Clinical correlates of mitochondrial function in Huntington's disease muscle

Clinical Correlates of Mitochondrial Function in Huntington’sDisease Muscle

Christopher Turner, MRCP,1 J. Mark Cooper, PhD,1 and Anthony H. V. Schapira, MD, DSc1,2

1University Department of Clinical Neurosciences, Royal Free and University College Medical School,University College London, London, United Kingdom

2Institute of Neurology, University College London, Queen Square, London, United Kingdom

Abstract: Huntington’s disease (HD) is caused by an abnor-mally expanded CAG repeat in the IT-15 gene, which encodesa widely expressed protein called huntingtin. Abnormalities ofmitochondrial respiratory chain function, specifically complexII/III, have been identified in HD striatum and defects of energymetabolism have been demonstrated in vivo in skeletal musclein both symptomatic and presymptomatic HD patients. Wehave investigated respiratory chain function using histochemi-cal and biochemical methods in HD skeletal muscle from 12patients and compared these with 12 age and sex-matchedcontrols. The data from the HD patients were related to clinicalparameters of HD including the Unified Huntington’s DiseaseRating Scale (UHDRS). There were positive correlations be-tween CAG repeat years (a product of CAG repeat length andage) and both motor (P � 0.002) and cognitive (P � 0.01)

scores of the UHDRS. There was no significant difference inthe activities of complexes I to IV compared to age-matchedcontrols. However, there were significant correlations for indi-vidual HD complex II/III activities with disease duration (P �0.017), repeat years (P � 0.032), and cognitive scores (P �0.019). There was also evidence from ultrastructural studiesthat inclusion formation may occur in HD muscle. These resultsprovide additional evidence that mutant huntingtin influencesmitochondrial complex II/III function in non-neuronal tissue(skeletal muscle) and suggest that muscle may be a potentialmarker of disease progression in HD. © 2007 MovementDisorder Society

Key words: Huntington’s disease; mitochondria; muscle;UHDRS; complex II/III

Huntington’s disease (HD) is a late-onset neurodegen-erative disease characterized clinically by a progressivemovement disorder and dementia. It is an autosomaldominant condition caused by the expansion of a CAGrepeat in exon 1 of the IT-15 gene encoding a 348-kDaprotein named huntingtin.1 There is widespread neuro-pathology, including neuronal loss and astrocytosis, inthe HD brain, which is most severe in the caudate nu-cleus where the medium �-aminobutyric acid (GABA)-ergic spiny neurons are particularly affected. The puta-men and cortex are less involved and the cerebellum is

relatively spared. Huntingtin is widely expressed through-out the brain and peripheral tissues and the relatively selec-tive loss of neurons remains unexplained. Intranuclear andcytoplasmic inclusions, as well as dystrophic neurites,which are immunocytochemically positive for ubiquitin andN-terminal epitopes of huntingtin, have been described inbrain regions affected by neuronal loss.2,3

The pathogenesis of HD is poorly understood butseveral hypotheses are currently under investigation. Inkeeping with other triplet repeat diseases, the pathogenichallmark is that the length of the CAG repeat correlatesstrongly with certain clinical features, such as age ofonset, but additional epigenetic and environmental fac-tors may also be important.4 At a molecular level, therole of intranuclear inclusions is still uncertain and thereis conflicting evidence that inclusions may be patho-genic, an epiphenomenon, or protective.5 Proteosomaldysfunction, transcriptional dysregulation, and abnormalvesicular trafficking have all been described and may bethe direct effect of mutant huntingtin or secondary to

No author has any conflict of interest.*Correspondence to: Professor A. Schapira, University Department

of Clinical Neurosciences, Royal Free and University College MedicalSchool, Rowland Hill Street, London NW3 2PF, United Kingdom.E-mail: [email protected]

Received 21 November 2006; revised 15 March 2007; accepted 28March 2007

Published online 7 June 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mds.21540

Movement DisordersVol. 22, No. 12, 2007, pp. 1715-1721© 2007 Movement Disorder Society

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downstream toxic effects of the mutant protein.6 The lossof the possible protective role of wild-type huntingtin hasnot been excluded as contributing to the disease process.7

It is a well recognized but unexplained observationthat HD patients have relatively severe muscle wasting,especially late in the disease, which is neither explainedby an inadequate calorie intake nor excessive energyexpenditure due to the movement disorder.8 In this con-text, it is of note that there is increasing evidence for therole of defects in energy metabolism in HD, especiallymitochondrial dysfunction.9 There is a severe deficiencyin the activity of complexes II/III in postmortem HDstriatum.10,11 Magnetic resonance spectroscopy (MRS)using 31P has demonstrated a defect of ATP synthesis inskeletal muscle in vivo in symptomatic and presymptom-atic HD patients12 that correlates with the length of theCAG repeat.13 A complex I deficiency has also beendescribed in three of four muscle biopsies from symp-tomatic HD patients.14 HD lymphoblasts have demon-strated increased mitochondrial depolarization in re-sponse to cyanide, a complex IV inhibitor, but not inresponse to complex I, II, or III inhibitors.15 The degreeof depolarization was correlated with the CAG repeatlength. There is an inverse relationship between CAGrepeat length and [ATP/ADP] in HD lymphoblasts, dem-onstrating a regulatory role for the polyglutamine tract inoxidative phosphorylation.16 Therefore, there is evidencefor widespread peripheral, as well as CNS, defects ofmitochondrial function in HD and this supports a directrole for an expanded huntingtin CAG repeat causingmitochondrial dysfunction in HD independently ofexcitotoxicity.

To investigate these changes in HD skeletal muscle,we have analyzed mitochondrial respiratory chain activ-ities in 12 HD patients biopsied at various disease stages,as well as 12 age and sex-matched controls. These resultswere related to specific clinical parameters within theUnified HD Rating Scale (UHDRS) as well as age,disease duration, and repeat length. Our results demon-strate that levels of activity of skeletal muscle complexII/III correlate with cognitive scores, age, a compound ofage and CAG repeat length (repeat years) and diseaseduration.

METHODS

Patient Details

These studies were performed with the approval of theRoyal Free Hospital Research Ethics Committee. A mus-cle biopsy from the left vastus lateralis was performed on12 HD patients and 12 “controls.” The “control” biopsieswere taken as a diagnostic procedure from patients with

nonspecific muscle symptoms, and showed no clinicalevidence of a mitochondrial or neurodegenerative dis-ease. Control biopsies were analyzed from vastus latera-lis to match exactly the source for the HD patients. Othermuscles may vary in their proportion of type I and IIfibers and in their mitochondrial assays.

The severity of the HD patient’s disease was ratedclinically using the UHDRS.17 The motor subscale wasscored out of 124 with increasing scores representingincreasing disease severity. The cognitive subscale wasthe total score of the combination of a verbal fluency test,symbol digit modality test, and Stroop interference testwith higher scores representing better cognitive function.The behavioral subscale was out of a total of 80 withincreasing scores representing worse behavior. The func-tional checklist was scored between 25 and 50 with ascore of 25 representing no effect on daily function anda score of 50 representing full functional dependence ofthe patient. The independence subscale was a percentagescore with 100% representing full independence and10% representing total bed care and tube feeding. Thefunctional capacity was scored from 0 to 13 with higherscores representing higher functional dependence. TheCAG repeat number was quantified by comparative PCRwith known repeat lengths by the Department of Neuro-genetics, Institute of Neurology, London, UK and theKennedy-Galton Centre, Harrow, UK.

Histochemistry, Immunocytochemistry, andUltrastructural Studies

Cytochrome oxidase (COX) and succinate dehydroge-nase (SDH) histochemical stains and immunocytochem-ical analyses were performed on frozen muscle sectionsaccording to standard procedures.18 The following anti-bodies were used; a polyclonal antibody to the first nineamino acids of huntingtin (Ab 675, a gift from Dr. L.Jones, Cardiff, diluted 1 in 1,000, incubated overnight at4°C) and a monoclonal antibody to ubiquitin (Chemicon,1 in 300, 4°C overnight). These antibodies were detectedwith either an antimouse or an antirabbit HRP conju-gated secondary antibody (DAKO, diluted 1 in 1,000 for1 hour). Antibodies were visualized with diaminobenzi-dine (DAB). Electron microscopy was performed onresin embedded sections of HD muscle.

Spectrophotometric Analyses

The spectrophotometric analyses of aconitase, citratesynthase, and complexes I, II/III, and IV activities wereperformed on muscle homogenates as previously de-scribed.19,20 All assays were performed blinded and theenzyme activities were expressed as nmols/min/mg ex-cept complex IV, which was expressed as the first order

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Movement Disorders, Vol. 22, No. 12, 2007

rate constant K (/min/mg). Protein was determined by thebicinchoninic acid (BCA) protein assay using bovineserum albumin as the standard.21 Statistical analyseswere performed using the unpaired two-tailed Mann–Whitney U test and the Spearman correlation test.

RESULTS

Clinical Scores

The controls were sex (5 males in each group) andage-matched to the HD patients (control mean 47.9 �11.8 years; HD mean 46.7 � 11.1 years). The creatinekinase and neurophysiological studies were within nor-mal limits in all HD patients and controls. The controlmuscle biopsies showed no evidence of any pathology onextensive histochemical and immunohistochemical ex-amination. The controls and HD patients had no evidenceof focal muscle weakness and had similar levels ofmobility, although this was not quantified. The UHDRSclinical scores were collected for each of the 12 HDpatients (Table 1). The patients were taken from a widespectrum of disease severities ranging from asymptom-atic in two patients, with respect to the motor score, toseverely affected and unable to perform the cognitivetests in one patient. There was a significant positivecorrelation between motor scores and age (r � 0.698,P � 0.01) and a significant negative correlation betweencognitive scores and age (r � �0.597, P � 0.04) (Fig.1). There was no correlation between cognitive or motorscores with CAG repeat length (data not shown). Toallow for the role of increased disease duration upon theclinical score, the cognitive and motor subscales of theUHDRS were related to the product of the age of thepatient and their number of CAG repeats or “CAG repeatyears.” This demonstrated a highly significant correlationbetween “repeat years ” and both motor (r � 0.823, P �0.002) and cognitive (r � �0.727, P � 0.01) scores(Fig. 1).

Histochemistry, Immunocytochemistry, andUltrastructural Studies

There were no detectable abnormalities on musclehistology and histochemistry in any patient or controlwith the exception of HD Patient 12. This patient was theoldest (66 years) and one of the more severely affectedpatients. There was an excess of SDH positive and COXnegative fibers (10%), beyond the number that mighthave been expected for her age (Fig. 2). Immunocyto-chemical analysis did not clearly demonstrate any pe-rinuclear inclusions and the 675 antibody to huntingtinalso did not demonstrate any inclusion formation. How-ever, ultrastructural studies, in HD Patient 10, who was alsoseverely clinically affected, demonstrated nuclear accumu-lations of electron dense material (Fig. 3) consistent withintranuclear inclusion formation. No intranuclear inclusionswere seen in the muscle of the controls in this study or inadditional age-matched controls (data not shown).

Spectrophotometric Analyses

The citrate synthase activities were not significantlydifferent between HD patients and controls (Table 2). Ona standard “group to group” analysis, the activities ofcomplexes I, II/III, and IV in the HD patients were notstatistically different from the control group, whetherexpressed as a specific activity or as a ratio with citratesynthase activity (Table 2).

Given the diversity of the HD group with respect totheir age, disease duration, and clinical status at analysis(Table 1), correlations were performed to determinewhether reductions in mitochondrial activities were as-sociated with specific clinical markers of disease pro-gression. Complex I, II/III, and IV citrate synthase ratioswere therefore correlated with patient age, CAG repeatyears, disease duration, and the component clinicalscores within the UHDRS. There was no decline inrespiratory chain CS ratios over the age range studied inthe control patients (data not shown). In the HD patients,complex II/III:CS ratios correlated positively with thecognitive subscale of the UHDRS (r � 0.676, P �0.019), and negatively with the age of the patient (r ��0.647, P � 0.026, data not shown), disease duration(r � �0.681, P � 0.017) and with repeat years (r ��0.631, P � 0.032) (Fig. 4) There was a trend for themotor subscale to be positively correlated with II/III:CSbut this did not reach significance (data not shown). Ofthe four subscales of the UHDRS that measure behav-ioral and functional change, there was a significant neg-ative correlation between the functional checklist (r ��0.656, P � 0.024), and a positive correlation betweenthe independence scale (r � 0.593, P � 0.046) withcomplex II/III:CS (data not shown).

TABLE 1. Summary of HD patient clinical details andUHDRS scores

Clinical parameter Range Mean S.D.

Age 33–66 46.7 11.1Disease duration (years) 0–10 3.69 3.28CAG repeat 40–49 44.2 2.48UHDRS scales

Motor 0–69 36.5 29.1Cognitive 0–260 165 78.1Behavior 0–18 5.92 6.1Functional checklist 25–50 31.8 8.67Independence 50–100 81.3 22.6Total functional capacity 1–13 8.25 5.55

CLINICAL CORRELATES OF MITOCHONDRIAL FUNCTION 1717


DISCUSSION

Mitochondrial function analyses on HD postmortembrain have demonstrated a severe defect of mitochondrialcomplex II/III activity in the striatum.10,11 Studies havealso shown abnormal bioenergetics in the HD brain invivo, with elevated lactate and reduced N-acetyl aspar-tate within the striatum.22 Huntingtin expression is notrestricted to the CNS and has been documented in skel-

etal muscle and myoblast cultures from the R6/2 trans-genic mouse model of HD.23 Using MRS, we havepreviously reported a defect of mitochondrial ATP syn-thesis capacity in HD skeletal muscle in vivo, whichcorrelated inversely with the length of the CAG repeati.e. the longer the repeat, the worse the defect.13 Further-more, gene expression profiles of R6/2 HD transgenicmice and HD skeletal muscle have also demonstrated

FIG. 1. Correlation between HD patient age (A, B) and repeat years (C, D) versus cognitive and motor scores on UHDRS in HD patients. The linesrepresent linear regression lines and statistical analyses are Spearman’s correlation coefficients (r) and statistical significance (P).

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changes with a transition from fast to slow-twitch fibersin the mutants, suggesting an adaptive response to mus-cle that is less dependent on oxidative phosphorylation.24

Our study demonstrates that individual HD skeletalmuscle complex II/III activity correlates with clinicaldisease progression and with the cognitive score on theUHDRS, two measures of functional status, the age ofthe patient, disease duration and the repeat years score.In contrast to Arenas et al.,14 who found a complex Idefect in HD muscle compared to age-matched controls,we did not find specific respiratory chain deficits com-pared to age-matched controls. This difference may beexplained by the size of the respective studies (Arenas etal.14 n � 3, Turner et al. n � 12) and the broad range ofclinical severities within the HD patients in our study.Skeletal muscle mitochondrial respiratory chain activityhas been shown to decline with increasing age,25 al-though, a wide age range is required to demonstrate thiseffect. The controls in our study did not demonstrate areduction in mitochondrial respiratory chain activity.

This probably reflected the relatively narrow age rangeof the control group (47.9 � 11.8 years). However, overa similar age range (46.7 � 11.1 years), there was asignificant negative correlation between age and com-plex II/III:CS ratios in the HD group. The specificity ofthe complex II/III:CS changes observed with age in HDbut not the control samples mitigates against a non-specific age-related effect which would be expected toinclude complexes I and IV, and suggests that mutanthuntingtin either directly affects skeletal muscle complexII/III or is a secondary consequence of disease progres-sion unrelated to huntingtin expression e.g. progressiveimmobility The correlations of complex II/III:CS ratioswith repeat years, disease duration, the cognitive sub-scale and two measures of functional status in the UH-DRS with complex II/III:CS ratios, in the absence ofage-related changes in the control samples, also suggeststhat this is not simply related to age but a consequence ofdisease progression and increasing CAG repeats.

Neuropsychological impairment in HD has beenshown to occur 2 years prior to manifestation of themotor features.26 The significant correlation with thecognitive subscale and the trend, albeit not significant,with the motor subscale and complex II/III activity mayalso be because the cognitive changes are more sensitiveand associated with earlier stages of the disease than themotor features. This may reflect the sensitivity of the

FIG. 2. Histochemical staining of serial sections of muscle from HDPatient 12 for succinate dehydrogenase (SDH or Complex II) (A) andcytochrome oxidase (COX or complex IV) (B). A SDH positive andCOX negative fiber can be seen in the center (see *) indicative ofmitochondrial dysfunction.

FIG. 3. Ultrastructural detail of HD muscle from Patient 10 in TableII. The electron dense mass (arrow) points to an inclusion within thenucleus of a muscle cell.

TABLE 2. Mitochondrial citrase synthase ratios forcomplexes I, II/III, and IV, and enzyme specific activities for

citrate synthase (CS nmol/min/mg) in HD and controlpatient’s muscle samples

Age I II/III IV (�103) CS

Patient1 33 0.281 0.421 40.5 1062 35 0.260 0.288 21.3 84.93 36 0.264 0.220 14.2 1324 36 0.291 0.328 20.7 1055 37 0.268 0.293 18.1 1306 46 0.347 0.167 38.3 45.67 51 0.222 0.147 16.1 1028 52 0.211 0.240 10.6 1049 56 0.280 0.268 28.0 95.310 56 0.293 0.165 16.9 84.911 57 0.281 0.193 19.5 10412 66 0.142 0.200 17.6 83.1Mean 46.8 0.262 0.244 21.8 98.1SD 11.1 0.0511 0.0796 9.23 22.7SEM 3.10 0.0147 0.0230 2.67 6.56

ControlMean 47.9 0.281 0.245 21.7 97.2SD 11.8 0.0854 0.106 7.71 35.0SEM 3.39 0.0247 0.0306 2.23 10.1P values 0.885 0.443 0.887 0.630 0.671

Patient and control groups were compared using Mann–Whitney Utest and P values stated.



respective assessments (cognitive versus motor) or beadaptive changes within muscle that ameliorate the bio-chemical effects of mutant huntingtin expression. Thismay be seen in the gene expression profile changesdescribed above.24

Two clinical factors that are associated with clinicalprogression in HD include the number of CAG repeatsand the age of the patient.4 We attempted to incorporatethese factors to reflect disease severity by correlating theproduct of age and CAG repeats or “repeat years” withclinical parameters. The cognitive and motor subscalesof the UHDRS correlated substantially more signifi-cantly with repeat years than age alone (Fig. 1). CAGrepeats did not correlate with these clinical scales (datanot shown). This suggests that repeat years may be abetter objective marker of disease progression than agealone although the exact mathematical relationship be-tween age, CAG repeats, and disease status has yet to bedetermined.

The present study demonstrated that huntingtin inclu-sion formation may occur in skeletal muscle and sug-gests that non-CNS tissues can also be affected by in-clusion formation as observed in the R6/2 mouse6 andR6/2 myoblasts.23

We demonstrated that a reduction in complex II/IIIfunction correlates with disease progression on severalclinical scales independently of an age-related phenom-enon. We would speculate that a progressive reduction incomplex II/III activity occurs in HD patient muscle andthat this could cause a sequence of biochemical eventsthat may induce damage, as is proposed for HD stria-tum.27,28 An excitotoxic mechanism in HD brain hasbeen suggested to lead to an increase in nitric oxide,which inhibits complex IV and subsequently complex

II/III via free radical production.28 The mechanism bywhich mutant huntingtin leads to a specific reduction incomplex II/III activity in muscle remains unknown butthere is now accumulating evidence that dysfunction incomplex II/III is an important step in HD pathogene-sis.10,28 A recent study in a yeast cell model suggestedthat a disturbance in mitochondrial trafficking by solubleor insoluble mutant huntingtin aggregates was associatedwith free radical production and a specific reduction incomplex II/III activity.29 There was subsequent recoveryof respiratory chain function by quenching with resvera-trol, a free radical scavenger.30 A reduction in complexII/III activity with disease progression could putativelybe associated with a secondary reduction in the levels ofubiquinone. The CARE HD study investigated the use ofremacemide, an antiglutaminergic, and coenzyme Q10 inthe treatment of HD over 2.5 years.30 There was a smallnonsignificant reduction in the rate of functional declinein the patients taking coenzyme Q10 over this period. Iflevels of ubiquinone in HD muscle are subsequentlyfound to be reduced, then a further clinical trial ofcoenzyme Q10 over a longer period would be justified.

Our data support the hypothesis that mitochondrialdysfunction and specifically complex II/III activity areinvolved in the sequence of HD pathogenesis and thiscan be correlated with the worsening of several clinicalparameters. Prolonged treatment with ubiquinone andfree radical scavengers may ameliorate the progressiveloss of complex II/III activity and modify diseaseprogression.

Acknowledgments: Thanks go to Jane Workmann (RoyalFree Hospital, London) for her help with muscle processing,immunocytochemistry, and histochemistry and to Dr. D.

FIG. 4. Relationship between complexes II/III:CS ratios and cognitive subscale of UHDRS, repeat years and disease duration in HD patients. Valuesrepresent mean � SEM, the lines represent linear regression lines, and statistical analyses are Spearman’s correlation coefficients (r) and statisticalsignificance (P).

1720 TURNER ET AL.


Landon (Institute of Neurology) for his help with the ultrastruc-tural study. This work was supported by the Wellcome Trust.CT was in receipt of a Wellcome Training Fellowship.

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Clinical correlates of mitochondrial function in Huntington's disease muscle