Yonago Acta medica 2014;57:23–35

Original Article

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
Atsushi Yokoyama,*† Kousaku Ohno,†‡ Asao Hirano,§ Masayuki Shintaku, Masako Kato,¶ Kazuhiko Hayashi¶and Shinsuke Kato**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, NY 10461, United States, Department of Pathology, Osaka
Red Cross Hospital, Osaka 543-8555, Japan and ¶Division of Molecular Pathology, Department of Pathology, School of Medicine,
Tottori University Faculty of Medicine, Yonago, 683-8503, Japan

ABSTRACTof 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 werepositive for HNE, acrolein and hsp 32, respectively.
Conclusion In MD patients, about 50% of the Purkinje cells have been lost due to maldevelopment anddegeneration. 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.

BackgroundTo clarify the pathogenesis of cerebellar Purkinje cell death in patients with Menkes kinky
hair disease (MD), a disorder of copper absorption, weinvestigated the morphological and functional abnormalities of residual Purkinje cells in MD patients andthe mechanism of cell death.
Methods Seven MD patients and 39 neurologicallynormal autopsy cases were studied. We performed histopathological and quantitative analyses of the Purkinjecells. In addition, we used immunohistochemistry todetect copper-dependent enzymes [cytosolic Cu/Znsuperoxide dismutase (SOD1) and copper chaperone forsuperoxide dismutase (CCS)], oxidative stress markers[4-hydroxy-2-nonenal (HNE) and acrolein] and heatshock protein (hsp) 32.
Results
The surviving MD Purkinje cells showedabnormal development, such as somatic sprouts andheterotopic 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 ofapproximately 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

Key words

copper chaperone for superoxide dismutase; cytosolic Cu/Zn-superoxide dismutase; immunohistochemistry; Menkes kinky hair disease; Purkinje cell
Menkes kinky hair disease (MD) is a sex-linked inherited disorder that is characterized by the onset ofseizures, developmental retardation and typical changesof 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 Purkinjecells 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 theintestine, 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, andis mainly localized to the cytoplasm. Each SOD1 monomer contains one Cu ion and one Zn ion. SOD1 is anantioxidant metalloenzyme that catalyzes the conversionof 2 superoxide radicals (O2•–) to 1 molecule each of

Corresponding author: Shinsuke Kato, MD, PhD
kato@med.tottori-u.ac.jp
Received 2013 November 8
Accepted 2013 November 19
Abbreviations: ABC, avidin-biotin-immunoperoxidase complex;
ANOVA, analysis of variance; BSA-PBS, phosphate-bufferedsaline with 1% bovine serum albumin; CCS, copper chaperone
for superoxide dismutase; HE, hematoxylin and eosin; HNE,
4-hydroxy-2-nonenal; MD, Menkes kinky hair disease; SOD1,
cytosolic Cu/Zn-superoxide dismutase; hsp, heat shock protein

23

A. Yokoyama et al.

H2O2 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 inthe 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 Cu2+ of SOD1 reacts with an O2•–,
it undergoes reduction to a Cu+ by gaining an electronfrom the superoxide radical, while the radical that reacted with the Cu2+ of SOD1 is converted to O2. Then theCu+ of SOD1 is oxidized again to return to its original
divalent state as it supplies an electron to another O2•–.
This other O2•– that receives an electron from the Cu+ of
SOD1 is converted to H2O2 by reacting with 2H+. Thus,
SOD1 catalyzes the conversion of 2 O2•– to 1 moleculeof H2O2 and 1 molecule of O2.12, 13 The H2O2 generated
by this reaction is ultimately transformed into H2O andO2 by enzymes such as catalase, glutathione peroxidaseand peroxiredoxin.13–15
The yeast copper chaperone (LSY7: 249 aminoacids) 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 singlecopy 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 thecytoplasm by copper transporter 1 located in the cell
membrane.21, 22 CCS has 3 domains, among which domain I contains a copper-binding site for the copper ionsthat it transports. Copper ions that bind to domain I aretransferred 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 thecopper-binding site on domain I to the copper-bindingsite 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 absorptionof 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. Fur-thermore, 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 ascopper-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 toclarify the morphological and functional abnormalities
of Purkinje cells in MD patients and the pathogenesis ofthese Purkinje cell changes.
MATERIALS AND METHODS
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 brainsfrom 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 thecerebrum at the midbrain level. Routine coronal slicesof 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 HiranoBielschowsky 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).
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 hemisphereand vermis on glass slides (Fig. 1A). The number of Purkinje cells was counted within a 3 mm length, as shownin Fig. 1B. For determining the 3 mm 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 withthe stage micrometer (AX0001, OB-M, Olympus). To
determine the number of Purkinje cells, we counted thenumber 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,
24

Immunohistochemistry of MD Purkinje cells
Table 1. Characteristics of the 7 patients with Menkes kinky hair diseaseAge at death
Brain weight (g)Family history
Reference number

Patient 1

Patient 2

Patient 3

Patient 4

Patient 5

Patient 6

Patient 7

1 yr 1 mo
650–
4, 5, 36, 37

1 yr 4 mo
500
–
4, 5

1 yr 6 mo
700–
4, 5, 36, 37

1 yr 6 mo
580+*
4, 5, 36, 37

1 yr 9 mo
450+
4, 5, 36, 37

2 yr 1 mo
720+*
4, 5, 36, 37

6 yr 4 mo
1,130?
4, 5, 38

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

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 foreach autopsy case.

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 wereincubated with the primary antibodies for 18 h at 4 ˚C andsections 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 BX50 F4) and mapped, and photomicrographs were obtainedusing 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 andthe proportion of antibody-positive cells was expressed asa percentage.

Immunohistochemistry

2 mm

1 mm

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

10

15

1

20

m
3m

0

5

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 circledarea 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.

25

A. Yokoyama et al.

2A

2B

GC

GC

2C

2D

C

2E

2G

2F

H
H

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 decreasein 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 (Aand 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) (HEstain). 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.

26

Immunohistochemistry of MD Purkinje cells

Fig. 3. Quantitative analysis of the number of Purkinje cells in the cerebellum. The number of Purkinje cells in each autopsy case isshown 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 ofvariance, followed by the Wilcoxon rank-sum test.

dendrites was observed (Fig. 2D). With respect to abnormal dendritic arborization in the residual Purkinjecells, 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, theresidual Purkinje cells of MD patients displayed axonal
expansions that are known as torpedoes (Fig. 2G).

Statistical analysis

IBM SPSS Statistics software (IBM, Chicago, IL) wasused for statistical analysis. The number of Purkinjecells is presented as the mean ± SE. For analysis of alldata, non-parametric Kruskal-Wallis analysis of variance (ANOVA) was performed, followed by a non-parametric Wilcoxon rank-sum test. Statistical significancewas accepted at P < 0.05.
RESULTS
Neuropathological findings

Quantitative analysis of Purkinje cell changes

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 patientswas accompanied by prominent Bergmann gliosis in the
Purkinje cell layer. In all 7 MD patients, the width of themolecular 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 granulecells in the MD patients.
The chief neuropathological characteristic in thecytoplasm of the residual Purkinje cells of MD patientswas somatic sprouts (Fig. 2C). The neuropathologicalabnormalities of the dendrites of the residual Purkinjecells in MD patients included expansion and cactuslike changes, i. e., partial enlargement of Purkinje cell

The mean number (± SE) of residual Purkinje cells inall 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 decreaseof Purkinje cells in the MD patients compared with thenumber 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 wasa 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 the27

A. Yokoyama et al.

4B

4A

D

D

GC
C

C

4C

4D

sB

T

sB
sB

S
P
C
AX

4E

4F

S

S

P
C

P

C

C

C

C
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 granulecells (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 (arrowheadwith 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.

MD patients with the number of Purkinje cells in thenormal control autopsy cases from age 10 to 19, fromage 20 to 29, from age 30 to 39, from age 40 to 49 andfrom age 50 to 59 years. All of these analyses revealed28

Immunohistochemistry of MD Purkinje cells

5A

5B

5C

5D

5E

5F

Fig. 5. Immunostaining of the cerebellum of a normal control with antibodies for HNE (A), acrolein (C) and hsp 32 (E). Normal Purkinjecells (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: hsp32 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.

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 thenormal control autopsy cases showed no significant difference across the age groups from 1 to 59 years (P > 0.2,
Kruskal-Wallis test with ANOVA).

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.
Purkinje cells of the normal cerebellum were also
immunostained by the anti-SOD1 antibody, with the cellbodies and the main dendrites (including primary andsecondary 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).
The surviving cerebellar Purkinje cells of MD
patients differed from normal Purkinje cells in termsof both morphology and immunoreactivity. Residual
Purkinje cells in MD patients showed immunostaining
by the anti-CCS antibody at varying intensities and ap-

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
29

A. Yokoyama et al.

6A

6B

6C

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) arenegative (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/Znsuperoxide dismutase.

proximately 77% (773 ± 49.5/1,000) of these cells werepositive for CCS (Fig. 6A). The most frequent immunostaining pattern was varying levels of CCS positivity inthe cell bodies and the proximal parts of the dendrites of
the Purkinje cells.
Immunostaining for SOD1 was positive in some ofthe surviving Purkinje cells of the MD patients, withthe positive rate for anti-SOD1 antibody being about
23% (232 ± 89.0/1,000) but the staining intensity waslow (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 showedthat many of the residual Purkinje cells were positivefor HNE in MD patients. A variable staining intensity
was observed (Fig. 7A), and the positive rate for antiHNE antibody among the surviving Purkinje cells
was approximately 56% (564 ± 55.8/1,000). When
HNE-positive and HNE-negative Purkinje cells werecompared 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
30

Immunohistochemistry of MD Purkinje cells

7A

7B

7C

7D

7E

7F

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) arenegative (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 (arrowin 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.

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. Whenacrolein-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 in-tensity (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 werecompared by HE staining, there were no clear differences between them (Fig. 7F).
DISCUSSION
In the present neuropathological study of MD patients,
marked loss of Purkinje cells and granule cells was ob31

A. Yokoyama et al.

served in the cerebellum. The residual Purkinje cells had
the following neuropathological characteristics: somatic
sprouts, cactus-like dendritic expansions, weeping willow extension of dendrites, heterotopia and axonal torpedoes.
Somatic sprouts are observed in Purkinje cells of
the normal fetus around 27 weeks at autopsy.4, 5 However, somatic sprouts are not observed in the normal
fetus much past 27 weeks of gestation, so the sprouts
disappear with increasing maturation.4, 5 Thus, somatic
sprouts are part of the normal developmental process,but should not be seen after birth. The fact that somatic
sprouts were noted in all of the 7 MD autopsy cases (agedfrom 1 year and 1 month to 6 years and 4 months) suggests that the Purkinje cells of patients with MD havebeen affected by maldevelopment or developmental disorder.
Ectopic Purkinje cells were present in the granulecell layer of the cerebellum in MD autopsy cases, but notin the normal control autopsy cases. Purkinje cells originate from the neural tube and pass through the granulecell layer while migrating toward the Purkinje cell layerof the cerebellum.30 Thus, normal migration results in
Purkinje cells reaching the Purkinje cell layer, so theobservation that MD patients have ectopic Purkinje cells
suggests that these cells were arrested in the process of
migrating toward the Purkinje cell layer and also indicates maldevelopment. Furthermore, based on the finding that ectopic Purkinje cells with somatic sprouts were
present, cerebellar Purkinje cells in MD patients show
evidence of maldevelopment.
The finding of weeping willow and cactus-likechanges indicated abnormal enlargement of some dendrites. Dendritic expansion and cactus-like changes are
assumed to arise during the process of dendrites extending from the Purkinje cell body. From this point of view,neuropathological findings such as dendritic expansion
and cactus-like changes can be interpreted as further
evidence of maldevelopment. On the other hand, it isalso possible that after the Purkinje cell dendrites had developed fully, some dendrites underwent expansion andcactus-like changes due to degeneration. Thus, the neuropathology of dendritic expansion and cactus-like changes
could be related to both maldevelopment and degeneration.
When development is normal, the dendrites of Purkinje cells extend from the cell bodies through the cerebellar molecular layer toward the side of the subarachnoid space. In contrast, the neuropathological weeping
willow pattern is characterized by downwardly extending
dendrites, and can be considered to arise from failure of
the normal development of Purkinje cell dendrites. In thiscase, the weeping willow pattern is regarded as evidenceof maldevelopment. On the other hand, the weeping willow pattern can also be interpreted as evidence of degeneration. There is a possibility that after the dendrites of
Purkinje cells have fully developed, downward extensionof some dendrites occurs as a result of shrinkage of theirarborization. In other words, the weeping willow pattern
could be regarded as due to maldevelopment and/or degeneration.
Axonal torpedoes of Purkinje cells like those in MD
patients are frequently observed in autopsy cases of olivo-ponto-cerebellar atrophy, which is one of the neurodegenerative diseases.31 Therefore, the axonal torpedoes
seen in MD patients may be an indicator of degeneration.
Taken together, these findings indicate that cerebellar MD Purkinje cells undergo cell death due to bothmaldevelopment and degeneration. Comparison of thenumber of Purkinje cells in the 7 MD patients with thatin the 39 normal controls revealed that almost half of the
Purkinje cells had been lost in the MD patients. Thus,
we clearly demonstrated that 50% of the Purkinje cells in
MD patients died due to the effects of maldevelopment
and degeneration.
There have been few reports about quantitative
measurement of the number of Purkinje cells in the cerebellum. To the best of our knowledge, there is only one
report by Hausmann et al.32 concerning quantitative measurement of the number of Purkinje cells in normal autopsy cases. They studied 52 autopsy cases aged between
5 and 82 years and reported that the number of Purkinjecells per 1 mm length was 9.21 cells in 12 cases of sudden death and 7.60 cells in 18 cases of drowning or asphyxia that resulted in death within 10 min. In our study,
the number of Purkinje cells was approximately 20.1
cells per 3 mm length in 39 normal control autopsy casesin the aged between 1 and 59 years. In the present study,
patients exhibiting normal cerebellar histology who diedbecause of organ failure and disorders outside the central
nervous system were used as normal controls. None ofthem were cases of sudden deaths due to unknown etiology. When we assess the data of Hausmann et al. fromthis standpoint, our normal control group is similar totheir 18 patients who died within 10 min due to drowning or asphyxia. Hausmann’s data regarding the numberof Purkinje cells could be converted to 22.8 cells per 3
mm length and this number is very close to our result
for measurement of the number of Purkinje cells in thecontrols. Such concordance suggests the validity of theresults of our quantitative analysis of the number of cerebellar Purkinje cells in normal controls and MD patients.
In addition, we found that the number of Purkinje cells
showed no significant difference among the age groups,
32Immunohistochemistry of MD Purkinje cells

from the group less than 10 years old to the group in their
50s. Based on our data, the number of Purkinje cells in
the normal cerebellum remains almost constant from 1
year to 59 years old.
Immunohistochemistry showed clear CCS staining
from the cell bodies to the peripheral spiny branchlets of
Purkinje cells. This immunohistochemical pattern suggests that CCS is an abundant protein in normal Purkinjecells. Similarly, SOD1 protein was abundant in normal
Purkinje cells and was expressed from the cell body tothe major dendrites. On the other hand, in MD patientswith copper deficiency, the percentage of residual Purkinje cells positive for SOD1 was only about 23% and theintensity of staining was weak. In contrast, 77% of the remaining Purkinje cells were positive for CCS, which was
higher than the rate for SOD1. In addition, the staining
intensity for CCS was similar to that of normal control
Purkinje cells. In other words, expression of CCS protein
was maintained in MD Purkinje cells, but SOD1 protein
was decreased.
There have been several reports about the levels
of SOD1 and CCS protein in fresh tissues of copperdeficient animals. Bertinato et al.33 fed one group ofmale Wistar rats a copper-deficient diet, while anothergroup received a normal diet without copper deficiency,
and they examined the levels of SOD1 and CCS proteinin the liver and red blood cells of both groups by thewestern blotting method. SOD1 protein levels in boththe liver and red blood cells were lower in the group
receiving the copper-deficient diet compared with thegroup receiving a normal diet. In contrast, CCS protein
levels were higher in both the liver and red blood cells of
the group receiving the copper-deficient diet than in thegroup receiving a normal diet.33 Prohaska et al.34 used
Holtzman rats and ND4 Swiss Webster mice to semiquantitatively examine the levels of SOD1 and CCS protein in the liver and brain of copper-deficient diet groups
and non-copper-deficient normal diet groups by western
blot analysis. SOD1 protein levels were lower in the liverand brain of rats and mice from the copper-deficientgroups than in those of rats and mice from the normal
diet groups. SOD1 protein levels in the livers of rats
and mice were 30% and 70%, respectively, while brain
SOD1 protein levels were 83% and 22%, respectively.
In contrast, CCS protein levels in the livers and brainsof rats and mice from the copper-deficient diet groups
were increased by approximately 3-fold in the rat liver,approximately 1.7-fold in the mouse liver, approximately
1.8-fold in the rat brain and approximately 2.8-fold in themouse brain compared with the normal diet groups.34
Furthermore, Gybina and Prohaska35 compared cerebellar levels of CCS between Holtzman rats in the copper-

deficient diet and normal diet groups. Compared with
the CCS protein level in the normal diet group, the CCS
protein level in the cerebellum of rats from the copperdeficient diet group was about 2-fold higher.35
In MD patients, Purkinje cells were exposed to copper deficiency for a very long period ranging from 1
year and 1 month to 6 years and 4 months, because MD
is a copper absorption disorder. Copper deficiency is assumed to lead to a decrease of SOD1 protein, which is acopper-dependent enzyme. The duration of copper deficiency was only 42 days in the study of Bertinato,33 23
to 30 days in that of Prohaska34 and 24 to 27 days in thatof Gybiana.35 Even with such short periods of copper
deficiency, a decrease of SOD1 protein was observed inthese animal experiments.33–35 In our study, immunohistochemical staining showed that SOD1 expression was
decreased in the residual Purkinje cells of MD patients.
This reflected the fact that MD Purkinje cells had been
subjected to severe damage caused by free radicals dueto the chronic decrease of SOD1 activity. Under suchcircumstances, it would be difficult for the Purkinje cellsof MD patients to maintain normal functions. On theother hand, while cell damage caused by free radicals
secondary to the decrease of SOD1 could have occurredin the animal models of short-term copper deficiency,
cell death was not observed.33–35 This suggested that
Purkinje cells were protected and survived as a result of
up-regulating CCS, which specifically transports copper
to SOD1. This up-regulation of CCS in the animals withshort-term copper deficiency might correspond to theprotective response of CCS in the remaining Purkinjecells of MD patients with chronic copper deficiency. According to our immunohistochemical results, even in theremaining Purkinje cells of MD patients that were close
to the moribund state, CCS was maintained at almost
the normal level. Based on this finding, the mechanismof CCS up-regulation still had a protective effect in theresidual Purkinje cells of MD patients.
Our immunohistochemical analysis of HNE, acrolein and hsp 32 demonstrated that normal cerebellar
Purkinje cells did not express any of HNE, acrolein or
hsp 32. In the Purkinje cells of MD patients, however,
we observed expression of HNE, acrolein and hsp 32
at varying intensities. The rate of positive staining for
HNE, acrolein and hsp 32 in the remaining Purkinjecells of MD patients was 56%, 42% and 40%, respectively. HNE is a cytotoxic product that is generated when
polyunsaturated fatty acids such as arachidonic acidare subjected to oxidative stress, and acrolein is anothertoxic metabolite generated when lipid membranes are
subjected to oxidative stress. Therefore, immunohistochemical detection of both HNE and acrolein in the sur33

A. Yokoyama et al.

viving Purkinje cells of MD patients is strong evidenceof oxidative stress. On the other hand, the immunohistochemical study also showed that hsp 32 was induced in
the surviving Purkinje cells of MD patients. This could
be regarded as evidence that hsp 32 was up-regulated in
the surviving Purkinje cells as a self-protective mechanism against oxidative stress to promote survival. Katoet al.4, 5 studied the expression of both hsp 27 and hsp 72
in the residual Purkinje cells of MD patients, and theirresults indicated that induction of both hsp 27 and hsp
72 occurs via a self-protective mechanism similar to thatof hsp 32.4, 5
In conclusion, the present study demonstrated that i)
in MD, approximately 50% of the Purkinje cells in the
cerebellum die due to maldevelopment and degeneration.
ii) In the residual Purkinje cells of patients with this disease, CCS expression is close to normal as a protective
response to a decrease of SOD1 activity due to chronic
copper deficiency. iii) In response to the increase of oxidative stress due to decreased SOD1 activity, hsp 32 is
also induced as another protective mechanism.

6 Becker LE, Yates AJ. Metabolic disease. In: Davis RL,
Robertson DM, editors. Textbook of Neuropathology, 3rd ed.
Baltimore: Williams & Wilkins; 1997. p. 407-91.
7 Ince PG, Wharton SB. Disease of movement and systemdegenerations. In: Love S, Louis DN, Ellison DW, editors. Greenfield’s Neuropathology, 8th ed. London: Edward
Arnold; 2008. p. 889-981.
8 Ghatak NR, Hirano A, Poon TP, French HJ. Trichopoliodystrophy. II. Pathological changes in skeletal muscle and nervous
system. Arch Neurol. 1972;26:60-72. PMID: 4108802.
9 Purpura DP, Hirano A, French JH. Polydendritic Purkinjecells in X-chromosome linked copper malabsorption: a Golgi
study. Brain Res. 1976;117:125-9. PMID: 990927.
10 Troost D, van Rossum A, Straks W, Willemse J. Menkes’
kinky hair disease. II. A clinicopathological report of threecases. Brain Dev. 1982;4:115-26. PMID: 7091568.
11 Shibata N, Hirano A, Kobayashi M, Umahara T, Kawanami
T, Asayama K. Cerebellar superoxide dismutase expression in
Menkes’ kinky hair disease: an immunohistochemical investigation. Acta Neuropathol. 1995;90:198-202. PMID: 7484097.
12 Takikawa M, Kato S, Esumi H, Kurashima Y, Hirano A,
Asayama K, et al. Temprospatial relationship between theexpressions of superoxide dismutase and nitric oxide synthasein the developing human brain: immunohistochemical andimmunoblotting analyses. Acta Neuropathol. 2001;102:572-8.
PMID: 11761717.
13 Fridovich I. Superoxide dismutases. Adv Enzymol Relat
Areas Mol Biol. 1986;58:61-97. PMID: 3521218.
14 Kato S, Kato M, Abe Y, Matsumura T, Nishino T, Aoki M,
et al. Redox system expression in the motor neurons in amyotrophic lateral sclerosis (ALS): immunohistochemical studies
on sporadic ALS, superoxide dismutase 1 (SOD1)-mutatedfamilial ALS, and SOD1-mutated ALS animal models. Acta
Neuropathol. 2005;110:101-12. PMID: 15983830.
15 Kato S, Saeki Y, Aoki M, Nagai M, Ishigaki A, Itoyama Y, etal. Histological evidence of redox system breakdown causedby superoxide dismutase 1 (SOD1) aggregation is common to
SOD1-mutated motor neurons in humans and animal models.
Acta Neuropathol. 2004;107:149-58. PMID: 14648077.
16 Culotta VC, Klomp LW, Strain J, Casareno RL, Krems B,
Gitlin JD. The copper chaperone for superoxide dismutase. J
Biol Chem. 1997;272:23469-72. PMID: 9295978.
17 Rothstein JD, Dykes-Hoberg M, Corson LB, Becker M,
Cleveland DW, Price DL, et al. The copper chaperon CCS
is abundant in neurons and astrocytes in human and rodent
brain. J Neurochem. 1999;72:422-9. PMID: 9886096.
18 Casareno RL, Waggoner D, Gitlin JD. The copper chaperone
CCS directly interacts with cupper/zinc superoxide dismutase.
J Biol Chem. 1998;273:23625-8. PMID: 9726962.
19 Rae TD, Schmidt PJ, Pufahl RA, Culotta VC, O’Halloran
TV. Undetectable intracellular free copper: the requirementof a copper chaperone for superoxide dismutase. Science.
1999;284:805-8. PMID: 10221913.
20 Wong PC, Waggoner D, Subramaniam JR, Tessarollo L,
Bartnikas TB, Culotta VC, et al. Copper chaperone for superoxide dismutase is essential to activate mammalian Cu/Zn superoxide dismutase. Proc Natl Acad Sci USA. 2000;97:288691. PMID: 10694572.
21 Peña MM, Lee J, Thiele DJ. A delicate balance: homeostatic control of copper uptake and distribution. J Nutr.
1999;129:1251-60. PMID: 10395584.
22 Petris MJ, Smith K, Lee J, Thiele DJ. Copper-stimulated endocytosis and degradation of the human copper transporter,

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 expenditurefrom the president of Tottori University (S.K.).
The authors declare no conflict of interest.

REFERENCES
1 Menkes JH, Alter M, Steigleder GK, Weakley DR, Sung JH.
A sex-linked recessive disorder with retardation of growth,
peculiar hair, and focal cerebral and cerebellar degeneration.
Pediatrics. 1962;29:764-79. PMID: 14472668.
2 Aguilar MJ, Chadwick DL, Okuyama K, Kamoshita S. Kinky
hair disease. I. Clinical and pathological features. J Neuropathol Exp Neurol. 1966;25:507-22. PMID: 5922550.
3 Danks DM, Campbell PE, Stevens BJ, Mayne V, Cartwright E.
Menkes’ kinky hair syndrome. An inherited defect in copper
absorption with widespread effects. Pediatrics. 1972;50:188201. PMID: 5045349.
4 Kato S, Ito M, Ohama E, Mikoshiba K, Maeda N, Yen SH, etal. Immunohistochemical studies on cerebellar Purkinje cells
of Menkes’ kinky hair disease. Neuropathology. 1993;13:15966.
5 Kato S, Ito M, Ohama E, Mikoshiba K, Maeda N, Hirano A.
Immunohistochemical investigation on cerebellar Purkinjecells of Menkes’ kinky hair disease: disappearance of inositol
1,4,5-triphosphate receptor protein, and expression of phosphorylated neurofilament proteins, B-crystallin and stressresponse proteins. Neuropathology. 1993;13:305-9.

34

Immunohistochemistry of MD Purkinje cells
hCtr1. J Biol Chem. 2003;278:9639-46. PMID: 12501239.
23 Kato S, Sumi-Akamaru H, Fujimura H, Sakoda S, Kato M,
Hirano A, et al. Copper chaperone for superoxide dismutase
co-aggregates with superoxide dismutase 1 (SOD1) in neuronal Lewy body-like hyaline inclusions: an immunohistochemical study of familial amyotrophic lateral sclerosis with SOD1
gene mutation. Acta Neuropathol. 2001;102:233-8. PMID:
11585247.
24 Sumi-Akamaru H, Fujimura H, Kato S, Takayasu S, Eto M,
Ohama E, et al. Immunohistochemical analysis of copper
chaperone for superoxide dismutase in mouse central nervous
system: a comparative study with paraffin and frozen section.
Acta Histochem Cytochem. 2002;35:33-8.
25 Kato S, Horiuchi S, Liu J, Cleveland DW, Shibata N,
Nakashima K, et al. Advanced glycation endproductomodified superoxide disumutase-1 (SOD1)-positive inclusions
are common to familial amyotrophic lateral sclerosis patientswith SOD1 gene mutations and transgenic mice expressing human SOD1 with a G85R mutation. Acta Neuropathol.
2000;100:490-505. PMID: 11045671.
26 Kato S, Esumi H, Hirano A, Kato M, Asayama K, Ohama
E. Immunohistochemical expression of inducible nitric oxide
synthase (iNOS) in human brain tumors: relationships ofiNOS to superoxide dismutase (SOD) proteins (SOD1 and
SOD2), Ki-67 antigen (MIB-1) and p53 protein. Acta Neuropathol. 2003;105:333-40. PMID: 12624786.
27 Kato S, Shimoda M, Watanabe Y, Nakashima K, Takahashi
K, Ohama E. Familial amyotrophic lateral sclerosis with a
two base pair deletion in superoxide dismutase 1: gene multisystem degeneration with intracytoplasmic hyaline inclusions
in astrocytes. J Neuropathol Exp Neurol. 1996;55:1089-101.
PMID: 8858006.
28 Kato S, Hayashi H, Nakashima K, Nanba E, Kato M, Hirano A,
et al. Pathological characterization of astrocytic hyaline inclusions in familial amyotrophic lateral sclerosis. Am J Pathol.
1997;151:611-20. PMID: 9273821.
29 Kato S, Horiuchi S, Nakashima K, Hirano A, Shibata N,
Nakano I, et al. Astrocytic hyaline inclusions contain ad-vanced glycation endproducts in familial amyotrophic lateralsclerosis with superoxide dismutase 1 gene mutation: immunohistochemical and immunoelectron microscopical analyses.
Acta Neuropathol. 1999;97:260-6. PMID: 10090673.
30 Friede RL. Gross and microscopic development of central
nervous system. In: Friede RL, editor. Developmental neuropathology, 2nd ed. Berlin: Springer-Verlag; 1989. p.12-6.
31 Kato S, Hayashi H, Mikoshiba K, Hirano A, Yen SH, Ohama
E. Purkinje cells in olivopontocerebellar atrophy and granulecell-type cerebellar degeneration: an immunohistochemical
study. Acta Neuropathol. 1998;96201:67-74. PMID: 9678515.
32 Hausmann R, Seidl S, Betz P. Hypoxic changes in Purkinjecells of the human cerebellum. Int J Legal Med. 2007;121:17583. PMID: 17031692.
33 Bertinato J, Iskandar M, L’Abbé MR. Copper deficiency induces the upregulation of the copper chaperone for Cu/Zn superoxide dismutase in weanling male rats. J Nutr. 2003;133:2831. PMID: 12514262.
34 Prohaska JR, Broderius M, Brokate B. Metallochaperone for
Cu,Zn-superoxide dismutase (CCS) protein but not mRNA
is higher in organs from copper-deficient mice and rats. Arch
Biochem Biophys. 2003;417:227-34. PMID: 12941305.
35 Gybina AA, Prohaska JR. Variable response of selected cuproproteins in rat choroid plexus and cerebellum following
perinatal copper deficiency. Genes Nutr. 2006;1:51-9. PMID:
18850220.
36 Iwata M, Hirano A, French JH. Thalamic degeneration in
X-chromosome-linked copper malabsorption. Ann Neurol.
1979;5:359-66. PMID: 443770.
37 Iwata M, Hirano A, French JH. Degeneration of the cerebellar
system in X-chromosome-linked copper malabsorption. Ann
Neurol. 1979;5:542-9. PMID: 475349.
38 Inagaki M, Hashimoto K, Yoshino K, Ohtani K, Nonaka I,
Arima M, et al. Atypical form of Menkes kinky hair disease
with mitochondrial NADH-CoQ reductase deficiency. Neuropediatrics. 1988;19:52-5. PMID: 2452375.

35