Case report

Protracted juvenile neuronal ceroid lipofuscinosis—An autopsy report

and immunohistochemical analysis

Yuki Anzai a,b,*, Masaharu Hayashi b, Noboru Fueki c, Kiyoko Kurata d, Tatsuo Ohya e

a Department of Pediatrics, National Rehabilitation Center of Disabled Children, 1-1-10, Komone, Itabashi-ku, Tokyo 173-0037, Japanb Department of Clinical Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan

c Department of Neurology, Nagano Children Hospital, Nagano, Japand Department of Pediatrics, Tokyo Metropolitan Medical Center for SMID, Tokyo, Japan

e Rikkyo University Clinic, Tokyo, Japan

Received 12 August 2005; received in revised form 3 December 2005; accepted 22 December 2005

Abstract

The juvenile form of neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene, and is characterized by progressive

loss of vision and development of motor deficits. A few patients exhibit a more protracted clinical course and are diagnosed with protracted

JNCL (PJNCL). Here, we report the autopsy in a case of PJNCL in a 55-year-old male and immunohistochemical examination of the

involvement of oxidative stress and glutamate excitotoxicity in neurodegeneration. The patient was born to consanguineous parents (I assume

this means that the parents were related. If not, then the sentence will need to be changed again.) and had brothers with similar neurological

disease. He showed mental retardation and visual impairment in the first decade which gradually developed along with motor dysfunction for

over 40 years. At autopsy, the cerebral pyramidal neurons revealed deposition of lipopigments, which demonstrated ‘finger print’ and

curvilinear profiles on electron microscopy. He also exhibited cerebellar cortical atrophy, fibrillary gliosis in the white matter, and rarefication

in the globus pallidus. Immunohistochemically, the number of neurons immunoreactive for advanced glycation end product was elevated in the

cerebellar cortex and midbrain. Immunoreactivity for excitatory amino acid transporter 1 was reduced in the cerebellar dentate and inferior

olivary nuclei. These findings suggest that oxidative damage to proteins and disturbed glutamate transport can be involved in PJNCL.

q 2006 Elsevier B.V. All rights reserved.

Keywords: Neuronal ceroid lipofuscinosis; Protracted juvenile form; Autopsy; Immunohistochemistry; Oxidative stress; Glutamate transporter

1. Introduction

Neuronal ceroid lipofuscinosis (NCL) is a progressive,

neurogenetic disease that occurs in children and occasionally

in adults. The juvenile form of NCL (JNCL) is caused by

mutations in the CLN3 gene, and is clinically characterized

by progressive loss of vision commencing between the ages

of 4 and 7 [1]. Patients usually become blind within 2–10

years. Mental disturbance and epilepsy follow, and

additional motor symptoms complete the clinical picture. A

few patients exhibiting a more benign and protracted clinical

0387-7604/$ - see front matter q 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.braindev.2005.12.004

* Corresponding author. Address: Department of Pediatrics, National

Rehabilitation Center of Disabled Children, 1-1-10, Komone, Itabashi-ku,

Tokyo 173-0037, Japan. Tel.: C81 3 3974 2146; fax: C81 3 3554 6176.

E-mail address: y-annzai@ryouiku.or.jp (Y. Anzai).

progression are diagnosed as having protracted JNCL

(PJNCL) [2]. In PJNCL, vision deteriorates gradually for

decades, and cognitive and/or motor dysfunction may appear

during or after middle age. Recently, it has been established

that PJNCL is associated with compound heterozygous

deletions of the CLN3 gene [3].

Here we report the autopsy in a case of PJNCL in a 55-

year-old male and immunohistochemical examination of the

deposition of oxidative products and expression of

glfcutamate transporters to investigate possible mechanisms

of neurodegeneration.

2. Case report

The subject was born to consanguineous parents with

normal delivery after uneventful pregnancy. His older sister

died of undetermined causes in the neonatal period.

Brain & Development 28 (2006) 462–465

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Y. Anzai et al. / Brain & Development 28 (2006) 462–465 463

The eldest brother is healthy, whereas his older and younger

brothers suffered from progressive visual impairment and

motor disability in the second decade, and the former died of

pneumonia in his fifties. The case showed normal infantile

development, but mild mental retardation became obvious

as the child became old enough to attend school. At the age

of nine, he showed visual impairment, which gradually

progressed with the retinal involvement. At the age of 15, he

became completely blind. He developed a spastic gait in his

20s, was gradually reduced to uttering words, and could not

stand at around the age of 50. He was admitted to Tokyo

Metropolitan Fuchu Medical Center at the age of 55 years.

He was bedridden, having spasticity and limited abduction

of eyeballs, but could utter two word-sentences. He was

blind and ophthalmologic examination demonstrated reti-

nochorioid degeneration. The tentative diagnosis was

familial spastic paraplegia with retinal degeneration,

although serum level of very long chain fatty acids, blood

amino acid profiles, urinary organic acid profile, lactate and

pyruvate in the cerebrospinal fluid and titers of antibody

against human immunodeficiency viruses were all normal.

He died of pneumonia. During the course of his illness, he

never exhibited convulsions. Genetic analysis was not

granted by his family.

Autopsy was performed 3 h after death. The brain

weighed 884 g. The cerebrum, cerebellum, and brainstem

were small, but there were no anomalous changes. The

cerebral sulci were mildly enlarged. On cross sections, the

ventricles were enlarged, the optic tracts were thin, and

melanin pigmentation was attenuated in the substantia nigra

and locus ceruleus. Microscopically, cytoarchitectures

Fig. 1. Representative illustrations in routine histochemistry. (A) Granular lipopi

frontal lobe cortex, Kluver–Barrera staining, scale barZ20 mm. (B). Fibrillary glio

the globus pallidus, Holzer staining. (C). Rarefication in the bilateral globus pallid

cortex, hematoxylin-eosin staining, scale barZ40 mm.

including lamination were spared in the cerebral cortex,

and there was no severe neuronal loss except in area CA2 of

the hippocampus. However, the pyramidal cells showed

storage of lipofuscin-like materials predominantly in the

third and fifth layers (Fig. 1A). The deep cerebral white

matter showed fibrillary gliosis (Fig. 1B), while astrocytosis

was observed in the fifth and sixth layers in addition to the

subcortical white matter. Gliosis was marked in the

temporal lobe. The striatum revealed neuronal deposition

of lipopigments and mild astrocytosis. Rarefication with

loss of myelinated fibers in the absence of gliosis was found

in the medial and lateral segments in the oral and caudal

globus pallidus (Fig. 1C), respectively. There was fibrillary

gliosis in the thalamus, but the subthalamic nucleus was

spared. The cerebellar cortex showed moderate loss of the

Purkinje cells (Fig. 1D). Both basket and Golgi cells had

lipopigment deposition. The dendate nucleus exhibited

fibrillary gliosis in the hilus and neuronal deposition of

lipopigments. There was mild neuronal loss in the

ventromedial part of the substantia nigra, with no

lipopigment deposition. The nuclei of cranial nerves

revealed mild neuronal storage of lipopigments. The inferior

olivary nucleus showed mild neuronal loss, lipopigment

deposition and moderate gliosis. The gracil fasciculus

demonstrated mild reduction of the myleinated fibers with

gliosis in the cervical segment of the spinal cord. Neither

neuronal loss nor lipopigment deposition was detected in the

ventral horn, Clark’s column, intermediolateral and Onuf’s

nuclei. Upon examination by electron microscopy, both

‘finger print’ and curvilinear profiles were recognized in the

neurons (Fig. 2).

gments were stained by luxol fast blue stain in the cortical neurons in the

sis in the temporal white matter (the lower half of the photograph) but not in

us, hematoxylin-eosin staining. (D). Loss of Purkinje cells in the cerebellar

Fig. 2. Representative illustrations on electron microscopic examination. Finger print profiles (A) and curvilinear profiles (B) were found.

Y. Anzai et al. / Brain & Development 28 (2006) 462–465464

We performed immunohistochemistry for markers of

oxidative stress and glutamate excitotoxicity in this case

subject and in a 61-year-old male control with no brain lesions.

Serial sections of the cerebral cortex, basal ganglia,

cerebellum, midbrain and medulla oblongata were treated

with mouse monoclonal antibodies against 8-hydroxy-2-

deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (4-HNE)

(Wako Chemical, Osaka, Japan; diluted to 1:2000), advanced

glycation end product (AGE) (Kumamoto Immunochemical

Laboratory, Kumamoto, Japan; diluted to 1:2000), and

excitatory amino acid transporters 1 and 2 (Novocastra

Laboratories, Newcastle, UK; diluted to 1:40) overnight

after antigen retrieval. A few hippocampal neurons and glial

cells demonstrated immunoreactivity for 8-OHdG, whereas

the control did not. In the case subject and control, 4-HNE

deposition was visualized in some neurons in the cerebral

cortex, the globus pallidus, and the cerebellar dendate nucleus.

In the case subject and control, neuronal immunoreactivity for

AGE was observed in neurons of the cerebral cortex, the

globus pallidus and the dentate nucleus. However, the number

of neurons immunoreactive for AGE increased in the

remaining Purkinje cells, the mesencephalic central gray

matter and substantia nigra (Fig. 3A). In the case subject and

control, the expression of EAAT1 and EAAT2 was visualized

in the neuropil of the cerebral cortex and basal ganglia, the

cerebellar molecular layer, the dentate nucleus, and the gray

matter of brainstem. Tissue from the case subject showed mild

to severe reduction of immunoreactivity for EAAT1 in the

dentate and inferior olivary nuclei (Fig. 3B).

Fig. 3. Representative illustrations in routine immunohistochemistry. (A) The rem

glycation end product, scale barZ20 mm. (B). Immunoreactivity for excitatory glut

in comparison with the neighboring structures, scale barZ400 mm.

3. Discussion

The case subject had a family history, developed visual

impairment with retinal involvement in the second decade,

and showed progressive motor and mental disturbances

without epileptic seizures. Neuropathologically, he demon-

strated cerebellar cortical degeneration, fibrillary gliosis in

the cerebral white matter and thalamus, and neuronal

lipopigment deposition in addition to the optic tract lesion.

These clinical and neuropathological features in addition to

typical findings on electron microscopy led to the diagnosis

of PJNCL, although the CLN3 gene abnormality was not

verified. Irrespective of preserved neuronal density in

histochemistry, the decrease in brain weight suggested

neuronal loss in the cerebral cortex, which can be involved

in psychomotor retardation in this case. Gliosis in the

cerebral white matter and lipopigment deposition in the

abducens nucleus might be related to spastic gait and limited

abduction of eyeballs, respectively. The lesions in the

Purkinje cells, the dentate and inferior olivary nuclei in this

case are usually correlated with epilepsy in JNCL. However,

the cause of lack of epileptic seizures still remains to be

determined. Rarefication with loss of myelinated fibers in

the absence of gliosis in the globus pallidus is uncommon in

JNCL, and partly mimics extrapontine myelinolysis [4].

We have shown that oxidative stress can be implicated in

child-onset neurodegenerative disorders such as hereditary

nucleotide repair disorders, subacute sclerosing panence-

phalitis and spinal muscular atrophy [5–7]. AGE and

aining Purkinje cell (arrow) demonstrated immunoreactivity for advanced

amate transporter 1 was reduced in the inferior olivary nucleus (arrowheads)

Y. Anzai et al. / Brain & Development 28 (2006) 462–465 465

8-OHdG are considered markers of protein damage by

glycoxidation and oxidative stress to nucleosides,

respectively. In our case, protein glycoxidation was

augmented in the cerebellar cortex and midbrain, although

oxidative stress to DNA was minimally visualized only in

the hippocampus. In three cases of the late infantile form of

NCL (LINCL), we found that lipid peroxidation, assessed

by the degree of 4-HNE deposition, was predominant and

widely spread (our unpublished data), in sharp contrast with

our current case subject. Glial glutamate transporters

modulate neurotransmission by maintaining low concen-

trations of extracellular glutamate and preventing glutamate

excitotoxicity [5]. Constitutive expressions of EAAT1 and

EAAT2 by astrocytes were well visualized in the neuropil of

the gray matter in our case subject. Of particular interest,

our subject showed a disturbed expression of EAAT1 in the

cerebellar dentate and inferior olivary nucleus, which may

be related to moderate to severe gliosis, because gliosis can

reduce the EAAT expressions in cases of West syndrome

[8]. In the aforementioned three cases of LINCL, we found

commonly disturbed expression of both glutamate trans-

porters in the cerebellar molecular layer, and there seems to

be a difference in the degree and distribution of glutamate

excitotoxicity among LINCL and PJNCL.

The immunohistochemical data presented herein suggest

that oxidative damage to proteins in addition to mild

disturbance of glutamate transport may be involved in

neurodegeneration of PJNCL. Cases of PJNCL with

autopsies are rare, and the immunohistochemical analysis

in our case will offer clues to delineate the pathogenesis of

NCL in general.

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