793-799 Yamamoto

Journal http://jcn.sagepub.com/ of Child Neurology

Oxidative Stress in the Brain of Fukuyama Type Congenital Muscular Dystrophy: Immunohistochemical Study on Astrocytes Tomoko Yamamoto, Noriyuki Shibata, Makio Kobayashi, Kayoko Saito and Makiko Osawa J Child Neurol 2002 17: 793 DOI: 10.1177/08830738020170110701 The online version of this article can be found at: http://jcn.sagepub.com/content/17/11/793

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Original Article

Oxidative Stress in the Brain of Fukuyama Type Congenital Muscular Dystrophy: Immunohistochemical Study on Astrocytes Tomoko Yamamoto, MD, PhD; Noriyuki Shibata, MD, PhD; Makio Kobayashi, MD, PhD; Kayoko Saito, MD, PhD; Makiko Osawa, MD, PhD

ABSTRACT Astrocytes in the cerebrum and medulla oblongata of cases of Fukuyama type congenital muscular dystrophy were examined by immunohistochemistry of oxidative modification products and free-radical scavenging enzymes because abnormal glia limitans formed by astrocytic end feet is considered to be involved in the genesis of brain lesions of Fukuyama type congenital muscular dystrophy. The study was performed on two fetal cases of Fukuyama type congenital muscular dystrophy of 18 and 20 weeks’ gestation and seven patients with Fukuyama type congenital muscular dystrophy ranging in age from 2 to 27 years. Eight age-matched control cases were used. Polymerase chain reaction (PCR) was performed to ascertain the gene phenotype of two child cases, in which prenatal gene analysis was not performed. Astrocytes, especially layer I astrocytes, of postnatal cases of Fukuyama type congenital muscular dystrophy were weakly positive for N-(carboxymethyl)lysine and argpyrimidine, suggesting that they were sensitive to oxidative stress, and the accumulation may be related to the abnormal glia limitans. Secondary increase of manganese (Mn) superoxide dismutase against the increase of free radicals was considered in patients with Fukuyama type congenital muscular dystrophy more than 14 years old considered to be homozygous for founder haplotype: homozygosity was suggested by PCR in two cases. In contrast, expression of Mn superoxide dismutase was decreased in 2- and 6-year-old children with Fukuyama type congenital muscular dystrophy that were heterozygous. Moreover, accumulation of argpyrimidine was exclusively found in astrocytes of the 2-year-old child that exhibited severe brain lesions. Function of astrocytes might be impaired or immature in severe or heterozygous cases. These results may confirm that astrocytes play an important role in the etiology of the brain lesion. (J Child Neurol 2002;17:793–799).

Fukuyama type congenital muscular dystrophy is an autosomal recessive disease with the gene locus on chromosome 9q31,1 exhibiting muscular dystrophy and brain lesions rep-

Received April 9, 2002. Received revised June 7, 2002. Accepted for publication June 18, 2002. From the Departments of Pathology (Drs Yamamoto, Shibata, Kobayashi), Surgical Pathology (Dr Yamamoto), and Pediatrics (Drs Saito and Osawa), Tokyo Women’s Medical University, Tokyo, Japan. This study was supported, in part, by a Grant-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 11670195). Address correspondence to Dr Tomoko Yamamoto, Department of Pathology, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Tel: +81-3-3353-8111; fax: +81-3-5269-7408; e-mail: [email protected].

resented by micropolygyria.2–4 The basement membrane of the central nervous system and skeletal muscle is abnormal in cases of Fukuyama type congenital muscular dystrophy.5–7 In the central nervous system, basement membrane covers the glia limitans, which separates nervous tissues from leptomeninges. The glia limitans is established by end feet of radial glia by 6 to 7 weeks’ gestation in humans.8 After the completion of neuronal migration, the end-feet of radial glia are progressively replaced by those of layer I astrocytes, which become the basic component of the glia limitans from the late prenatal period.9 The glia limitans is disrupted in the fetal Fukuyama type congenital muscular dystrophy brain,10,11 and the disruption probably caused by abnormal basement membrane may be involved in the genesis of the brain lesions of Fukuyama type congenital muscular dystrophy.5–7,10,11 In postnatal cases of Fukuyama type congenital muscular dystrophy, superficial gliosis is observed 793

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Journal of Child Neurology / Volume 17, Number 11, November 2002

Table 1. Brief Summary of Cases Cases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Age

Sex

18 gw 20 gw 2 yr 6 yr 14 yr 14 yr 15 yr 17 yr 27 yr 22 gw 23 gw 1 yr 6 yr 13 yr 17 yr 29 yr 31 yr

M F M F M F F F M F F M M F F M F

Postmortem Time* 4:13 0:40 4:17 20:00 3:30 8:40 1:33 5:29 1:32 Unknown 1:40 3:14 1:46 9:02 14:50 1:37 7:04

Brain Weight (g) 29.5 41.6 1030 1386 1336 1260 1230 1340 1311 55.7 79.0 950 1370 1464 1420 1418 1415

Diseases (Haplotype†) FCMD (homozygous) FCMD (homozygous) FCMD (heterozygous) FCMD (heterozygous) FCMD (homozygous) FCMD (unknown) FCMD (unknown) FCMD (homozygous) FCMD (homozygous) Cardiopulmonary hypoplasia CCAM Down syndrome Ventricular septal defect Ovarian tumor Systemic lupus erythematosus VAHS Marfan syndrome

*Interval between death and autopsy (hr:min). † Genetic analysis was performed before death with informed consent. gw = gestational week; FCMD = Fukuyama type congenital muscular dystrophy; CCAM = congenital cystic adenomatoid malformation; VAHS = virus-associated hemophagocytic syndrome.

in the cerebral surface.2,4 We previously reported that astrocytes showed abnormal morphology in child and adult cases of Fukuyama type congenital muscular dystrophy,12 and that fukutin, a gene responsible for Fukuyama type congenital muscular dystrophy, was expressed in astrocytes as well as immature neurons of cases of non–Fukuyama type congenital muscular dystrophy.13 If astrocytes cannot form a normal glia limitans owing to a genetic defect, they might be weak to oxidative stress, and secondary loss of astrocytic function and morphologic alteration might be induced. An immunohistochemical examination for oxidative modification products and free radical scavenging enzymes was performed in the cerebrum and medulla oblongata of cases of Fukuyama type congenital muscular dystrophy. MATERIALS AND METHODS Histologic Examination This investigation was carried out on the frontal or parietal lobe of the cerebrum and medulla oblongata of cases of Fukuyama type congenital muscular dystrophy from the autopsy file of Tokyo Women’s Medical University: two fetuses of 18 and 20 weeks’ gestation and seven patients ranging in age from 2 to 27 years. Eight age-matched cases of non–Fukuyama type congenital muscular dystrophy including two fetuses, were used as controls (Table 1). Specimens were fixed in 10% buffered formalin or UFIX (Sakura, Tokyo), processed to paraffin embedding and cut into sections in a routine manner. Sections were stained with hematoxylin and eosin (H&E). Immunohistochemical Examination The avidin-biotin complex method was employed for the immunohistochemical staining. The paraffin-embedded tissues were sectioned at 6 m. All sections were treated with 3% H 2O 2 in phosphate buffered saline solution for 10 mininutes to block endogenous peroxidase. The sections were then rinsed in phos-

phate buffered saline solution and transferred to normal goat serum for 30 minutes to block background antibody binding. After washing, the tissue was transferred to phosphate buffered saline solution containing the primary antibody at 4°C and left overnight. The slides were then washed in phosphate buffered saline solution and transferred to a secondary antibody for 30 minutes at room temperature. After washing in phosphate buffered saline solution, the tissue was treated with avidin-biotin (AB) complex for 30 minutes at room temperature. The slides were then washed, and chromogen diaminobenzidine tetrahydrochloride was applied. The slides were stained with hematoxylin for nuclear detail. The dilutions and sources of the primary antibodies were as follows: 8-hydroxy-2´-deoxyguanosine (1:100, Panapharm Laboratories, Kumamoto, Japan), 4-hydroxy-2-nonenal-histidine (1:100, Nippon Oil Forces, Tokyo, Japan), crotonaldehyde-lysine (1:200, Nippon Oil Forces), N-(carboxymethyl)lysine (1:1500, Panapharm Laboratories), argpyrimidine (1:200, Nippon Oil Forces), pyrraline (1:1000, from Dr Horiuchi, Department of Biochemistry, Kumamoto University), Cu/Zn superoxide dismutase (1:5000, from Dr Asayama, Department of Pediatrics, University of Occupational and Environmental Health, Japan), and manganese (Mn) superoxide dismutase (1:5000, from Dr Asayama). Microwave antigen retrieval was performed for argpyrimidine (APM) immunostaining. Immunostaining of glial fibrillary acidic protein (1:1000, DAKO, Carpinteria, CA) was performed to determine astrocytes using serial sections. Double-immunostaining was also done (data not shown). Evaluation Intensity of immunostaining was evaluated as negative, weakly positive, and positive and the percentage of positively stained glial cells was calculated. In the cerebrum, glial cells in the subcortical white matter were counted. In the medulla oblongata, the average between the tegmentum and the olivary nuclei was calculated. More than 1000 cells were counted, and weakly stained cells were omitted from calculation.

Oxidative Stress in Fukuyama Type Congenital Muscular Dystrophy / Yamamoto et al

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Table 2. Results in the Cerebral White Matter

Fetal cases FCMD (18–20 gw) Control (22–23 gw) Postnatal cases FCMD (2–6 yr) Control (1–6 yr) FCMD (14–27 yr) Control (13–31 yr)

8-OHdG (%)

4-HNEH (%)

CRAL (%)

CML (%)

APM (%)

PY (%)

Cu/Zn SOD (%)

Mn SOD (%)

– –

n n

– –

– –

– –

––+ ––+

+ +

––+ ––+

23.8 (0–47.6) 2.7 (0–5.3) 5.9 (0–27.6) 7.8 (0–33.1)

– – – –

– – – –

– – + –

+ – – –

––+ ––+ ––+ ––+

0.8 (0–1.6) + 11.9 (0–56.6)* 1.6 (0–8.0)

2.5 (0–4.9) 23.0 (10.0–35.9) 36.4 (15.8–49.5) 17.3 (0–35.4)

– = negative; + = weakly positive; n = predominant nuclear staining; FCMD = Fukuyama type congenital muscular dystrophy; 8-OHdG = 8-hydroxy-2’-deoxyguanosine; 4-HNEH = 4-hydroxy-2-nonenal-histidine, CRAL = crotonaldehyde-lysine; CML = N-(carboxymethyl)lysine; APM = argpyrimidine; PY = pyrraline; SOD = superoxide dismutase. *Only one case (case 4) showed 56.6% of positive cells, and less than 3.1% of cells were positive in other cases.

Polymerase Chain Reaction Polymerase chain reaction (PCR) was performed to presume the gene phenotype of cases 6 and 7, in which prenatal gene analysis was not performed. DNA was extracted from paraffin-embedded liver tissues of cases 4 to 9 and 14 to 17. Polymerase chain reaction was performed using Takara Ex TaqTM (Takara, Tokyo, Japan) with primers covering 5823-5978 bp (exon 10) of fukutin complementary DNA and covering intron 6 to exon 7 of fructose-1,6-bisphosphatase gene.14 The sequence of the primers for fukutin DNA was 5’-GGGTCAGAACATATTCCTTTACTC -3’ (coding sense) and 5’-GGAGGCAAACTGGTAATGTCTC-3’ (anticoding sense), and that for fructose-1,6-bisphosphatase DNA was 5’-CCCGGTCTTGTTTTCCAGCTG-3’ (coding sense) and 5’-GCAGAGTGCTTCTCATACACC-3’ (anticoding sense). The reaction mixture was amplified twice for 35 cycles in a Zymoreactor thermo-cycler (ATTO Co., Tokyo, Japan). The amplification profiles consisted of denaturing at 94°C for 1.5 minutes, annealing at 60°C for 1 minute, and extension at 72°C for 1 minute. RESULTS

In the cerebrum and medulla oblongata of fetal cases of Fukuyama type congenital muscular dystrophy, neuronal tissues overmigrated via disrupted glia limitans. Overmigration was more prominent in the cerebrum. Details including immunohistochemical and electron microscopic examination were described in the previous reports.6,11 In a 2-year-old child with Fukuyama type congenital muscular dystrophy, the cerebrum showed pachygyric or micropolygyric appearance with superficial gliosis, and the medulla oblongata showed conspicuous heterotopic glioneu-

ronal tissues in the leptomeninges. In cases of Fukuyama type congenital muscular dystrophy more than 6 years old, the cerebrum exhibited micropolygyria with superficial gliosis. The medulla oblongata looked almost normal with focal heterotopic glioneuronal tissues. Immunohistochemistry

The results of immunohistochemistry are summarized in Tables 2 and 3 and Figures 1 and 2. Differences between cases of Fukuyama type congenital muscular dystrophy and control cases were found in the immunostaining of N(carboxymethyl)lysine, argpyrimidine and Mn superoxide dismutase in postnatal cases. Fetal Cases

There was no difference between cases of Fukuyama type congenital muscular dystrophy and control cases. Glial cells were negative for 8-hydroxy-2´-deoxyguanosine, crotonaldehyde lysine, N-(carboxymethyl)lysine, and argpyrimidine in the cerebrum and medulla oblongata of all cases. 4-Hydroxy-2-nonenal-histidine was stained predominantly in the nuclei. Pyrraline, Cu/Zn superoxide dismutase, and Mn superoxide dismutase were negative or weakly positive. Cases of 1 to 6 Years of Age

The results of 8-hydroxy-2´-deoxyguanosine varied from case to case, and no difference between cases of Fukuyama type congenital muscular dystrophy and control cases was found. 4-Hydroxy-2-nonenal-histidine and crotonaldehyde lysine were negative in all cases. N-(carboxymethyl)lysine

Table 3. Results in the Medulla Oblongata

Fetal cases FCMD (18–20 gw) Control (22–23 gw) Postnatal cases FCMD (2–6 yr) Control (1–6 yr) FCMD (14–27 yr) Control (13–31 yr)

8-OHdG (%)

4-HNEH (%)

CRAL (%)

CML (%)

APM (%)

PY (%)

Cu/Zn SOD (%)

Mn SOD (%)

– –

n n

– –

– –

– –

––+ ––+

+ +

+ +

28.0 (0–56.0) 0.7 (0–1.4) 0.4 (0–1.1) 1.6 (0–5.7)

– – – –

– – – –

– – – –

+ – – –

––+ ––+ ––+ ––+

+ + + +

3.2 (2.9–3.4) 5.6 (3.1–8.1) 20.7 (1.7–37.5) 7.1 (2.5–14.0)

– = negative; + = weakly positive; n = predominant nuclear staining; FCMD = Fukuyama type congenital muscular dystrophy; 8-OHdG = 8-hydroxy-2’-deoxyguanosine; 4-HNEH = 4-hydroxy-2-nonenal-histidine; CRAL = crotonaldehyde-lysine; CML = N-(carboxymethyl)lysine; APM = argpyrimidine; PY = pyrraline; SOD = superoxide dismutase.

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Figure 1 Immunostaining of N -(carboxymethyl)lysine (CML) (A, B), argpyrimidine (APM) (C, D), and Mn superoxide dismutase (Mn SOD) (E, F) in a 2-year-old case of Fukuyama type congenital muscular dystrophy (A, C, E) and in a 1-year-old control case (B, D, F). Astrocytes and neuropil are weakly positive for N-(carboxymethyl)lysine and argpyrimidine in the case of Fukuyama type congenital muscular dystrophy, but are negative for Mn superoxide dismutase (original magnification, 500). WM = cerebral white matter.

was weakly positive in glial cells in layer I of cases of Fukuyama type congenital muscular dystrophy, although negative in the cerebral white matter and medulla oblongata. These glial cells were mainly astrocytes (see Figure 1). Argpyrimidine was weakly positive in glial cells, including astrocytes, in a 2-year-old child with Fukuyama type congenital muscular dystrophy (see Figure 1). Pyrraline was negative to weakly positive in all cases. Cu/Zn superoxide dismutase immunostaining showed no apparent difference. Mn superoxide dismutase–positive glial cells, including astrocytes, were decreased in the cerebrum of cases of Fukuyama type congenital muscular dystrophy (see Figure 1). Cases over 14 Years of Age

The results of 8-hydroxy-2´-deoxyguanosine varied from case to case and no difference between cases of Fukuyama type congenital muscular dystrophy and control cases was found. 4-Hydroxy-2-nonenal-histidine, crotonaldehyde lysine, and argpyrimidine were negative in all cases. N-(carboxymethyl)lysine was weakly positive in glial cells of the layer I and cerebral white matter of cases of Fukuyama type congenital muscular dystrophy, but negative in all con-

trol cases (see Figure 2). N-(carboxymethyl)lysine-positive glial cells included astrocytes. Pyrraline was negative to weakly positive in all cases. Cu/Zn superoxide dismutase immunostaining showed no apparent difference. Mn superoxide dismutase–positive glial cells, including astrocytes, looked increased in the cerebrum and medulla oblongata of cases of Fukuyama type congenital muscular dystrophy (see Figure 2). Polymerase Chain Reaction

In PCR for fructose-1, 6-biphosphatase (FBPase) DNA, all cases showed a band at 203 bp. In PCR for fukutin DNA, a band was observed at 156 bp in a heterozygous case (case 4) and control cases (cases 14–17), but no band was seen in a homozygous case (case 5, 8 and 9). Cases 6 and 7 showed no bands (Figure 3). DISCUSSION

Oxidative stress is suggested to be involved in normal aging and many diseases. Various oxidative modification products are formed under the effect of free radicals in proteins, lipids, and nucleotides. Recently, immunohisto-


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Figure 2 Immunostaining of N -(carboxymethyl)lysine (CML) (A–D) and Mn superoxide dismutase (Mn SOD) (E, F) in a 27-year-old case of Fukuyama type congenital muscular dystrophy (A, C, E) and a 31-year-old control case (B, D, F). Astrocytes and neuropil are weakly positive for N-(carboxymethyl)lysine and positive for Mn superoxide dismutase in the case of Fukuyama type congenital muscular dystrophy (original magnification, 500). WM = cerebral white matter; MO = medulla oblongata.

chemistry of oxidative modification products has been available. 8-Hydroxy-2´-deoxyguanosine is a major product of oxidative DNA damage.15,16 One of the good markers for detecting lipid peroxidation is 4-hydroxy-2-nonenalhistidine.17,18 Crotonaldehyde lysine is also formed by lipid peroxidation in vivo.19 N-(carboxymethyl)lysine is formed by oxidation of proteins20–22 and by lipid peroxidation.20,22 Argpyrimidine is an end product of protein modification caused by oxidative stress.20,23,24 In contrast, pyrraline is formed by nonoxidative reactions.20, 25 In the present study, slight accumulation of N-(carboxymethyl)lysine and argpyrimidine was observed in astrocytes of Fukuyama type congenital muscular dystrophy, but no accumulation was proved in pyrraline, 8-hydroxy-2´-deoxyguanosine, 4hydroxy-2-nonenal-histidine, and crotonaldehyde lysine immunostaining. Astrocytes seem sensitive to oxidative stress in Fukuyama type congenital muscular dystrophy, and the oxidative stress may influence more in proteins than in lipids or DNA. N-(carboxymethyl)lysine and argpyrimidine accumulated in postnatal cases of Fukuyama type congenital muscular dystrophy but not in fetal cases, suggesting that long exposure of oxidative stress is necessary for the accumu-

lation. In Fukuyama type congenital muscular dystrophy, the brain lesion represented by micropolygyria is prominent in the cerebrum, and the brain stem, including the medulla oblongata, generally exhibits only minor lesions.2–4,10,11 The effect of oxidative stress seems stronger in the area that showed more severe lesions, and it is possible that the accumulation of N-(carboxymethyl)lysine and argpyrimidine may be related to abnormal glia limitans because the accumulation is predominant in cerebral astrocytes, especially those in layer I. In addition, neurons might facilitate the accumulation in astrocytes because neurons are disorganized in the micropolygyric area with aberrant myelinated fibers in the superficial layer.4 Free radical scavenging enzymes such as Cu/Zn superoxide dismutase and Mn superoxide dismutase metabolize oxidative modification products. Cu/Zn superoxide dismutase and Mn superoxide dismutase are expressed in neurons, astrocytes, and microglial cells in the central nervous system.26–29 Expression of Mn superoxide dismutase looked increased in both the cerebrum and medulla oblongata of cases of Fukuyama type congenital muscular dystrophy more than 14 years of age, suggesting secondary reaction against the increase of free radicals. In contrast, expression

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Figure 3 In polymerase chain reaction (PCR) for FBP DNA, all cases show a band at 203 bp. In PCR for fukutin DNA, a band at 156 bp is observed in control cases (cases 14–17) and a heterozygous case (case 4), but no band is seen in homozygous cases (cases 5, 8, and 9). Cases 6 and 7 show no band.

of Mn superoxide dismutase was decreased in the 2- and 6year-old cases, and argpyrimidine was exclusively accumulated in the 2-year-old case. Decrease of Mn superoxide dismutase and accumulation of argpyrimidine suggest that function of astrocytes might be impaired or immature. The 3-kb-DNA is inserted in the fukutin gene between bases 5889 and 5890 in the Fukuyama type congenital muscular dystrophy chromosome carrying the founder haplotype.30 Most of the cases of Fukuyama type congenital muscular dystrophy are homozygous for the founder haplotype, and at least one chromosome in each patient with Fukuyama type congenital muscular dystrophy has the founder haplotype.31 In the PCR examination amplifying 5823 to 5978 bp of fukutin DNA, homozygous cases showed no band at 156 bp because of the insertion in both chromosomes, and a heterozygous case showed a band, probably because one of the chromosomes did not contain the insertion. Amplification of DNA in control cases proved that the method was appropriate, and DNA of each case seems to be preserved since a longer DNA fragment (FBPase DNA of 203 bp) was amplified. Severe cases of Fukuyama type congenital muscular dystrophy tend to be heterozygous,31 and brain lesions were prominent, especially in the 2-year-old patient with Fukuyama type congenital muscular dystrophy. Cases 6 and 7 showing no band were a common type of Fukuyama type congenital muscular dystrophy, clinically and pathologically (data not shown), and highly suspected to be homozygous for founder haplotype. Function of astrocytes might be impaired or immature in severe or heterozygous cases. These results may confirm that astrocytes play an important role in the etiology of the brain lesion. Acknowledgment

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6.

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Yamamoto T, Toyoda C, Kobayashi M, et al: Pial-glial barrier abnormalities in fetuses with Fukuyama congenital muscular dystrophy. Brain Dev 1997;19:35–42.

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The authors wish to thank Fumiaki Muramatsu and Noriko Sakayori for their excellent technical assistance.

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