagia; hypotrophic hypotrophy; 2mo for 1 y; high foot drop the weakness of fasciculations in distal episodic CK serum next year, upper and limbs; had dysphagia; levels then lower limbs; high CK high CK fasciculations dysphagia serum levels serum levels in the whole and body; used dysphonia wheelchair for 1y Histological Neurogenic Neurogenic Severe Moderate Great muscle Severe Very severe Moderate morphology aspect with aspect with neurogenic neurogenic fibers neurogenic neurogenic neurogenic atrophic and angular aspect; great aspect with variability; aspect; very aspect; many aspect with angular fibers and muscle fiber scattered many little atrophic and groups of angular fibers; large moderate variability; a angular fibers groups of angular very atrophic fibers, fiber type 1 muscle fiber lot of atrophic and great atrophic and fibers; many and angular moderate grouping variability fiber groups muscle fiber angular type II fibers; muscle fiber variability fibers; large grouping almost all the variability; fiber type II fibers are many type II grouping type II groupings Respiratory chain activity, nM/min/mg of proteinc CI 15.7 (3.2) 18.4 16.2 5.0b 10.8 17.1 3.9b 4.2b 7.3b CII 9.9 (2.3) 10.1 10.6 2.8b 8.9 14.5 4.2b 3.2b 3.5b CIϩIII 43.3 (12) 36.5 22.7b 31.3b 34.9 34.4 38.2 46.2 22.6b CIIϩIII 12.8 (3.5) 13.9 7.4b 2.5b 10.5 13.5 3.5b 3.1b 4.6b CIV 39.75 (7.5) 52.3 32.7 11.8b 44 51.2 12.1b 12.6b 13.1b CS 129.0 (17.8) 173 180.9 111.8 101.5 99.9 113 71.5 141.4 mtDNA Negative Negative Negative Negative Negative Negative Negative Multiple Southern deletionsa blot results SOD1 Negative p.Q22Rb Negative Negative Negative Negative Negative Negative mutation TARDBP Negative Negative Negative p.A382Tb Negative Negative Negative Negative mutation mtDNA Normal Normal Normal Normal Normal Normal Normal Normal sequence Age 61 38 27 58 31 28 28 74 at onset, y

Abbreviations: C, complex of mitochondrial respiratory chain; CK, creatine kinase; CS, citrate synthase; mtDNA, mitochondrial DNA. a Sequence analysis of the coding regions of the following genes was negative: ANT1, PEO1, POLG1, POLG2, and OPA1. b Activities reduced by at least 30% of the normal mean values. c Mean (SD) values of the respiratory chain complex activities are normalized to CS.

tivity in muscle and fibroblasts; the absence of muta- mellitus, and cardiac arrhythmia who died of cardiac ar- tions in SMN1 and the presence of cardiomyopathy rest during the study. Autopsy results showed a normal suggest that this child might have had mutations in the cortex and corticospinal tracts but numerous COX- COX assembly gene SCO2. Finsterer43 described a mother deficient motor neurons. Sequencing of mtDNA showed and 2 daughters with symptoms compatible with ALS. a heteroplasmic mitochondrial transfer RNAIle mutation All 3 patients showed COX-negative muscle fibers, ul- different from that reported by Finsterer.43 Recently, trastructurally abnormal mitochondria, and no muta- Hirano and colleagues34 reported on a 65-year-old man tions in SOD1, but harbored 3 mtDNA mutations, one with typical ALS in whom muscle biopsy specimens in the transfer RNAIle gene, a second in the cytochrome showed 10% ragged red fibers, which were unexpect- b gene, and the third in the adenosine triphosphatase 6 edly abundant even at his age and suggested mitochon- gene. Fetoni et al44 described a man with monomelic amy- drial dysfunction. otrophy, diabetes mellitus, and COX-negative ragged red Given the provocative but still anecdotal evidence of fibers in the muscle biopsy. Other family members had mitochondrial involvement in muscle of patients with ALS, maternally inherited hearing loss. A mutation in the trans- we decided to review systematically the remarkable col- fer RNASer(UCN) gene of mtDNA was found in the patient lection of muscle biopsy specimens from patients with and in a maternal niece. Borthwick et al45 described a pa- typical ALS in our possession. Our histochemical data tient with clinical features suggestive of ALS, diabetes in 50 patients showed variably severe but unequivocal

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 COX deficiency in 46% of patients, an unexpectedly high The partial oxidative defect in patients with patho- proportion. Patients can be divided into 4 groups ac- genic mutations in SOD1 (patient 2) and TARDBP (pa- cording to COX histochemical activity: 27 had normal tient 4) further suggests that the COX deficiency may be COX activity; and 8 had mild, 8 had moderate, and 7 had secondary to identifiable genetic defects. This concept severe COX deficiency. When respiratory chain enzyme is reinforced by the molecular findings in the single pa- activities were measured spectrophotometrically in whole tient with muscle multiple mtDNA deletions, though we muscle extracts, only 3 patients showed combined en- could not identify the nuclear gene responsible for this zyme defects (Table, patients 3, 6, and 7). In patient 6, defect of intergenomic signaling. COX activity worsened in a second biopsy taken 9 months after the first. The fact that respiratory enzyme analyses Accepted for Publication: December 18, 2009. in 4 of 7 patients with severe histochemical COX defi- Author Affiliations: Department of Neurological Sci- ciency were normal is not surprising because biochem- ences, “Dino Ferrari” Center, Università degli Studi di istry does not accurately reflect the extent of histochemi- Milano, Scientific Institute for Research and Treatment cal changes, which are nonetheless significant.34 (IRCCS) Foundation Ospedale Maggiore Policlinico, Man- Notably, among the patients with severe COX defi- giagalli and Regina Elena (Drs Crugnola, Lamperti, Luc- ciency, 1 (patient 2) had a mutation in SOD1 (c.65AϾG, chini, Ronchi, Peverelli, Prelle, Sciacco, Fassone, Corti, p.Q22R),41 another (patient 4) had a mutation in TAR- Bresolin, Comi, and Moggio; Ms Bordoni; and Mr For- DBP,13 whereas another patient (patient 7) with bio- tunato); and Department of Neurology and Laboratory chemically confirmed COX deficiency had multiple of Neuroscience, “Dino Ferrari” Center, Università de- mtDNA deletions detectable by Southern blot analysis, gli Studi di Milano, IRCCS Istituto Auxologico Italiano though sequencing all the known nuclear genes associ- (Dr Silani), Milan, Italy; and Department of Neurology, ated with multiple mtDNA deletions failed to show any Columbia University Medical Center, New York, New pathogenic mutation. The patient’s advanced age may ex- York (Dr Di Mauro). plain this finding, at least in part46; however, a substan- Correspondence: Maurizio Moggio, MD, Department of tial proportion of mtDNA multiple deletion syndromes Neurological Sciences, Universita` degli Studi di Milano, is not assigned to any known gene.47 Therefore, the pos- IRCCS Foundation Ospedale Maggiore Policlinico, Man- sibility of a mutation in a yet unidentified gene cannot giagalli and Regina Elena, Padiglione Ponti, Via Fran- be ruled out. Vielhaber et al48 described multiple dele- cesco Sforza 35, 20122 Milan, Italy (maurizio.moggio tions of mtDNA in 1 patient with sporadic ALS and at- @unimi.it). tributed the mtDNA alteration to the decreased activity Author Contributions: Study concept and design: Crugnola, of membrane-associated mitochondrial Mn-superoxide Silani, Bresolin, and Moggio. Acquisition of data: Lam- dismutase activity, but did not study nuclear genes as- perti, Lucchini, Ronchi, Peverelli, Prelle, Sciacco, Bor- sociated with multiple mtDNA deletions. doni, Fassone, Fortunato, Corti, Silani, Bresolin, and Our data confirm that varyingly severe histochemi- Comi. Analysis and interpretation of data: Lamperti, Ron- cal COX deficiency is a common finding in skeletal muscle chi, Prelle, Sciacco, Bordoni, Fortunato, Corti, Silani, from ALS patients and does not correlate with age. In fact, Bresolin, Di Mauro, and Comi. Drafting of the manu- it is noteworthy that most patients with severe COX de- script: Lamperti, Ronchi, Prelle, Sciacco, Bordoni, For- ficiency by histochemistry (group 4) were in their 30s, tunato, Corti, and Comi. Critical revision of the manu- 1 was aged 40 years, another was in his 60s, and only 1 script for important intellectual content: Crugnola, Lamperti, was aged 75 years. Paradoxically, the patient with the most Lucchini, Peverelli, Prelle, Sciacco, Fassone, Corti, severe histochemical COX deficiency was one of the Silani, Bresolin, Di Mauro, Comi, and Moggio. Adminis- youngest in our cohort (32 years of age at disease onset). trative, technical, and material support: Lucchini, Ron- We failed to detect a general correlation between se- chi, Peverelli, Bordoni, Fassone, Fortunato, Silani, and verity of the oxidative defect and duration of the dis- Bresolin. Study supervision: Crugnola, Lamperti, Prelle, ease. Most patients underwent muscle biopsy within 1 Sciacco, Corti, Silani, Bresolin, Di Mauro, Comi, and year from the onset of symptoms. However, in the only Moggio. patient who underwent a second muscle biopsy, we did Financial Disclosure: None reported. find a correlation between worsening of the respiratory Funding/Support: This research received support from chain defect and severity of symptoms. Echaniz-Laguna Associazione Amici del Centro Dino Ferrari. Dr Comi re- et al49 twice performed biopsies in 7 patients with spo- ceived funding from research grant Progetti Ricerca In- radic ALS and showed worsening of complex IV defi- teresse Nazionale (PRIN) 2006 2006069034 from the Ital- ciency in the later biopsies. ian Ministry of University and Research for the project In 7 patients, the oxidative defect was severe enough entitled “An Integrated Approach to the Study of the Etio- to support the hypothesis that, at least in some cases, pathogenesis of Mitochondrial Disorders.” Dr Comi was mitochondrial dysfunction plays a role in the pathogen- also funded by the Italian Ministry of Health for the 2009 esis of the disease. Although aging can cause mitochon- project entitled “Mitochondrial Disorders: From Medi- drial changes50,51 and onset is typically late in ALS, it is cal Genetics to Molecular Mechanisms, Toward the De- important to note that most of our ALS patients were velopment of Therapeutic Strategies.” The Telethon Net- still young and that the changes we observed were work of Genetics Biobanks (Genetic Telethon Biobank much more pronounced than those described in No. 07001E) was the source of muscle, DNA, and cells healthy elderly individuals and were not present in age- used in this study. EuroBioBank project QLTR-2001- matched controls. 02769 is also gratefully acknowledged.

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