Platelet mitochondrial evaluation during cytochrome c and dichloroacetate treatments of MELAS

Mitochondrion 5 (2005) 426–433 www.elsevier.com/locate/mito

Platelet mitochondrial evaluation during cytochrome c and dichloroacetate treatments of MELAS Kazutoshi Nakano*, Mikako Tarashima, Emiko Tachikawa, Naoko Noda, Tomohiro Nakayama, Kaori Sasaki, Eriko Mizoguchi, Mihoko Matsuzaki, Makiko Osawa Department of Pediatrics, Tokyo Women’s Medical University, 8-1, Kawada-cho, Shinjuku-ku, Tokyo 162 8666, Japan Received 23 March 2005; received in revised form 13 September 2005; accepted 12 October 2005 Available online 14 November 2005

Abstract We hypothesized that serial changes in platelet (PLT) mitochondrial enzyme (ME) activities might correspond to the effects of medications for mitochondrial encephalomyopathy and stroke-like episodes (MELAS). Cytochrome c and sodium dichloroacetate (DCA) were given to a 7-year-old girl with MELAS who had an A3243G mitochondrial DNA mutation. The effects were evaluated with whole PLT-ME assays, developed by our group, using a microplate-reader. During cytochrome c treatment, complex IICIII (IICIII), complex IV (IV) and citrate synthase (CS) activities showed gradual but statistically significant decrease. IICIII activity dropped below normal. IICIII/CS activity was initially below normal, followed by a transient improvement, then decreased again before the appearance of central nervous system symptoms. IICIII, IV, IICIII/CS and IV/CS activities reached their lowest levels in association with a stroke-like episode, then increased with DCA treatment. Our results suggest that progressive mitochondrial dysfunction may occur before the stroke-like episodes in MELAS and that DCA treatment may increase mitochondrial activities. Our whole PLT-ME assay system may be useful for serially evaluating mitochondrial functions in relation to clinical symptoms. q 2005 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Keywords: MELAS; Stroke-like episodes; Electron transport enzymes; Platelet; Sodium dichloroacetate; Cytochrome c

1. Introduction Abbreviations PLT, platelet; MELAS, mitochondrial encephalomyopathy and stroke-like episodes; DCA, sodium dichloroacetate; CS, citrate synthase; mtDNA, mitochondrial DNA; MRS, magnetic resonance spectrometry; NAA, N-acetyl asparate; SPECT, single photon emission computed tomography; PET, positron emission topography. * Corresponding author. Tel.: C81 3 3353 8111; fax: C81 3 5269 7338. E-mail address: [email protected] (K. Nakano).

Mitochondrial encephalomyopathy and stroke-like episodes (MELAS) is characterized clinically by intractable repeated stroke-like episodes with dementia. MELAS is associated with an A3243G mutation in mitochondrial DNA (mtDNA) (Goto et al., 1990), and decreased activities of complex I and/or complex IV among electron transport enzymes in muscle (Vilarinho et al., 1999) and platelets (PLTs) (Nakano et al., 1994).

1567-7249/$ - see front matter q 2005 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2005.10.002

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Although evaluating correlations between clinical symptoms and mitochondrial enzyme activities is important, no study has yet examined serial changes in mitochondrial electron transport enzyme activities, which occur in parallel with the stroke-like episodes and clinical progression. This is because it is very difficult to conduct repeat examinations of electron transport enzyme activities using muscle biopsy specimens. Assays for mitochondrial enzymes or complex I, complex IICIII, complex IV, and citrate synthase (CS) activities, with mitochondrial isolation from PLTs, have been examined in Parkinson disease (Shults et al., 1995; Haas et al., 1995; Shults et al., 1998). We developed miniaturized assays, using a microplate-reader in PLTs, without mitochondrial isolation, to assess the complex IICIII, complex IV and CS activities. As treatments with sodium dichloroacetate (DCA) (Kuroda et al., 1997; Saitoh et al., 1998; Pavlakis et al., 1998; Mori et al., 2004; Duncan et al., 2004) and cytochrome c (Nakagawa et al., 1996; Tanaka et al., 1997) are reportedly effective for mitochondrial encephalomyopathies, we evaluated the correlations between clinical features and serial changes in mitochondrial electron transport enzyme activities using our novel PLT assay system.

2. Subjects 2.1. Case report The patient, a 7-year-old girl, had an elder brother with Down syndrome. There was no family history of neurological disorders including her maternal relatives, except for her brother. Although she had developed normally until 3 years of age, she began to complain of fatigue after exercise and showed gradually worsening muscle weakness. As she was no longer able to maintain a standing posture by 5 years 1 month of age, she was admitted to the Department of Pediatrics, Second Hospital of Tokyo Women’s Medical University. As mitochondrial myopathy was suspected based on elevated lactate and pyruvate in blood and cerebrospinal fluid, she was referred to our department at 5 years 2 months of age. There was no central nervous system (CNS) involvement, i.e. she had no history of either headache or stroke-like episodes on admission. A

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quadriceps muscle biopsy revealed ragged red fibers on histochemical examination. Among mitochondrial electron transport enzyme activities, cytochrome c oxidase (complex IV) was decreased. MtDNA analysis of the patient and her mother revealed the A3243G mutation of MELAS.

2.2. Clinical course with cytochrome c and DCA treatments The patient, at 5 years, was unable to stand on admission to our hospital at the age. In an effort to improve her general fatigue and muscle weakness, injectable cytochrome c (Cytomack-P) was initiated at 5 years 2 months of age. This treatment was started at a dose of 0.5 mg/kg, with vitamin B1 (70 mg: 5 mg/kg) and vitamin B2 (30 mg: 2 mg/kg), daily for 4 weeks. Her general fatigue and muscle weakness improved and she was able to walk about a month after starting cytochrome c treatment. The cytochrome c injections were gradually shifted to oral cytochrome c (cytorest), which was given at a maximum dose of 120 mg/day with vitamin B1 (210 mg: 15 mg/kg per day) and vitamin B2 (60 mg: 4 mg/kg per day). During this gradual change from injected to oral medication, her muscle weakness was stable except for transient exacerbations with infection. However, a complex partial seizure occurred at the age of 5 years 3 months, and mild headache and photopsia, like those associated with migraines, occurred at 5 years 7 months, or 5 months after starting cytochrome c treatment. She suffered from severe protracted headaches and vomiting at the age of 5 years 11 months. Thus, the injectable cytochrome c was again administered daily at a dose of 0.5–1 mg/kg with vitamin B1 and vitamin B2, also daily, for a total of 12 days. The headache improved transiently 1 day after restarting injectable cytochrome c treatment, but severe headache recurred, despite continuous injection of cytochrome c, on the 10th day. We considered these symptoms to reflect a stroke-like episode and evaluated cytochrome c treatment as being ineffective for this manifestation of MELAS. DCA administration was started, in place of cytochrome c treatment, according to the protocol developed by the Department of Pediatrics, Tokushima University (Kuroda et al., 1997) with some modifications. Briefly, 50 mg/kg of DCA was administered orally three times, 12 h apart, followed by

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administration of 50 mg/kg per day of DCA in two divided doses. The DCA dose was controlled by monitoring blood lactate levels and blood concentrations of DCA. Vitamin B1 and vitamin B2 were also continuously administered orally at the same doses as those given before DCA administration: the dosages of vitamin B1 and B2 were 15 and 4 mg/kg per day, respectively. The severe headache promptly improved with two DCA administrations, while mild headache and photopsia appeared at 3–7-day interval during DCA treatment. Muscle strength was unaffected by DCA treatment. Informed consent was obtained from the patient’s parents prior to administration of DCA and cytochrome c. 2.3. Statistical analysis We statistically analyzed the results using regression analysis with Stat view software. A P-value less than 0.05 was considered significant.

3. Methods 3.1. Whole PLT mitochondrial enzyme assays 3.1.1. PLT isolation Five to 10 ml of venous blood are collected with a tourniquet and mixed at a 9:1 ratio with 3.8% w/v trisodium citrate. This mixture is centrifuged at room temperature at 200!g for 10 min. The PLT rich plasma (PRP) is collected and the residue is again centrifuged at 200!g for 10 min allowing further collection of PRP. The PRP is centrifuged at 1000!g at room temperature for 30 min producing a PLT pellet. This pellet is resuspended and washed twice in an equal volume of modified Tyrode’s buffer at pH 7.4 (150 mM NaCl, 5 mM HEPES, 0.55 mM NaH2PO4, 7 mM NaHCO 3, 2.7 mM KCl, 0.5 mM MgCl2 , 5.6 mM glucose, 1 mM EDTA(Di-K)) with centrifugation for 15 min at RT at 1000!g. Although the PRP pellet has a tendency to clump, it can be dissolved by resuspension and washing with Tyrode’s buffer. All subsequent procedures are carried out at 4 8C. The washed PLT pellet is gently resuspended in 6 ml of icecold buffer consisting of 250 mM sucrose, 1 mM EGTA, 10 mM Tris, and 1 mM ATP (pH 7.4),

followed by centrifugation at 3000!g for 10 min. The final pellet is suspended in ice-cold buffer mixed with glycerol and stored in liquid nitrogen. 3.1.2. Miniaturized mitochondrial assays Miniaturized assays were employed for the measurement of protein, complex IICIII (succinate cytochrome c oxidoreductase), complex IV (cytochrome c oxidase) and citrate synthase activities using an iEMS reader MF (Labsystem Corp.). The procedure developed for the miniaturized assays for complexes IICIII and IV is presented below. The citrate synthase (CS) assay was described previously (Shults et al., 1998). 3.1.2.1. Complex IICIII assay (succinate:cytochrome c oxidoreductase). The total working volume was 250 ml. The 25 ml diluted samples, which consisted of four different protein concentrations, were added to wells. Then, 175 ml of 71 mM potassium phosphate (pH 7.4), with 10.44 mg% NaCN and 2.84 mM rotenone, were added to each well. Twenty-five microliters of 200 mM succinate in water were also added. The mixture was preincubated in the dark for 10 min at 37 8C. The reaction was started by adding 25 ml of 1 mM cytochrome c (Sigma type IV) in water. The rate of the cytochrome c increase at 550 nm was measured for 4 min at 37 8C. For this calculation, the activity must be multiplied by a factor of 0.648 to correct for the length of the light path and microplate absorption. The coefficient of variation in complex IICIII reproducibility (assays were performed three) was 7.3%. 3.1.2.2. Complex IV (cytochrome c oxidase) assay. The total working volume was 225 ml. One hundred and seventy five microliters of 25 mM potassium phosphate (pH 7.0) were added to each well. Twentyfive microliters of diluted samples, with four different protein concentrations, were then added to the wells. After preincubation for 6 min at 37 8C, the reaction was initiated by adding 25 ml of 25 mM reduced cytochrome c in 20 mM potassium phosphate (pH 7.0). The decrease in absorption at 550 nm was measured for 120 s and maximally oxidized absorption was determined by adding potassium ferricyanide at completion. Twenty-five microliters of 10 mg/ml dodecylmaltoside were added before preincubation, As Box Cox

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analysis indicated inhomogeneity of variance, all analyses were performed using the natural logarithmic transformation of the data. The coefficient of variation in complex IV reproducibility (assays were performed is three) was 5.2%.

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3.2. Normal ranges of mitochondrial enzymes The data from 17 control samples were obtained for mitochondrial enzymes. The ages, in 1 year 1 month to 16 years, are matched to the patient’s age. The meanGSD of complex IICIII activity is 30.6G 8.9 (range: 15.4–54.5, nmol/min per mg protein), that of complex IV, 2.5G0.6 (range: 1.8–3.9, 10! 10K2 nmol/min per mg protein), that of CS, 109.6G 15.9 (range: 84.2–152.2, nmol/min per mg protein), that of complex IICIII/CS, 27.9G6.4 (range: 15.3– 35.8, 10!10K2) and that of complex IV/CS, 23.4G 5.3 (range: 17.1–35.8, 10!10K5). Levels below K2SD were considered to be outside the normal range.

3.1.2.3. Citrate synthase (CS) assay. The assay utilized is a modification of the technique described by Haas et al., (1995). CS was assayed in Triton X-100 solubilized PLTs. The total working volume was 250 ml. Sample wells contained 125 ml of buffer (0.16 M Tris (pH 8.0)), 2 ml of 10 mM fresh 5 0 ,5 0 -dithio-bis(2-nitrobenzoic acid (DTNB) in 0.1 M Tris (pH 8.3), 25 ml diluted samples, with four different protein concentrations, 25 ml of Triton X-100 0.8% in water, and 25 ml of acetyl coenzyme A (acetyl-CoA; 0.5 mM) in water. The reaction was started after a 3 min preincubation at 30 8C by adding 25 ml of freshly made oxaloacetate (5 mM in water). The increase in absorption of the 5-thio-2-nitrobenzoate ion at 412 nm was measured. The results of this assay are multiplied by a correction factor of 0.676 to correct for well dimensions and absorption. The coefficient of variation in CS reproducibility (assays were performed four) was 2.0%.

4. Results 4.1. Changes in mitochondrial enzyme activities with cytochrome c treatments During cytochrome c treatment, complex IICIII and IV activities initially fluctuated without declining, then gradually decreased, but stayed within

Table 1 Changes in mitochondrial enzyme activities Age (year:month:day)

Citrate synthase

IICIII

IV (10!10K2)

IICIII/CS (10!10-2)

IV/CS (10!10K5)

5:2:4 5:2:12 5:2:17 5:2:19 5:3:2 5:3:9 5:3:24 5:3:0 5:4:7 5:4:14 5:4:21 5:4:28 5:5:4 5:5:19 5:6:2 5:8:4 5:10:11 6:0:8 6:0:21 6:1:21 7:1:6

124.8 125.1 138.5 145.9 141.7 128.6 135.7 131.3 135.4 133.9 94.6 122.7 127.9 132 117.7 126.3 113.5 101.6 138.3 112.8 144.1

16.89 17.64 17.88 16.02 17.53 18.73 17.27 17.4 20.57 19.82 18.13 18.66 16.24 17.72 15.46 15.29 12.51 11.87 11.56 20.06 18.57

2.544 2.785 3.07 2.995 3.142 3.582 2.65 3.107 2.774 3.772 2.079 2.582 2.598 2.43 2.575 2.868 2.385 1.617 2.506 2.94 2.834

13.53 14.10 12.91 10.98 12.37 14.56 12.73 13.25 15.19 14.80 19.16 15.21 12.70 13.4 13.14 12.12 11.02 11.68 8.359 17.78 12.89

20.38 22.26 22.17 20.53 22.17 27.85 19.53 23.66 20.49 28.17 21.98 21.04 20.31 18.41 21.88 22.71 21.01 15.92 18.12 26.06 19.67

Unit of citrate synthase, complex IICIII and complex IV: nmol/min per mg whole PLT protein. Levels significantly below normal are underlined.

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their normal ranges. On the other hand, complex IICIII/CS activity was initially significantly below normal, followed by a transient improvement. This activity was again declined significantly starting at 5 years 5 months of age, followed by the appearance of CNS involvement, including mild headaches, vomiting and photopsia. The change in IV/CS activity was similar to that in complex IICIII/CS activity, but both levels were within normal ranges. The complex IICIII and IV activities reached their lowest levels around the time of a stroke-like episode. Most notably, complex IICIII activity decreased significantly at 5 years 10 months onward (Table 1). The complex IICIII/CS and IV/CS activities also reached their lowest levels around the time of this episode. Complex IICIII/CS activity was abnormally low, while IV/CS activity was within normal range (Table 1). Regression analysis during cytochrome c treatment revealed complex IICIII, IV and CS activities to be significantly decreased (Fig. 2). On the other hand, complex IICIII/CS and IV/CS activities did not decrease (Fig. 3).

4.2. Changes in mitochondrial enzyme activities with DCA treatment The complex IICIII, IV, IICIII/CS and IV/CS activities increased transiently after DCA treatment. In particular, those of complex IICIII and IICIII/CS reached their normal ranges. However, the complex IICIII/CS activity had again decreased significantly at one year (Fig. 1, Table 1).

5. Discussion Cytochrome c is reportedly effective for mitochondrial encephalomyopathies (Nakagawa et al., 1996; Tanaka et al., 1997). Cytochrome c given with vitamins B1 and B2 improved muscle strength in our patient, while her CNS symptoms, including headache and photopsia, appeared during cytochrome c treatment (Fig. 1). Complex IICIII/CS activity was initially significantly below normal but then showed a transient improvement. Before the CNS involvement appeared, this activity again fell

Fig. 1. Changes in mitochondrial enzyme activities and clinical course with cytochrome c and sodium dichloroacetate treatments. : injection of cytochrome c (Cytomack-P), vitamin B1 and vitamin B2. The dosages were 0.5, 5 and 2 mg/kg, respectively. : photopsia. : the short bar indicates mild headache, which was transient and tolerable. : the tall bar indicates severe headache, which was persistent and intolerable. 7: a complex partial seizure. ;: vomiting.

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(b) 21

14

20 19

13

Comp2+3

CS(10E+1)

(a) 15

12 11 10 9 –50

0

50

100

150 days

200

250

300

350

18 17 16 15 14 13 12 11 –50

0

(c)

40 37.5 35

Comp4(–10E–1)

Y = 13.393 – .009 * X; R^2 = .347

32.5 30 27.5 25 22.5 20 17.5 15 –50

0

50

431

100

150 days

200

250

300

350

Y = 18.709 – .02 * X; R^2 = .574

50

100

150 days

200

250

300

350

Y = 30.563 – .035 * X; R^2 = .342

Fig. 2. Changes in citrate synthase (a), complex IICIII (b) and complex IV (c) activities during cytochrome c treatment, as evaluated by regression analysis. Complex IICIII (correlation coefficient K0.758, P-value 0.0003), complex IV (correlation coefficient K0.585, P-value 0.0108) and CS (correlation coefficient K0.604, P-value 0.0079) activities showed statistically significant correlations with their cytochrome c treatment periods.

significantly. Long term cytochrome c treatment may be related to our patient’s CNS symptoms, although the natural disease course could not be ruled out. The complex IICIII and IV activities fluctuated initially, then gradually decreased. The decreases in complex IICIII, IV and CS activities were statistically significant. Most notably, complex IICIII activity was significantly below normal before a stroke-like episode (Table 1). Decreases in mitochondrial enzyme activities during cytochrome c treatment may raise the possibility of progressive mitochondrial dysfunction. Mitochondria play a critical role in mediating both apoptotic and necrotic cell death. Recently, it was revealed that changes in mitochondrial permeability transition lead to mitochondrial swelling, outer membrane rupture and the release of apoptotic mediators, including cytochrome c (Baines

et al., 2005), triggering apoptotic and necrotic cell death processes. These processes may relate to the mitochondrial dysfunction demonstrated during cytochrome c treatment in our present case. Complex IICIII, IV, IICIII/CS and IV/CS activities reached their lowest levels around the time of a stroke-like episode in this case. Most notably, the complex IICIII and IICIII/CS activities were significantly below normal. All enzyme activities improved after DCA treatment in this case. Our patient’s response suggests that DCA may improve mitochondrial enzyme functions during stroke-like episodes. However, the single episode in this case could not be confirmed to be a stroke-like episode. We need to study more patients to clarify this issue. As to the pathophysiology of the stroke-like episodes in MELAS, neuron involvement and cerebral vascular

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(b) 30

19

28

18 Comp4/CS(–10E–5)

CCmp2+3/CS(10E–2)

(a) 20

17 16 15 14 13 12

24 22 20 18 16

11 10 –50

26

0

50

100

150

200

250

300

350

14 –50

0

50

100

150

200

250

300

350

Fig. 3. Changes in complex IICIII/CS (a) and complex IV/CS (b) activities during cytochrome c treatment, as evaluated by regression analysis. There were no statistically significant correlations between complex IICIII/CS (correlation coefficient K0.315, P-value 0.2034) or complex IV/CS (correlation coefficient K0.396, P-value 0.1034).and their cytochrome c treatment periods.

dysfunction, as demonstrated by brain biopsy, have been reported (Gilchrist et al., 1996). Maruyama et al. (Maruyama et al., 1998) reported that proton MRS showed a lactate peak as well as a decreased NAA/ choline ratio in the involved area during stroke-like episodes. SPECT and PET also revealed metabolic abnormalities of neurons and vessels (Bohnen et al., 1998; Iizuki et al., 2002; Nariai et al., 2001). Accumulation of mitochondria in smooth muscle and endothelial cells of brain autopsy specimens (Ohama et al., 1988) and serial diffusion imaging (Ohshita et al., 2000) have suggest the involvement of cerebral vascular dysfunction in stroke-like episodes. The decrease in PLT mitochondrial enzyme activities during stroke-like episodes may reflect dysfunction of neurons or blood vessels in the brain. Mitochondrial abnormalities have been reported in neuronal degenerative diseases such as Parkinson (Shults et al., 1995; Haas et al., 1995; Shults et al., 1998; Parker and Parks, 2005), Alzheimer (Parker et al., 1994) and Huntington’s (Parker et al., 1990) diseases. PLT mitochondrial assays may be useful for evaluating mitochondrial function in neurological disorders. We have developed whole PLT mitochondrial enzyme assays of complex IICIII, IV, IICIII/ CS and IV/CS activities, employing a miniaturized assay system with a microplate-reader. We found that mitochondrial enzymes decrease before a stroke-like episode. Most notably, complex IICIII and IICIII/ CS declined significantly. This assay system may allow prediction of stroke-like episodes. This assay system may be useful not only for evaluating mitochondrial diseases but also for some

neurodegenerative disorders such as Parkinson, Alzheimer, and Huntington diseases, allowing serial evaluations of mitochondrial function in parallel with clinical symptoms.

Acknowledgements This work was supported in part by the T. Satake Scholarship Foundation and in part by grant #14Syouni-006, Clinical Research for Evidence Based Medicine, from the Ministry of Health, Labor and Welfare, Japan.

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