Yonago Acta Med 57(1): 23-35

PMC: PMC4110693

PMID: 25067875

Cerebellar Expression of Copper Chaperone for Superoxide, Cytosolic Cu/Zn-Superoxide Dismutase, 4-Hydroxy-2-Nonenal, Acrolein and Heat Shock Protein 32 in Patients with Menkes Kinky Hair Disease: Immunohistochemical Study

Background

To clarify the pathogenesis of cerebellar Purkinje cell death in patients with Menkes kinky hair disease (MD), a disorder of copper absorption, we investigated the morphological and functional abnormalities of residual Purkinje cells in MD patients and the mechanism of cell death.

Methods

Seven MD patients and 39 neurologically normal autopsy cases were studied. We performed histopathological and quantitative analyses of the Purkinje cells. In addition, we used immunohistochemistry to detect copper-dependent enzymes [cytosolic Cu/Zn-superoxide dismutase (SOD1) and copper chaperone for superoxide dismutase (CCS)], oxidative stress markers [4-hydroxy-2-nonenal (HNE) and acrolein] and heat shock protein 32 (hsp 32).

Results

The surviving MD Purkinje cells showed abnormal development, such as somatic sprouts and heterotopic location. Due to maldevelopment and degeneration, dendrites showed the cactus and weeping willow patterns. Axonal degeneration led to the formation of torpedoes. Quantitative analysis revealed loss of approximately 50% of the Purkinje cells in MD patients. Almost all of the normal Purkinje cells were positive for immunostaining by anti-CCS and anti-SOD1 antibodies, with staining of the cell bodies, dendrites and axons. Normal Purkinje cells were not stained by antibodies for HNE, acrolein or hsp 32. In MD patients, the majority of Purkinje cells were positive for CCS, but the positive rate for SOD1 was only about 23%. Approximately 56%, 42% and 40% of the Purkinje cells of MD patients were positive for HNE, acrolein and hsp 32, respectively.

Conclusion

In MD patients, about 50% of the Purkinje cells have been lost due to maldevelopment and degeneration. In the residual Purkinje cells, CCS expression seems to be nearly normal as a protective response to decreased SOD1 activity due to copper deficiency. Because oxidative stress is elevated secondary to decreased SOD1 activity, hsp 32 is induced as another protective mechanism.

Subjects and neuropathologic examination

This study was carried out on brain tissues obtained at autopsy from 7 patients with Menkes kinky hair disease (MD). The main characteristics of these 7 patients are summarized in Table 1. Histologically normal brains from 39 other autopsy patients (ages: 1–59 years) served as the controls. After fixation in 10% buffered formalin, the brain stem and cerebellum were removed from the cerebrum at the midbrain level. Routine coronal slices of the cerebrum and sagittal and transverse slices of the cerebellum were cut. The thickness of these slices was about 5 mm and each slice completely transected the cerebrum and cerebellum. Slices of the cerebellum were embedded in paraffin and then were cut into 6-μm thick sections, which were stained by using the following routine methods: hematoxylin and eosin (HE), Klüver-Barrera, Holzer, Bodian and modified Hirano-Bielschowsky stains. Representative paraffin sections were used for the immunohistochemical analyses. The protocol of this study was approved by the Ethics Committee of Tottori University (No. 1994: 2012).

Table 1.

Characteristics of the 7 patients with Menkes kinky hair disease

	Patient 1	Patient 2	Patient 3	Patient 4	Patient 5	Patient 6	Patient 7
Age at death	1 y 1 mo	1 y 4 mo	1 y 6 mo	1 y 6 mo	1 y 9 mo	2 y 1 mo	6 y 4 mo
Brain weight (g)	650	500	700	580	450	720	1,130
Family history	–	–	–	+*	+	+*	?
Reference number	4, 5, 36, 37	4, 5	4, 5, 36, 37	4, 5, 36, 37	4, 5, 36, 37	4, 5, 36, 37	4, 5, 38

*sibling; –, absent; +, present; ?, unknown.

Quantitative analysis of cerebellar Purkinje cells

We performed quantitative analysis of the number of Purkinje cells in the cerebellum using HE-stained sagittal and transverse sections of the cerebellar hemisphere and vermis on glass slides (Fig. 1A). The number of Purkinje cells was counted within a 3 mm length, as shown in Fig. 1B. For determining the 3mm length of the circle in Fig. 1A, we used a light microscope (Olympus BX-50 F4, Tokyo, Japan) equipped with an eyepiece micrometer grid (24-#10/10X10, Olympus), in combination with the stage micrometer (AX0001, OB-M, Olympus). To determine the number of Purkinje cells, we counted the number of these cells with nuclei. As shown in Fig. 1B, we counted the number of Purkinje cells within this 3 mm length using a measuring device (Erma, Tokyo, Japan). In each autopsy case, these circles including the 3 mm length (Fig. 1A) were selected randomly at 15 to 80 sites on HE-stained sections of the cerebellar hemisphere and vermis on glass slides. Then we determined the mean number of Purkinje cells per 3 mm length for each autopsy case.

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Fig. 1.

Diagram of the method for quantitative measurement of the number of Purkinje cells in the cerebellum.

A: Sagittal section of the cerebellar hemisphere. The circle shown in A involves a 3 mm length of the Purkinje cell layer. The circled area was used for quantitative determination of the number of Purkinje cells. In each autopsy case, at least 15 sites were randomly selected from a sagittal section of the cerebellar hemisphere, and the number of Purkinje cells was counted in each area.

B: An enlarged image of the circle shown in A. We measure a 3 mm length (indicated by 3 mm in B) in the cerebellar Purkinje cell layer and count the number of red-stained Purkinje cells within this 3 mm length. For example, there are 20 Purkinje cells in B.

Immunohistochemistry

The following primary antibodies were used: an affinity purified rabbit antibody directed against human copper chaperone for superoxide dismutase (CCS) [diluted 1:500 in phosphate-buffered saline with 1% bovine serum albumin (BSA-PBS), pH 7.4],23, 24 a rabbit polyclonal antibody for human SOD1(diluted 1:3,000),12, 14, 15, 23, 25–29 an affinity purified rabbit antibody targeting 4-hydroxy-2-nonenal (HNE) (diluted 1:400; JaICA, Fukuroi, Japan), a rabbit polyclonal antibody for acrolein (diluted 1:3,000; JaICA) and a rabbit polyclonal antibody directed against hsp 32 (diluted 1:200; Santa Cruz, Dallas, TX). The specificity of these antibodies for immunohistochemical detection of the respective epitopes in paraffin sections of human materials has been documented previously.12, 14, 15, 23–29

Sections were deparaffinized, and endogenous peroxidase activity was quenched by incubation for 30 min with 0.3% H₂O₂. Then the sections were washed in PBS (pH 7.4). Normal serum homologous to each secondary antibody was used as the blocking agent. Sections were incubated with the primary antibodies for 18 h at 4 ˚C and sections incubated with PBS served as controls. Bound antibodies were visualized by the avidin-biotin-immunoperoxidase complex (ABC) method using the appropriate Vectorstain ABC kit (Vector Laboratories, Burlingame, CA) and 3,3'-diaminobenzidine tetrahydrochloride (Dako, Glostrup, Denmark).

The cellular structures on immunostained sections were observed under a light microscope (Olympus BX-50 F4) and mapped, and photomicrographs were obtained using a digital camera (Olympus DP12). Then representative immunostained sections were also subjected to HE staining.

To assess immunostaining of the Purkinje cells in the cerebellum by each antibody, 1,000 Purkinje cells were examined. Cells that were immunoreactive to the respective primary antibodies were considered to be positive and the proportion of antibody-positive cells was expressed as a percentage.

Statistical analysis

IBM SPSS Statistics software (IBM, Chicago, IL) was used for statistical analysis. The number of Purkinje cells is presented as the mean ± SE. For analysis of all data, non-parametric Kruskal-Wallis analysis of variance (ANOVA) was performed, followed by a non-parametric Wilcoxon rank-sum test. Statistical significance was accepted at P < 0.05.

Neuropathological findings

Comparison between the 39 normal control autopsy cases (Fig. 2A) and the 7 MD autopsy cases (Fig. 2B) revealed severe loss of Purkinje cells and marked reduction of granule cells (Fig. 2B) in the cerebellum of the MD patients. The loss of Purkinje cells in MD patients was accompanied by prominent Bergmann gliosis in the Purkinje cell layer. In all 7 MD patients, the width of the molecular layer of the cerebellum, which is mainly composed of Purkinje cell dendrites and the parallel fiber axons of granule cells, was significantly decreased (Fig. 2B) in comparison with that of the control autopsy cases, reflecting the marked loss of Purkinje cells and granule cells in the MD patients.

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Fig. 2.

Histologic features of Purkinje cells in a normal control autopsy case (A) and an MD patient (B–G).

A: Cerebellum of a normal control autopsy case.

B: Cerebellum of an MD patient. Compared with the normal cerebellum, the cerebellum of the MD patient shows severe loss of Purkinje cells (arrowheads in A and B) and marked reduction of granule cells (GC in A and B). As a result, there is a marked decrease in the width of the molecular cell layer (arrows in A and B). Panels A and B are at the same magnification (HE stain). Bars = 200 μm (A and B).

C: The residual Purkinje cells of the MD patient show somatic sprouts (arrows) (HE stain). Bar = 50 μm.

D: Surviving Purkinje cells of the MD patient exhibit cactus-like change (arrowhead and C) and dendritic expansion (arrowhead) (HE stain). Bar = 50 μm.

E: Residual Purkinje cells of the MD patient display a weeping willow dendritic pattern (double arrowheads) (HE stain). Bar = 50 μm.

F: In the MD patient, ectopic Purkinje cells bearing somatic sprouts (arrows and H) are found in the granule cell layer (HE stain). Bar = 50 μm.

G: A residual Purkinje cell of the MD patient shows an axonal torpedo (arrowhead) (HE stain). Bar = 50 μm.

HE, hematoxylin and eosin; MD, Menkes kinky hair disease.

The chief neuropathological characteristic in the cytoplasm of the residual Purkinje cells of MD patients was somatic sprouts (Fig. 2C). The neuropathological abnormalities of the dendrites of the residual Purkinje cells in MD patients included expansion and cactus-like changes, i.e., partial enlargement of Purkinje cell dendrites was observed (Fig. 2D). With respect to abnormal dendritic arborization in the residual Purkinje cells, we observed downward extension of the dendrites, which has been called the weeping willow pattern by Kato et al. 5 (Fig. 2E). In the granule cell layer of MD patients, the presence of heterotopic Purkinje cells bearing somatic sprouts was noted (Fig. 2F). In addition, the residual Purkinje cells of MD patients displayed axonal expansions that are known as torpedoes (Fig. 2G).

Quantitative analysis of Purkinje cell changes

The mean number (± SE) of residual Purkinje cells in all 7 MD autopsy cases was 9.1 ± 2.1 cells/3 mm. In contrast, the mean number (± SE) of Purkinje cells in the normal cerebellum at the age of 1 to 9 years, 10 to 19 years, 20 to 29 years, 30 to 39 years, 40 to 49 years and 50 to 59 years was 20.1 ± 0.4 cells/3 mm, 20.1 ± 0.4 cells/3 mm, 20.0 ± 0.6 cells/3 mm, 19.9 ± 0.5 cells/3 mm, 19.9 ± 0.6 cells/3 mm and 19.2 ± 0.4 cells/3 mm, respectively (Fig. 3). There was a significant decrease of Purkinje cells in the MD patients compared with the number of cells in the controls (P < 0.01, Kruskal-Wallis test with ANOVA). We also performed a comparison between the number of residual Purkinje cells in MD patients and the number of Purkinje cells in normal children from the age of 1 to 9 years. As a result, there was a significant decrease in the number of Purkinje cells in the MD patients (P < 0.01, Wilcoxon rank-sum test). Furthermore, we performed between-group analyses to compare the number of residual Purkinje cells in the MD patients with the number of Purkinje cells in the normal control autopsy cases from age 10 to 19, from age 20 to 29, from age 30 to 39, from age 40 to 49 and from age 50 to 59 years. All of these analyses revealed a significant decrease in the number of Purkinje cells in the MD patients (P < 0.01, Wilcoxon rank-sum test) (Fig. 3). In contrast, the number of Purkinje cells in the normal control autopsy cases showed no significant difference across the age groups from 1 to 59 years (P > 0.2, Kruskal-Wallis test with ANOVA).

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Fig. 3.

Quantitative analysis of the number of Purkinje cells in the cerebellum. The number of Purkinje cells in each autopsy case is shown as mean ± SE. Compared with the number of Purkinje cells in the normal controls, the number of Purkinje cells was significantly low in the MD patients. Error bars represent SE. *P < 0.01 compared with the normal control autopsy cases by Kruskal-Wallis analysis of variance, followed by the Wilcoxon rank-sum test.

Immunohistochemical findings

When control tissue sections were incubated with PBS, no immunoreaction products were seen. In the normal controls, Purkinje cells were stained by the antibody for CCS. Staining of the cell bodies, dendrites, and axons was seen, with the primary, secondary and tertiary dendrites as well as spiny branchlets being clearly detected (Figs. 4A–D). The axons could be traced to the dentate nucleus. The glial cells, myelin sheaths, blood vessels, leptomeninges and other neuronal cells of the normal cerebellum did not react with the anti-CCS antibody.

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Fig. 4.

Immunohistochemical expression of CCS (A–D) and SOD1 (E) by the Purkinje cells of a normal control.

A: Almost all of the Purkinje cells in the normal cerebellum are stained by the antibody for CCS (arrowheads and arrow), while granule cells (GC) do not react with the anti-CCS antibody (CCS immunostaining without counterstaining). Bar = 500 μm.

B: Higher magnification of the Purkinje cells identified by arrowheads in A. The cell bodies (arrowheads with C) and dendrites (arrowheads with D) are readily seen (CCS immunostaining without counterstaining). Bar = 100 μm.

C: Higher magnification of the Purkinje cells indicated by an arrow in A. The cell body (arrowhead with C), primary dendrites (arrowhead with P), secondary dendrites (arrowhead with S) and tertiary dendrites (arrowhead with T), as well as an axon (arrowhead with AX), are clearly seen. CCS immunostaining without counterstaining. Bar = 100 μm.

D: High-powered view of the Purkinje cell dendrites indicated by the arrowhead with S and arrowhead with T in C. Many spiny branchlets (arrowhead with sB) are clearly observed. CCS immunostaining without counterstaining. Bar = 30 μm.

E and F: Preparations of the same section of normal control cerebellar cortex stained with anti-SOD1 antibody (E) or with HE (F). Almost all of the Purkinje cells in the normal control cerebellum are stained by the anti-SOD1 antibody with varying intensity. Purkinje cell bodies (arrowheads with C) are positive, as are their main dendrites, including the primary dendrites (arrowhead with P) and secondary dendrites (arrowhead with S) (E: SOD1 immunostaining without counterstaining. F: HE staining). Bars = 100 μm.

CCS, copper chaperone for superoxide dismutase; HE, hematoxylin and eosin; SOD1, cytosolic Cu/Zn-superoxide dismutase.

Purkinje cells of the normal cerebellum were also immunostained by the anti-SOD1 antibody, with the cell bodies and the main dendrites (including primary and secondary dendrites) being positive (Figs. 4E and F). Normal Purkinje cells did not show immunostaining by the antibodies for HNE (Fig. 5A), acrolein (Fig. 5C) or hsp 32 (Fig. 5E).

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Fig. 5.

Immunostaining of the cerebellum of a normal control with antibodies for HNE (A), acrolein (C) and hsp 32 (E). Normal Purkinje cells (arrowheads) are not stained by the antibodies for HNE (A), acrolein (C) and hsp 32 (E).

A: HNE immunostaining with weak hematoxylin counterstaining. Bar = 100 μm.

C: Acrolein immunostaining with weak hematoxylin counterstaining. Bar = 100 μm.

E: hsp 32 immunostaining with weak hematoxylin counterstaining. Bar = 100 μm.

B, D, F: HE staining. Bars = 100 μm.

HE, hematoxylin and eosin; HNE, 4-hydroxy-2-nonenal; hsp, heat shock protein.

The surviving cerebellar Purkinje cells of MD patients differed from normal Purkinje cells in terms of both morphology and immunoreactivity. Residual Purkinje cells in MD patients showed immunostaining by the anti-CCS antibody at varying intensities and approximately 77% (773 ± 49.5/1,000) of these cells were positive for CCS (Fig. 6A). The most frequent immunostaining pattern was varying levels of CCS positivity in the cell bodies and the proximal parts of the dendrites of the Purkinje cells.

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Fig. 6.

Immunostaining of the cerebellum from a patient with MD using antibodies for CCS (A) and SOD1 (B).

A: Many of the surviving Purkinje cells are positive for CCS at varying intensities (CCS immunostaining without counterstaining). Bar = 200 μm.

B and C: Some of the surviving Purkinje cells (arrow in B and C) are positive for SOD1, while other cells (arrowhead in B and C) are negative. (B: SOD1 immunostaining without counterstaining. C: HE staining). Bars = 200 μm.

CCS, copper chaperone for superoxide dismutase; HE, hematoxylin and eosin; MD, Menkes kinky hair disease; SOD1, cytosolic Cu/Zn-superoxide dismutase.

Immunostaining for SOD1 was positive in some of the surviving Purkinje cells of the MD patients, with the positive rate for anti-SOD1 antibody being about 23% (232 ± 89.0/1,000) but the staining intensity was low (Fig. 6B). When SOD1-positive and SOD1-negative Purkinje cells of the MD patients were compared by HE staining, it was impossible to clearly distinguish between them (Fig. 6C).

Immunostaining using anti-HNE antibody showed that many of the residual Purkinje cells were positive for HNE in MD patients. A variable staining intensity was observed (Fig. 7A), and the positive rate for anti-HNE antibody among the surviving Purkinje cells was approximately 56% (564 ± 55.8/1,000). When HNE-positive and HNE-negative Purkinje cells were compared by HE staining, it was not possible to observe any clear differences between them (Fig. 7B). Immunostaining with the anti-acrolein antibody demonstrated that some of the viable Purkinje cells in MD patients were positive for acrolein. A variable staining intensity was observed (Fig. 7C), and the positive rate for anti-acrolein antibody was approximately 42% (420 ± 94.1/1,000) among the residual Purkinje cells. When acrolein-positive and acrolein-negative Purkinje cells were compared by HE staining, no marked differences between them were observed (Fig. 7D). Immunostaining with the anti-hsp 32 antibody showed that some of the residual Purkinje cells of MD patients were positive for hsp 32. There was variation of the staining intensity (Fig. 7E), and the positive rate for anti-hsp 32 antibody was approximately 40% (396 ± 73.5/1,000) among the residual MD Purkinje cells. When hsp 32-positive and hsp 32-negative Purkinje cells were compared by HE staining, there were no clear differences between them (Fig. 7F).

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Fig. 7.

Immunostaining of the cerebellum from a patient with MD using antibodies for HNE (A), acrolein (C) and hsp 32 (E).

A and B: Some of the remaining Purkinje cells (arrowhead in A and B) are positive for HNE, while other cells (arrow in A and B) are negative (A: HNE immunostaining without counterstaining. B: HE staining). Bar = 100 μm.

C and D: Some residual Purkinje cells are positive for acrolein (arrowhead in C and D), while other residual Purkinje cells are not (arrow in C and D) (C: Acrolein immunostaining without counterstaining. D: HE staining). Bar = 100 μm.

E and F: Some of the remaining Purkinje cells (arrowhead in E and F) are immunoreactive for hsp 32, while other cells are not (arrow in E and F) (E: hsp 32 immunostaining without counterstaining. F: HE staining). Bar = 100 μm.

HNE, 4-hydroxy-2-nonenal; HE, hematoxylin and eosin; hsp, heat shock protein; MD, Menkes kinky hair disease.

*Division of Neuropathology, Department of Pathology, School of Medicine, Tottori University Faculty of Medicine, Yonago, 683-8503, Japan

†Division of Child Neurology, Department of Brain and Neurosciences, School of Medicine, Tottori University Faculty of Medicine, Yonago, 683-8503, Japan

‡Sanin Rosai Hospital, Yonago, 683-8605, Japan

§Division of Neuropathology, Department of Pathology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York, USA

‖Department of Pathology, Osaka Red Cross Hospital, Osaka 543-8555, Japan

¶Division of Molecular Pathology, Department of Pathology, School of Medicine, Tottori University Faculty of Medicine, Yonago, 683-8503, Japan

Corresponding author: Shinsuke Kato, MD, PhD

E-mail: kato@med.tottori-u.ac.jp

Corresponding author: Shinsuke Kato, MD, PhD

E-mail: kato@med.tottori-u.ac.jp

Received 2013 Nov 8; Accepted 2013 Nov 19.

Abstract

Background

Methods

Results

Conclusion

Keywords: copper chaperone for superoxide dismutase, cytosolic Cu/Zn-superoxide dismutase, immunohistochemistry, Menkes kinky hair disease, Purkinje cell

Abstract

Menkes kinky hair disease (MD) is a sex-linked inherited disorder that is characterized by the onset of seizures, developmental retardation and typical changes of the hair in early infancy.1–5 MD patients develop neuropathological abnormalities throughout the central nervous system.4–7 In the cerebellar cortex, there is marked loss of Purkinje cells, while the surviving Purkinje cells display characteristic somatic sprouts and bizarre thickening of the dendritic tree.4, 5, 8–10 Several studies have shown that severe hypocuprinemia occurs in MD patients due to impaired absorption of copper from the intestine, which causes inactivation of copper-dependent enzymes such as cytosolic Cu/Zn-binding superoxide dismutase (SOD1).11

SOD1 is usually a dimer of 2 identical monomers with a molecular weight of approximately 16 kDa, and is mainly localized to the cytoplasm. Each SOD1 monomer contains one Cu ion and one Zn ion. SOD1 is an antioxidant metalloenzyme that catalyzes the conversion of 2 superoxide radicals (O₂^{) to 1 molecule each of H}₂O₂ and O₂.12, 13 An O₂^{is formed when an oxygen molecule accepts an additional electron. Superoxide radicals cause oxidative damage to the nuclei, organelles and enzymes of cells, by reacting with nucleic acids in the nuclei, proteins that compose cytoplasmic organelles and proteins of enzymes, respectively. Under normal conditions, the copper ion of SOD1 exists in the divalent state. When this Cu}^{of SOD1 reacts with a O}₂^{, it undergoes reduction to a Cu}^{by gaining an electron from the superoxide radical, while the radical that reacted with the Cu}^{of SOD1 is converted to O}₂. Then the Cu^{of SOD1 is oxidized again to return to its original divalent state as it supplies an electron to another O}₂^{. This other O}₂^{that receives an electron from the Cu}^{of SOD1 is converted to H}₂O₂ by reacting with 2H^{. Thus, SOD1 catalyzes the conversion of 2 O}₂^{to 1 molecule of H}₂O₂ and 1 molecule of O₂. 12, 13 The H₂O₂ generated by this reaction is ultimately transformed into H₂O and O₂ by enzymes such as catalase, glutathione peroxidase and peroxiredoxin. 13–15

The yeast copper chaperone (LSY7: 249 amino acids) and the mammalian copper chaperone for superoxide dismutase (CCS: 274 amino acids) have both been identified.16 The RNA for CCS is encoded by a single-copy gene on chromosome 11, and CCS expression has been found in all human tissues and cells examined.16, 17 CCS specifically transfers copper ions to SOD1, resulting in its activation.16, 18 Since there are no free copper ions in vivo,19 SOD1 cannot obtain copper without CCS and cannot be activated, so the delivery of copper to SOD1 by CCS is essential for its normal activity.20

Extracellular copper ions are transported into the cytoplasm by copper transporter 1 located in the cell membrane.21, 22 CCS has three domains, among which domain I contains a copper-binding site for the copper ions that it transports. Copper ions that bind to domain I are transferred to domain III via domain II. Domain II of CCS shows structural similarity to SOD1 at the molecular level and contains the coupling region for binding with SOD1. After the copper ion is transferred from the copper-binding site on domain I to the copper-binding site on domain III, it is supplied to SOD1.

Therefore, CCS has an important, though indirect, role in fighting oxidative stresses. Immunohistochemical studies have shown that CCS is extensively distributed throughout the normal central nervous system, including the cerebellar Purkinje cells, similar to SOD1.17 Decreased SOD1 activity due to impaired absorption of copper could cause cellular damage in MD patients, with oxidative stress leading to oxidation of membrane lipids and the production of toxic aldehyde metabolites such as 4-hydroxy-2-nonenal (HNE) or acrolein. Furthermore, heat shock protein (hsp) 32 would be induced in response to the increase of oxidative stress.

In MD patients, copper deficiency could be expected to lead to Purkinje cell damage due to oxidative stress. In the present study, we performed an immunohistochemical analysis of cerebellar CCS and SOD1 as copper-dependent enzymes, HNE and acrolein as markers of oxidative stress and hsp 32 as a stress protein. We also carried out a quantitative analysis of the number of Purkinje cells in the cerebellum. Our objectives were to clarify the morphological and functional abnormalities of Purkinje cells in MD patients and the pathogenesis of these Purkinje cell changes.

Acknowledgments:

The authors express their appreciation to Dr. K. Asayama for kindly providing the antibody to SOD1.

This study was supported in part by a Grant-in-Aid for Scientific Research (c) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (S.K.: 24500420); a Grant-in Aid from the Scientific Committee of CNS Degenerative Diseases from the Ministry of Health, Labour and Welfare of Japan (S.K.); a Grant-in Aid for the physician training project in Shimane Prefecture (S.K.) and a Grant-in Aid for discretionary expenditure from the president of Tottori University (S.K.).

Acknowledgments:

Notes

The authors declare no conflict of interest.

Notes

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