Higuchi Et Al. 2012. Enzyme Replacement Therapy -ERT- Procedure for Mucopolysaccharidosis Type II -MPS II- By Intraventricular Administration -IVA- In Murine MPS II

8/11/2019 Higuchi Et Al. 2012. Enzyme Replacement Therapy -ERT- Procedure for Mucopolysaccharidosis Type II -MPS II- By I

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Enzyme replacement therapy (ERT) procedure for mucopolysaccharidosis type II(MPS II) by intraventricular administration (IVA) in murine MPS II

Takashi Higuchi a, Hiromi Shimizu a,b,Takahiro Fukuda c,Shiho Kawagoe a, Juri Matsumoto a,Yohta Shimada e, Hiroshi Kobayashi a,d,e, HiroyukiIda a,d,e, Toya Ohashi a,d,e, Hideto Morimoto f,Tohru Hirato f, Katsuya Nishino f, Yoshikatsu Eto a,a Department of Genetic Diseases & Genomic Science, The Jikei University School of Medicine, Tokyo, Japanb Department of Physics, Faculty of Science and Engineering, Waseda University, Tokyo, Japanc Neuropathology, The Jikei University School of Medicine, Tokyo, Japand Department of Pediatrics, The Jikei University School of Medicine, Tokyo, Japane Department of Gene Therapy, Institute of DNA Medical, The Jikei University School of Medicine, Tokyo, Japanf JCR Pharmaceuticals Co., Ltd. Hyogo, J apan

a b s t r a c ta r t i c l e i n f o

Article history:

Received 8 March 2012

Received in revised form 1 May 2012

Accepted 2 May 2012

Available online 18 May 2012

Keywords:

Hunter syndrome

Mucopolysaccharidosis

Lysosomal storage disease

Iduronate-2-sulfatase

IntraventricularEnzyme replacement therapy

Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is a lysosomal storage disorder caused by a de-

ciency of iduronate-2-sulfatase (IDS) and is characterized by the accumulation of glycosaminoglycans

(GAGs). MPS II has been treated by hematopoietic stem cell therapy (HSCT)/enzyme replacement therapy

(ERT), but its effectiveness in the central nervous system (CNS) is limited because of poor enzyme uptake

across the bloodbrain barrier (BBB). To increase the efcacy of ERT in the brain, we tested an intraventric-

ular ERT procedure consisting of repeated administrations of IDS (20 g/mouse/3 weeks) in IDS-knockout,

MPS II model mice. The IDS enzyme activity and the accumulation of total GAGs were measured in mouse

brains. The IDS activity was signicantly increased, and the accumulation of total GAGs was decreased in

the MPS II mouse brains treated with multiple administrations of IDS via intraventricular ERT. Additionally,

a high level of IDS enzyme activity was appreciated in other MPS II mouse tissues, such as the liver, spleen,

testis and others. A Y-maze was used to test learning and memory after repeated intraventricular ERT withIDS. The IDS-treated mouse groups recovered the capacity for short-term memory and activity. Although

large and small vacuoles were found at the margin of the cerebellar Purkinje cells in the disease-control

mice, these vacuoles disappeared upon treated with IDS. Loss of vacuoles was also observed in other tissues

(liver, kidney and testis). These results demonstrate the possible efcacy of an ERT procedure with intraven-

tricular administration of IDS for the treatment of MPS II.

Crown Copyright 2012 Published by Elsevier Inc. All rights reserved.

1. Introduction

Mucopolysaccharidosis type II (MPS II), also known as Hunter syn-

drome, is an X-linked, recessive inborn error of metabolism caused by

mutations in the gene coding for the lysosomal enzyme, iduronate-2-

sulfatase (IDS)[1]. The enzyme IDS removes the sulfate group from

heparin sulfate and dermatan sulfate. In the absence of IDS, these sul-

fated glycosaminoglycans (GAGs) accumulate in the lysosomes. In

Japan, MPS II is the most common type of mucopolysaccharidosis, ac-

counting for 60% of documented cases [2]. MPS II may occur in mild or

severe forms, and there is wide variability in symptoms for individual

patients. The severe form is characterized by progressive somatic and

neurological disease. Early treatment of MPS II patients with enzyme

replacement therapy (ERT)/hematopoietic stem cell therapy (HSCT)

is very important[3].

Treatment with ERT is one of the therapeutic approaches for pa-

tients who cannot be treated with HSCT with at least 6 different lyso-

somal storage diseases (LSDs) such as Gaucher disease, Pompe

disease, Fabry disease, and MPS I, II, and VI[49]. Usually, ERT treat-

ments are performed by intravenous (IV) infusion of the enzyme.

ERT has been successful in treating hepatosplenomegaly, card-

iomegaly, and skin, etc., ERT has not been proven to be ineffective

in CNS disease, but may have limited effects on the CNS and skeletal

system. The neuropathic forms of LSD, those that affect the CNS, are

generally difcult to treat with ERT because of limited enzyme uptake

Molecular Genetics and Metabolism 107 (2012) 122128

Abbreviations:MPS II, mucopolysaccharidosis type II; ERT, enzyme replacement

therapy; IDS, iduronate-2-sulfatase; BBB, bloodbrain barrier; CNS, central nervous

system; HSCT, hematopoietic stem cell therapy; LSD, lysosomal storage disease; IV, in-

travenous infusion; GAG, glycosaminoglycan; CT, computed tomography; H&E, hema-

toxylin and eosin.

Corresponding author at: Department of Genetic Diseases and Genomic Science,

The Jikei University School of Medicine, 3-25-8, Nishi-Shinbashi, Minato-ku, Tokyo

105-0003, Japan. Fax: +81 3 5400 1293.

E-mail address:[email protected](Y. Eto).

1096-7192/$ see front matter. Crown Copyright 2012 Published by Elsevier Inc. All rights reserved.

doi:10.1016/j.ymgme.2012.05.005

Contents lists available at SciVerse ScienceDirect

Molecular Genetics and Metabolism

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http://dx.doi.org/10.1016/j.ymgme.2012.05.005http://dx.doi.org/10.1016/j.ymgme.2012.05.005http://dx.doi.org/10.1016/j.ymgme.2012.05.005mailto:[email protected]://dx.doi.org/10.1016/j.ymgme.2012.05.005http://www.sciencedirect.com/science/journal/10967192http://www.sciencedirect.com/science/journal/10967192http://dx.doi.org/10.1016/j.ymgme.2012.05.005mailto:[email protected]://dx.doi.org/10.1016/j.ymgme.2012.05.005


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across the bloodbrain barrier (BBB) [10]. In response to this chal-

lenge, many trials have been established to evaluate potential treat-

ments for the neuropathic forms of LSDs[1113].

Here, we treated theIdsgene-knockout, MPS II mouse model with

direct, repeated intraventricular administrations of IDS. This proce-

dure bypasses the BBB, allowing for direct uptake of the IDS enzyme

by mouse neuronal cells. The IDS enzyme activity, accumulation of

total GAGs, and histopathology were examined in the MPS II mouse

tissues. This examination was conducted after repeat administrationsof IDS via intraventricular ERT, and it demonstrated that IDS enzyme

activity was maintained highly and total GAGs were decreased in the

MPS II mouse brain. Furthermore, the mice that underwent treatment

showed improved short-term memory and activity during the Y-

maze test. In addition, histological ndings demonstrated a loss of

vacuoles at the margin of Purkinje cells and other cell types (liver

cells, tubule cells, and Leydig cells). These results strongly suggest

the potential of intraventricular ERT as an effective treatment for

MPS II.

2. Materials and methods

2.1. Animals

Male MPS II model mice, iduronate-2-sulfatase knockout (IDS-KO)

mice, were produced by JCR Pharmaceuticals Co., Ltd. (Hyogo, Japan).

In these mice, the Ids gene was deleted from exon 2 to exon 5. The

IDS-KO mouse was bred from a C57BL/6 background strain and has

a null mutation in the Ids gene. The normal control, wild-type mice

were bred from a C57BL/6 strain. Mice were housed in groups of 3

per cage in a colony room under a 12-hour lightdark cycle. Rodent

food and water were available ad libitum. All animal studies were ap-

proved by the Animal Experiments Committee at The Jikei University.

2.2. Computed tomography (CT) scan experiment

The skulls of the mice were scanned using a Latheta LCT-200 ex-

perimental animal CT system (Hitachi-Aloka Medical, LTD, Tokyo,

Japan).

2.3. Foot print pattern analysis

The treated MPS II mice and the control, wild-type mice were test-

ed during the same sessions to minimize variability. Analysis of the

walking footprint pattern was performed as previously described

[14]. Gait abnormalities were identied by painting the mouse paws

with non-toxic, washable paint and then by placing the mice in a cor-

ridor lined with white paper.

2.4. Intraventricular ERT administration

Mice were anesthetized with pentobarbital sodium (Abott Japan

Co., Ltd., Tokyo, Japan) and mounted in a stereotaxic apparatus(Narishige Co., Ltd., Tokyo, Japan). A total of 20 g of recombinant

human iduronate-2-surfatase (JCR Pharmaceuticals Co., Ltd., Hyogo,

Japan) or saline (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) was

injected into the lateral ventricle which was repeated 4, 6 and

8 times every 3 weeks with a 30-gauge needle [coordinates relative

to bregma: anteroposterior 3.0 mm; lateral +2.0 mm (midline);

dorsoventral3.0 mm from skull surface]. The IDS dose was deter-

mined by reference to the previous report[15].

2.5. Tissue collection

Tissue samples were collected from the treated and untreated

IDS-KO and wild-type mice at the end of each treatment session.

The mice were anesthetized and sacriced by cardiac perfusion

using 40 ml of phosphate-buffered saline (PBS()). Thetissue samples

were collected, and half of each sample was xed for histopathological

analysis (H&E staining, Alcian blue staining, and immunostaining),

while the other half was frozen in dry ice prior to processing with the

IDS activity assay and total GAG accumulation analysis. The brains

were divided into three regions: front section cerebral, posterior section

cerebral, and cerebellum.

2.6. IDS activity analysis

IDS activity was determined with homogenized tissues in water,

as described using the articial substrate 4-methylumbelliferyl-

alpha-iduronide-2-sulfate (MU-Idu-2S) [Moscerdam Substrates,

Oegstgeest, Netherlands]. Tissue protein concentrations were deter-

mined using the BCA protein assay kit (Wako Pure Chemical Indus-

tries, Ltd., Osaka, Japan). The measurement of IDS activity was

conducted as previously described [15]. A total of 15 g of protein

was incubated with 20l of the substrate solution (1.25 mM

MU-Idu-2S-Na2, 0.1 M Na-acetate/0.1 M acetic acid buffer (pH

5.0), 10 mM Pb-acetate) for 4 h at 37 C in a dark room. Next, 40 l

of Pi/Ci buffer (0.4 M Na-phosphate/0.2 M citrate buffer (pH 4.5),

0.02% Na-azide) and 10l LEBT(puried Lysosomal Enzymes from

Bovine Testis) were added to the incubation samples, which were

then incubated for an additional 24 h at 37 C. Finally, the reaction

was stopped by adding 200l of stop buffer(0.5 M NaHCO3/0.5 M

Na2CO3 buffer (pH 10.7),0.025% Triton X-100) to themixture. Thelib-

erated uorescence of the 4-methylumbelliferone was then measured

with a RF-5300PC spectrouorophotometer at 365 nm excitation and

460 nm emission (SIMADZU Co., Kyoto, Japan). The enzyme activity

was expressed as nmol/4 h/g protein, as calculated with the standard

curve of the uorogenic substrate, 4-methylumbelliferyl--iduronide

(Sigma-Aldrich Japan Co., LLC. Tokyo, Japan). We took the average of

34 experiments or more.

2.7. Tissue total GAG accumulation assay

Treated and untreated MPS II and wild-type mouse tissues were

homogenized in an NaCl-T buffer (0.9% NaCl, 0.2% Triton X-100).The samples were shaken overnight at 4 C and microcentrifuged

for 5 min at 3000 rpm, and the supernatants were collected. Total

GAG levels were measured using the Wieslad sGAG quantitative

Alcian blue-binding assay kit (Euro-Diagnostica, Malm, Sweden)

[15]. First, 50 l of homogenized samples was incubated with 50 l

of 8 M Guanidine-HCl at room temperature (RT) for 15 min. Then,

50l of STA solution (0.3% H2SO4, 0.75% Triton X-100) was added

for 15 min at RT, with a solution of Alcian blue working in the solution

for 15 min. The samples were then centrifuged for 15 min at

12,000 rpm and rinsed with DMSO solution (40% DMSO, 0.05 M

MgCl2). Finally, 500l of Gu-prop solution (4 M Guanidine-HCl,

33% 1-propanol, 0.25% Triton X-100) were added to the pellets, and

the mix was allowed to completely dissolve. Alternatively, absor-

bance was read in the SH-9000 spectrophotometer (Hitachi High-Technologies Co., Tokyo, Japan) at 600 nm. GAG was normalized to

the protein concentration. Protein concentration was measured

with BCA Protein Assay kits. The GAG ratio was expressed as mg of

GAG/mg of protein, as calculated through the standard curve of the

GAG substrate, chondroitin sulfate-6 (Euro-Diagnostica Co., Malm

Sweden). We took the average of 34 experiments or more.

2.8. Spontaneous alternation behavior

Spontaneous alternation behavior requiring attention and work-

ing memory was assessed using a Y-maze [16,17]. Each arm of the

Y-maze was 40-cm long, ranging from 3 to 10 cm wide from the bot-

tom to the top and 12.5 cm high. The arms were labeled A, B, or C, and

both arms were positioned at equal angles (diverging at a 120). The

123T. Higuchi et al. / Molecular Genetics and Metabolism 107 (2012) 122128


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animal was placed in the arm facing the center (Arm A) and was

allowed to freely explore for an 8-minute test session without the re-

inforcement of food, water, or electric foot shock. An arm entry was

dened as the entry of all four paws into one arm. The sequence of

arm entries was manually recorded. Alternation behavior (actual al-

ternations) was dened as consecutive entry into the three arms

(i.e., Arm A to B to C). The maximum number of alternations was

calculated by measuring the total number of arm entries minus 2,

and the percentage of alternation behavior was calculated as (actualalternations/maximum alternations)100. Each mouse was tested

four times with the experiment. The total number of arms entered

during the sessions, which reects locomotor activity, was also

recorded. We took the average of 34 experiments or more.

2.9. Pathologically analysis

Tissues (brain, liver, kidney and testis) were dissected and xed

with a 4% formaldehyde neutral buffer solution (Wako Co., LTD,

Osaka, Japan) and embedded in parafn. The sliced sections were

then stained with H&E, toluidine blue stain, and immunostaining

against the mouse lysosomal marker, lysosomal-associated mem-

brane protein 2 (Lamp2) [18,19]. Immunostaining was performed

according to a procedure previously described[20]. Briey, the slicedsections were made permeable with PBS () containing 0.1% Triton

X-100, and the slides were blocked with peroxidase and locked with

3% H2O2 in methanol. They were then blocked with PBS containing

10% normal goat serum (Vector Laboratories, CA, USA). The tissues

were incubated with the anti-LAMP2 rabbit polyclonal antibody

(AnaSpec Inc., catalog # 54234, CA, USA,). Biotinylated anti-rabbit

IgG (H+L) antibody (Vector Laboratories, CA, USA) in PBS was then

applied, and the slides were serially stained with an avidinbiotin

complex using the ABC elite kit (Vector Laboratories, CA, USA) with

3, 3-diaminobenzidine as a chromogen. Counterstaining for cell nu-

clei was performed with Mayer hematoxylin.

3. Results

3.1. Iduronate-2-sulfate-decient mice have a coarse face because of

bone deformation

We studied the faces of the MPS II mice using X-ray computed to-

mography (CT) (Fig. 1A). The IDS-knockout, MPS II model mice

exhibited a phenotype characterized by a coarse face, large round

eyes, and a rounded nose when compared with the wild-type mice.

Furthermore, the X-ray CT analysis showed hyperostosis and nose

dysostosis in the MPS II mouse head. The walking footprint patterns

of the MPS II mice and the wild-type controls at 21 weeks of age are

illustrated (Fig. 1B). Most wild-type mice walked in a straight line

away from the investigator. By contrast, MPS II mice walked unstead-

ily and walked with a short-stepped gait that had approximately half

the stride length of the wild-type mice.

3.2. Intraventricular administration of ERT for the MPS II murine model

The IDS enzyme was injected into the MPS II mouse (21 week old)lateral ventricle 4 times. Mouse body weights were measured during

intraventricular ERT or administration of normal saline (Fig. 2A).

Body weights in the intraventricular ERT group were similar to

those in the control animals. Mice brains were divided into three re-

gions (front cerebral, posterior cerebral, and cerebellum) (Fig. 2B).

In the disease-control, MPS II mice, the frontal and posterior regions

of the brain showed almost negligible IDS enzyme activity. In con-

trast, in the intraventricular ERT group, cerebral IDS enzyme activity

increased approximately three-fold compared with the wild-type

group. Additionally, IDS activity in the cerebellum was elevated to

levels comparable to those in the wild-type group (Fig. 2C). The accu-

mulation of total GAGs was measured in the brain. Although the total

GAGs in the disease-control group was elevated, this elevation was

inhibited in the three brain regions after treatment with IDS via intra-ventricular ERT (Fig. 2D). We analyzed IDS enzyme activity following

IDS administration with intraventricular ERT not only in the brain but

also in other tissues (eye, lung, heart, liver, kidney, spleen, and testis).

A deciency in IDS enzyme activity was also found in other tissues of

the disease-control, MPS II mice. In the intraventricular ERT group,

IDS enzyme activity was increased to levels comparable to those in

the wild-type group (Fig. 2E).

3.3. Effects of treatment with multiple administration s of IDS via

intraventricular ERT for young adult MPS II mice

3.3.1. Analysis of alternation behavior and learning with the Y-maze test

in MPS II mice treated with intraventricular ERT

An IDS was injected into the MPS II mice (16 week old) brain,which was repeated 6 times. Although alternation behavior in the

disease-control, MPS II mouse group was signicantly decreased

compared with wild-type mice, this behavior was recovered in the

IDS-treated MPS II mouse group (Table 1). MPS II mice exhibited an

impairment in spontaneous alternation and behavior in a Y-maze

compared with all other groups (one-way ANOVA *P0.05). The

arm entries in the MPS II control group were elevated, while the

arm entries in the intraventricular ERT mice returned to normal

levels.

under side

Wild-typemice

MPSIImice

left side walking footprint patterns

Wild-typemice

MPSIImice

A B

Fig. 1. The upper column shows thewild-type mouse, and the lower column shows the MPSII mouse. (A) Pictures show the left side ofthe face and an X-rayCT image of the head in

a 21-week-old mouse. The arrow shows nose dysplasia in the MPS II mouse. (B) Walking footprint patterns show the forefoot (red) and hindfoot (black) in a 21-week-old mouse.

124 T. Higuchi et al. / Molecular Genetics and Metabolism 107 (2012) 122128


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3.3.2. Pathological analysis of the MPS II mouse treated with IDS

administration via intraventricular ERT

Additional injections of the IDS were performed 2 times in the

previous MPS II mouse (total 8 times). A pathologic analysis of the

mouse brain, liver, and testis was performed (Fig. 3). In the MPS II

mouse brain, many vacuolations were identied in the cytosol of

the Purkinje cells and the margin cells of the cerebellum. These vacu-

oles were visualized with H&E and toluidine blue staining. Lamp2

MPS II mice + IDS (n=3)

MPS II mice + saline (n=4)

Wild-type mice + saline (n=4)

(g)

(week-old)

33

31

29

27

2520 3321 2724 30

mouse body weight

IDS administrationpoint

cerebrum

posterior section cerebellumfront section

bregma

cerebrum

posterior section cerebellum

IDS enzyme activity in the brain

IDSenzymeactivity(nmol/4hr/mgp

rotein)

**

front section

**

0

300

600

900

1200




accumulation of total GAGs in the brain

cerebrum

posterior section cerebellumTheratioofMPSIImicevaluetoW

ild

typemicevalue(%)

** **




**

front section

17.68 0.61

22.66 1.18

20.28 0.45

17.76 0.83

24.02 0.75

19.32 0.20

16.23 2.05

21.00 0.38

18.51 0.95

front cerebral posterior cerebral cerebellum

(g / mg protein)

50

0

100

150

GAGs measurement value

A

C

E

B

D

IDSenzymeactivity(nmol/4hr/mgprotein)

eye lung heart liver kidney spleen testis

IDS enzyme activity in the tissues

100

0

200

300

Wild-type mice + saline (n=4) MPS II mice + saline (n=4) MPS II mice + IDS (n=3)

Fig. 2.(A) Average body weight change in MPS II mice treated with/without IDS. Error bars denote SEM. (B) Three brain regions of the cerebrum (front and posterior) and cere-

bellum. (C) Measurement of IDS enzyme activity in the brain treated with/without IDS using the MU-Idu-2S enzyme activity assay. All the mice were sacriced by transcardial

perfusion with PBS. Error bars denote SEM. **: P b0.01 (D) measurement of total GAGs in the brain treated with/without IDS using Alucian blue-binding assay (control wild-

type mice are 100%). The actual measurement value is shown in the gure. Error bars denote SEM. **: Pb0.01 (E) measurement of IDS enzyme activity in the tissues treated

with/without IDS using MU-Idu-2S enzyme activity assay. All the mice were sacriced by transcardial perfusion with PBS. Error bars denote the SEM.



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protein overexpression was observed in the cytosol of these cells. The

liver cells also had many vacuoles, and Lamp2 was overexpressed

throughout the entire liver. In the testis, many vacuoles were visual-

ized, and Lamp2 protein was overexpressed in the Leydig cells. Fol-

lowing administration of IDS with intraventricular ERT, the number

of vacuolations and the amount of Lamp2 protein were reduced in

the brain, liver, and testis. The changes in the mouse face were also

determined with/without administration of IDS via intraventricularERT. The wild-type mice had a sharp nose, normal eyes, and good-

quality hair, whereas the MPS II model mice had a coarse face and

poor-quality hair. The MPS II mice treated with IDS had also a coarse

face and poor-quality hair. However they were a little better than

non-treated MPS II mice (data not shown).

4. Discussion

We used an iduronate-2-sulfatase-knockout, Hunter syndrome

(mucopolysaccharidosis type II, MPS II) model mouse with a deletion

in theIds gene from exon 2 to exon 5. As a corollary, MPS II patients

generally exhibit bone and joint disorders. In most cases, the face of

the MPS II patient is coarse. X-ray analysis shows the characteristic

features of MPS, such as marked skeletal hyperostosis and nose defor-mation [21]. The iduronate-2-sulfatase-knockout, MPS II model

mouse has been reported to exhibit widespread bone deformities

[14]. Our MPS II model mice had large round eyes, hyperostosis in

the head and nose dysplasia (Fig. 1A). Furthermore, they walked

with a short-stepped gait and a funny, waddling walk when com-

pared with the wild-type mice (Fig. 1B). These results suggest that,

similar to humans, their symptoms were caused by bone and joint

dysfunctions. X-ray CT analysis and walking footprint analysis were

appropriate methods for evaluating bone and joint diseases in the

MPS II model mouse. The coarse face of the MPS II mice is similar to

that of human MPS II patients. The cause of the nose dysostosis is

total GAG accumulation in the bones.

The 21-week-old MPS II mice were injected with IDS into the lat-

eral ventricle, which was performed 4 times over a period of12 weeks. The body weights of mice treated with intraventricular

ERT were measured (Fig. 2A). There was no signicant difference in

the weights in the intraventricular ERT group compared with those

in the control group. We did not nd any abnormal inltration of in-ammatory cells in the brain, liver, and spleen with pathological anal-

ysis (data not shown). In this experiment, mice that treated IDS had

good coat and were fed normal chow. There was no signicant

change in body weight in mice. So we suspected that large stress of

administration and serious immunological response against IDS had

not occurred in the MPS II mouse. The IDS enzyme activity in the ce-

rebrum of the IDS-treated group was signicantly elevated compared

with the disease-control mice (Fig. 2C). The enzyme activity in the

front cerebral region was almost equal to that in the posterior cere-

bral region. It indicates that IDS was distributed through the entire

cerebrum via ventricular uid. Although IDS enzyme activity in the

cerebellum also increased, this level was equal to the level of IDS en-

zyme activity in the wild-type mouse. In the cerebellum, uptake of

the IDS enzyme may be poor compared with uptake in the cerebrum.

There might be some other mechanism about the poor uptake of IDS

in the cerebellum. The contact area of IDS might be the difference be-

tween cerebrum and cerebellum. Or there might be a difference in the

expression level of receptors (i.e. mannose-6-phosphate receptor)

Table 1

Analysis of alternation behavior and number of arm entries for 32-week-old MPS II

mice using the Y-maze test. IDS intraventricular ERT protocol: IDS 20g/3 weeks6

times for a 16-week-old mouse. Each mouse was tested four times per experiment.

Data were expressed as M SEM. One-way ANOVA *: P0.05. N.S.: not signicant.

Effects of multiple IDS intraventricular ERT on Y-maze behavior

Alternation behavior (%) Number of arm entries

Wild-type mice

(n=9)

MPS II mice+saline

(n=7)

MPS II mice+IDS

(n=3)

Wild-type

MPS II+ saline

MPS II

+IDS

brain(cerebellum)

H&E stain toluidine blue stain Lamp2 immunostaining

Wild-type

MPS II+ saline

MPS II

+IDS


liver


Wild-type

MPS II

+ saline

MPS II+IDS

testis

Fig. 3.H&E stain, toluidine blue stain and Lamp2 immunostaining analysis of 40-week-

old mouse tissues treated with IDS via intraventricular ERT (brain (cerebellum), liver,

and testis). Arrowhead design () is a vacuole in the Purkinje cell, or Leydig cell.

Arrow design () is a vacuole in the margin cell of the Purkinje cell.

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between cerebrum and cerebellum. We measured the accumulation

of total GAGs in three regions of the brain (Fig. 2D). Total GAGs in

the disease-control MPS II group was elevated by 2030% of the

wild-type group. However, following treatment with IDS via 4 times

intraventricular ERT, this accumulation was decreased by more than

50% of storage growth. In other tissues (eye, lung, heart, liver, kidney,

spleen, and testis), IDS activity was increased to levels comparable to

those in the wild-type mouse (Fig. 2E). The IDS injected into the lat-

eral cerebral ventricle was distributed in the ventricular

uid. Theuid ows through to the spinal uid. Thus, neuronal cells could di-

rectly uptake IDS instead of via BBB. We had expected an increase

of IDS enzyme activity in brain-specic manner. However, contrary

to expectations, the enzyme activity was also up in the tissues other

than the brain. There is a report that showed a systemic effect of

GDNF that was administered intracerebroventricular injections of

GDNF [22]. So, there might be possibly some sort of drug transfer

pathway from the brain to the body. The cerebrospinal uid con-

taining IDS might communicate with the systemic venous system

via the subarachnoid space. So we hypothesize that the enzyme was

carried via the bloodstream to many tissues.

We analyzed spontaneous alternation behavior in the MPS II

mouse treated with multiple administrations of intraventricular ERT

with the Y-maze test (Table 1). In many cases, MPS II patients have

neurological involvement of their disease[23]. There are many previ-

ously published studies using MPS II murine models [2427]. Until

now, however, the study to analyze the MPS II mouse behaviors has

not been done. Our study provides novel insight into MPS II mouse

behavior and the effects of treatment with IDS via intraventricular

ERT in the MPS II murine model. Alternation behavior (i.e., short-

term working memory) was signicantly decreased, and the number

of arm entries (i.e., physical exertion or activity) was signicantly in-

creased in disease-control mice compared with wild-type mice. These

data indicate that the MPS II model mouse may have short-term

working memory dysfunction and overactivity. These data showed

that administration of IDS via intraventricular ERT is an efcient

method to improve brain functions in the MPS II mouse. The results

of intraventricular enzyme administration in MPS II mice might be

able to offer treatment methods for other mucopolysaccharidosis(MPS I) and also for many other LSD (Gaucher disease, Krabbe disease

or metachromatic leukodystrophy) that exhibit neurologic symptoms

[2831].

The assessment of mice exposed to treatment with IDS via intraven-

tricular ERT demonstrated that the appearance of the coarse face and

hair was improved compared with untreated mice. Intraventricular

ERT could treat neurological ndings as well as systemic clinical fea-

tures, such as hepatosplenomegaly, and skin, among others. There

was no weight loss in the IDS-treated mouse, so we suspected that ad-

verse events, such as an IDS-immunoreactive response, were not cau-

sed by IDS administration. Untreated MPS II mice exhibited a coarse

face and looked older than treated mice. The round nose did not im-

prove in the treated mice. After 16-weeks of age mice were less re-

sponsive to ERT for bone involvement. Moreover, we analyzed manymouse tissues with histological procedures, such as hematoxilin-eosin

staining, toluidine blue staining and Lamp2 immunostaining (Fig. 3).

Although the cerebrum of MPS II mice exhibited changes compared

with wild-type and treated mice, there was a signicant pathological

difference in the cerebellum. There were massive vacuolations in the

cell body of Purkinje cells and in the margin cells. Additionally, the

Lamp2 protein, a lysosome marker, was also overexpressed in those

cells. The cerebellum might have more pathological involvement than

the cerebrum. Hepatomegaly is one major symptom in MPS II patients

[32]. There were large and massive vacuoles, and overexpression of the

Lamp2 protein, in a widespread area of the liver in the disease control

mouse. The liver had a tendency to become heavier when compared

with wild-type and IDS-treated mice (data not shown). Our

iduronate-2-sulfatase-decient, MPS II model mouse displays an

increased incidence of male infertility. The HOS (Hunter Outcome Sur-

vey) study also indicated that a higher incidence of infertility has been

reported in MPS II patients [33]. The genitourinary complications in

MPS II have been noted because the testis also shows pathological in-

volvement. The testis contained massive vacuoles and stained heavily

with Lamp2, especially in the tubular epithelial cells and the Leydig

cells. Although histological ndings in the brain, liver and testis cells

were signicantly improved by treatment with IDS via intraventricular

ERT. It is intriguing that intraventricular administration of the IDS en-zyme clearly stimulated enzyme activity in the CNS cells in addition

to the various systemic tissues, such as the liver, spleen, testis and kid-

ney cells. Therefore, intraventricular ERT can treat neurological ndings

as well as systemic clinical features, such as hepatosplenomegaly, skin,

and among others.

5. Conclusions

The recombinant human iduronate-2-sulfatase administration via

intraventricular ERT led to stimulation of the enzyme in thebrain in ad-

dition to other tissues (liver, spleen, testis, etc.) in the mouse. Further-

more, administration of the IDS via intraventricular ERT improved

brain functions including short-term memory and behavior. These re-

sults demonstrate the possible efcacy of an ERT procedure with intra-ventricular administration of IDS for the treatment of MPS II.

Acknowledgments

This work was supported by a Grant for Research on Measures for

Intractable Diseases from the Japanese Ministry of Health, Labor, and

Welfare. We are grateful to our colleagues in the Department of

Genetic Diseases & Genomic Science and the Department of Gene

Therapy for their excellent technical assistance. T.H. is main experi-

menter in this project. H.S., S.K., and J.M. help to measure IDS activity

and total GAGs. T.F. supports to analyze pathologically. Y.S., H.K., H.I.,

and T.O. take an active part in the discussion in this paper. H.M, T.H.,

and K.N. advise me a method for rearing MPS II mouse and take part

in the invaluable discussion in this project. Y.E is a director in thisproject.

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Higuchi Et Al. 2012. Enzyme Replacement Therapy -ERT- Procedure for Mucopolysaccharidosis Type II -MPS II- By Intraventricular Administration -IVA- In Murine MPS II