Oxidative Stress in Skin Fibroblasts Cultures of Patients with Huntington’s Disease
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- del Hoyo, P., García-Redondo, A., de Bustos, F. et al. Neurochem Res (2006) 31: 1103. doi:10.1007/s11064-006-9110-2
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Oxidative stress and mitochondrial dysfunction should play a role in the neurodegeneration in Huntington’s disease (HD). The most consistent finding is decreased activity of the mitochondrial complexes II/III and IV of the respiratory chain in the striatum. We assessed enzymatic activities of respiratory chain enzymes and other enzymes involved in oxidative processes in skin fibroblasts cultures of patients with HD.
We studied respiratory chain enzyme activities, activities of total, Cu/Zn- and Mn-superoxide-dismutase, glutathione-peroxidase (GPx) and catalase, and coenzyme Q10 (CoQ10) levels in skin fibroblasts cultures from 13 HD patients and 13 age- and sex-matched healthy controls.
When compared with controls, HD patients showed significantly lower specific activities for catalase corrected by protein concentrations (P < 0.01). Oxidized, reduced and total CoQ10 levels (both corrected by citrate synthase (CS) and protein concentrations), and activities of total, Cu/Zn- and Mn-superoxide-dismutase, and gluthatione-peroxidase, did not differ significantly between HD-patients and control groups. Values for enzyme activities in the HD group did not correlate with age at onset and of the disease and with the CAG triplet repeats.
The primary finding of this study was the decreased activity of catalase in HD patients, suggesting a possible contribution of catalase, but not of other enzymes related with oxidative stress, to the pathogenesis of this disease.
KeywordsHuntington’ diseaseOxidative stressMitochondrial respiratory chainGlutathione-peroxidaseSuperoxide-dismutaseCoenzyme Q10CatalaseEtiopathogenesis
Huntington’s disease (HD) is the more frequent cause of hereditary chorea. It is an autosomal dominant neurodegenerative disease caused by an expanded CAG repeat in the IT-15 gene, located at the short arm of the chromosome 4. The abnormal gene encodes the mutant protein huntingtin, characterized by an expansion of polyglutamines in the N-terminal [1–3].
The mechanism by which huntingtin causes neurodegeneration is not well known, although oxidative stress and mitochondrial dysfunction should play a role. There have been found increased levels of oxidative damage products such as malondialdehyde 8-hydroxydeoxyguanosine, 3-nitrotyrosine, and hem-oxygenase in areas of degeneration in HD brain, and increased free radical production in some animal models [4–7].
It is very interesting that 3-nitropropionic acid, an irreversible inhibitor of the complex II of the mitochondrial respiratory chain, induces dose and age-dependent neurodegeneration of the striatum, hippocampus and thalamus in rat and of caudate-putamen in non-human primates and humans, which replicates many of the characteristic histological and neurochemical features of HD [8–10]. Nitropropionic acid is able to induce oxidative stress in the striatum , and triggers a reduction in glutathione content and catalase and glutathione-peroxidase (GPx) activities , and an enhancement in superoxide-dismutase (SOD) activity, in striatal nucleus synaptosomes of rats . Intrastriatal injection of the reversible inhibitor of complex II malonate, also induces age-dependent striatal lesions .
Most studies on mitochondrial respiratory chain function in the brain of patients with HD showed decreased activity of complexes II/III and IV [15, 16]. GPx activity has been found normal in the brain of patients with HD . Loomis et al.  found normal SOD activity, and Browne et al.  described a slight reduction of cytosolic and normality of particulate SOD in the parietal cortex and cerebellum of HD patients.
GPx activity is normal in transgenic models of HD  but mice deficient in cellular GPx have shown increased vulnerability to 3-nitropropionic acid and malonate . Santamaría et al.  found increased SOD activity in young mice transgenic for the HD mutation, but decreased in transgenic old mice and after 3-nitropropionic acid intrastriatal injections. Hansson et al.  found normal SOD activity in the striatum of transgenic HD mice.
Coenzyme Q10 (CoQ10) is the electron acceptor for mitochondrial complexes I and II and a powerful antioxidant . Shults et al.  reported correlation between mitochondrial CoQ10 levels and activities of complexes I and II/III. Andrich et al.  found decreased serum CoQ10 levels in untreated HD patients when compared with treated HD patients and controls. To our knowledge, brain CoQ10 levels have not been measured in HD brain.
The aim of this study was to assess the enzymatic activities of respiratory chain enzymes and other enzymes involved in oxidative processes in skin fibroblasts cultures of patients with HD. The study was carried out in skin fibroblasts because the specimens were easily accessible and should be free of influence from medication, environmental hazards and other possible factors contributing to oxidative stress.
Patients and controls
Clinical data of Huntington’s disease and control patients groups
Huntington’s disease (n = 13)
Controls (n = 13)
8 M/5 F
6 M/7 F
Age at onset HD
Duration of HD
The following exclusion criteria were applied both to patients and controls: (A) Ethanol intake higher than 80 g/day in the last 6 months. (B) Previous history of chronic hepatopathy or diseases causing malabsorption. (C) Previous history of severe systemic disease. (D) Atypical dietary habits (diets constituted exclusively by one type of foodstuff, such as vegetables, fruits, meat, or others, special diets because of religious reasons, etc) (F) Intake of drugs which modify lipid absorption. (G) Therapy with vitamin supplements in the last 6 months.
Skin fibroblast cultures
Human skin fibroblasts were obtained from the dorsal region of the upper arm of each HD patient or control. Fibroblasts from the biopsy specimens were cultured in Dulbecco’s modified Eagle’s medium containing penicillin (100 UI/ml), streptomycin (100 mg/dl), L-glutamine (4 mM) and supplemented with heat-inactivated foetal calf serum at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were grown to confluence, harvestested by trypsinization at 37°C, washed with culture medium and resuspended with phosphate buffer 20 mM, and then sonicated to obtain the cell homogenate. Care was taken not to use cultures with a passage number greater than 12.
Respiratory chain enzymes assay
Respiratory chain enzymes and citrate synthase (CS) activities were measured by duplicate in a DU-68 spectrophotometer (Beckman), applying 35–150 μg mitochondrial protein per 1 ml test volume. Incubation temperatures were 30°C for NADH coenzyme Q oxidoreductase (complex I), rotenone-sensitive NADH cytochrome c reductase (complexes I + III), succinate cytochrome c reductase (complexes II + III), succinate dehydrogenase (complex II) and CS, and 38°C for cytochrome c oxidase (complex IV). Complex I was measured using the oxidation of NADH at 340 nm in 100 mM Tris–HCl pH 7.4, 500 mM sucrose, 2 mM EDTA, 5 mM KCN, 100 μM NADH, and 50 μM DB (2,3-dimethyl-5-decyl-6-methylbenzoquinone) . The activities of complexes II, I + III, II + III, IV, and CS were determined as reported elsewhere . Complex V was monitorized measuring 2.5 mM ATP extinction in a mean with 50 mM Hepes-Mg buffer at pH 8.0, 0.2 mM NADH, and phosphoenol-pyruvate 2.5 mM, and then adding 5 μl of pyruvate-kinase (10 mg/ml) and 10 μl of lactate-dehydrogenase (5 mg/ml) in presence of 10 μl of antimycin A (0.2 mg/ml in 50% ethanol). The oligomycin sensitive fraction was measured by adding 10 μl of oligomycin (0.2 mg/ml in 50% ethanol).
To correct for mitochondrial volume, all respiratory chain enzyme activities were normalized to the activity of CS, that was measured using the change of absorbance at 412 nm produced by the reaction of 100 μM DTNB (5–5′ dithio bis 2-nitrobenzoic acid) with the free coenzyme A formed by the condensation of acetylCoA (350 μg/ml) with 0.5 mM oxalacetate in a solution with a 75 mM Tris–HCl buffer at pH 8.0 and 0.1% triton X-100. Protein was measured by the method of Lowry et al. . Specific activities were expressed as nmol × min−1 × mg protein−1, and referred to the specific activities of CS to correct for mitochondrial volume. All chemicals were from Boehringer Mannheim (Boehringer Mannheim, Germany) and Sigma Chemicals (St. Louis, MO).
Glutathione-peroxidase, catalase and superoxide-dismutase isoenzymes determination
GPx specific activity was determined according to the method described by Flohé and Günzler  based on NADPH oxidation followed at 340 nm at 37°C.
Catalase activity was determined according to the method described by Aeby  based on H2O2 decomposition followed at 240 nm at room temperature. Catalase specific activity was determined by calculating the rate constant of a first order reaction.
Total and Mn-SOD activities were determined according to the method described by Spitz and Oberley  based on nitroblue tetrazolium reduction by superoxide radicals followed at 560 nm at room temperature. Mn-SOD was distinguished from cyanide-sensitive Cu/Zn-SOD by the addition of 5 mM NaCN. Cu/Zn-SOD activity was calculated by substracting the cyanide-resistant SOD activity from the total SOD activity. One unit of SOD activity is defined as the amount of enzyme that inhibits the reaction rate by 50%.
All enzyme activities were expressed as values normalized to total cellular protein.
Coenzyme Q10 determinations
Oxidized, reduced and total coenzyme Q10 levels were determined by high performance liquid chromatography with electrochemical detection. The method used was that of Langedijk et al.  with some modifications. The stationary phase was a reverse phase column (HR-80 RP-C18, 80 × 4.6 mm. ESA Inc). The mobile phase was prepared dissolving 7 g of NaClO4 · H2O in 1000 ml of methanol/propanol/HClO4 70%, 700.8:200:0.2 (v/v), and the flow rate was set at 0.8 ml/min. The programmed conditions for the electrochemical detector and the post-column valve were similar to those of Langedijk et al. . The system was entirely controlled by a computer (Kromasystem 2000, Kontron Instruments). Injections were made in a 50 μl injection valve (Model 7161, Rheodyne, Cotaty, USA) with a 100 μl syringe from Hamilton (Bonaduz, Switzerland). The calibration method used ubiquinone as external standard. The within-run coefficients of variation for CoQ10 and CoQH2 were, respectively, 5 and 3.2%, and the day to day precisions were 9.2 and 6.3%. CoQ10 recovery ranged between 88 and 93%. The measurements of CoQ10 were expressed in nmol/g of protein.
Results were expressed as mean ± SD. Statistical analysis was done by the SPSSWIN Packet (12.0 version) and included the two-tailed student’s t test, and calculation of Pearson’s correlation coefficient when appropriate.
Mean (SD) respiratory chain enzymes activities (expressed as nmol/min/mg protein) in skin fibroblast cultures of patients with Huntington’s disease (HD) and controls (CS = citrate synthase)
Huntington’s disease (n = 13)
Controls (n = 13)
Complex V (ATPase)/CS
Complex I + III/CS
Complex II + III/CS
Complex V (ATPase)/protein
Complex I + III/protein
Complex II + III/protein
Mean (SD) superoxide-dismutase (SOD), glutathione-peroxidase (GPx), and catalase activities (expressed as units/mg protein); and concentrations of coenzyme Q10 (expressed in nmol/g protein) in skin fibroblast cultures of patients with Huntington’s disease (HD) and controls (CS = citrate synthase)
Huntington’s disease (n = 13)
Controls (n = 13)
Oxidized Q10/Reduced Q10
The possible role of mitochondrial dysfunction in the pathogenesis of HD is not well established. The first study of respiratory chain enzymes activity performed on brain tissue was reported by Brennan et al. , who found a significant decrease in HD caudate mitochondrial respiration, cytochrome oxidase (complex IV) activity and cytochromes b, cc1 and aa3. Mann et al. found a marked decreased of the activity of complexes II/III [33, 34] and complex IV  in the caudate nucleus. Browne et al. [4, 5] found a marked reduction of complex II–III activity in both HD caudate and putamen and of complex IV in HD putamen. Finally, Tabrizi et al.  found decreased activity of aconitase and complexes II/III in the putamen and cortex.
Mitochondrial dysfunction in peripheral tissues is a controversial issue. Parker et al.  reported a marked decreased of complex I activity in the platelets of five patients with HD, but this finding that has not been confirmed in other recent study . Arenas et al.  reported a variable defect of complex I activity in muscle of patients with HD, which was correlated with the number of CAG triplets. Analysis of complex II/III activity in HD fibroblasts has been previously found normal .
A potentially interesting finding is the decreased membrane potential in lymphoblast mitochondria from patients with HD . There have been found increased concentrations levels of lactate in the cortex by using 1H magnetic resonance spectroscopy, and increased cerebrospinal fluid lactate/piruvate ratio, suggesting a deficit of energetic metabolism, in patients with HD . Finally, Lodi et al. , using 31P magnetic resonance spectroscopy, found a significant reduction of the ratio ATP/phosphocreatine + inorganic phosphate in the muscle from HD patients, suggesting a deficit of in vivo mitochondrial oxidative metabolism, and therefore, mitochondrial dysfunction.
It has been reported that treatment with coenzyme Q10 alone or combined with the NMDA antagonist remacemide improved motor performance, without prolonging survival, in mice models of HD [41, 42]. Despite that serum CoQ10 levels have been found decreased in untreated HD patients , and the description that treatment with coenzyme Q10 decreases cortical lactate concentrations, the results of chronic treatment with this drug in patients with HD were unsuccessful [43, 44].
The results of the present study showed that in HD patients the activity of catalase, corrected by protein concentrations in skin fibroblast cultures, was decreased. However, mitochondrial respiratory chain complexes activities, corrected by CS, and activities of total, Cu/Zn- and Mn-SOD, and GPx, corrected by protein concentrations, were also normal. In addition, enzyme activities and CoQ10 were not correlated with the clinical features of HD. When compared with controls, HD patients had similar serum oxidized, reduced and total CoQ10 levels in skin fibroblasts. These data do not rule out the possibility that there may be regional deficiencies of enzyme activities and of CoQ10 in some areas of the brain.
The significance of the decreased catalase activity found in the present study seems uncertain. To our knowledge, this enzyme has not been measured to date in brain tissue from HD patients. In addition, the references to catalase activity in experimental models of HD are scarce. Pérez-Severiano et al.  found a very low catalase activity in the striata of both control and transgenic for the HD mutation mice. These authors could not detect catalase activity in 11-week-old animals and in control 35-week old animals, although for 19-week-old animals the catalase activity was 11-fold lower in transgenic mice compared with controls. Túnez et al.  described a reduction in catalase activity in the striatum of rats induced by 3-nitropropionic acid.
Catalase descomposes hydrogen peroxide (H2O2) into H2O and O2. Although H2O2 is a weak oxidant, in the presence of some metals such as Fe2+ it can decompose to hydroxyl radical, a powerful reactive species that causes oxidative damage to DNA, proteins and lipid membranes. It has been suggested a possible influence of catalase activity on apoptosis as well. Catalase should have both an antiapoptotic and a proapoptotic role. The antiapoptotic action should be related with removing H2O2 (a mediator for the apoptotic program). However, the decrease in H2O2 by upregulation of catalase activity also supports apoptosis, possible because of a supportive role of H2O2 in a survival pathway . Interestingly, catalase has a protective action against neurotoxicity induced by 3-hydroxykinurenine (an endogenous neurotoxin that have been found to be increased in HD brain] in primary neuronal cultures from the striatum of rats . The metabolism of 3-hydroxykinurenin produces quinolinic acid as a metabolic intermediate, and injections of this acid into the striatum appear to replicate the histopathological and neurochemical features of the brains of HD patients . If this hypothesis is true, a deficiency of catalase could facilitate the neurotoxicity through this metabolic pathway.
In conclusion, the findings of the present study in skin fibroblasts cultures suggest a possible contribution of a deficiency of catalase (although it must be taken in consideration that it is unknown whether catalase activity in skin fibroblasts should reflect that of brain tissue), but not of other enzymes related with oxidative stress, to the pathogenesis of HD.
This work was supported in part by the grant of the Fondo de Investigaciones Sanitarias 99/0518.