Cyclosporin A, an immune suppressor, enhanced neurotoxicity of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice

Neurochem. Int. Vol. 15, No. 2, pp. 249-254, 1989 Maxwell Pergamon Macmillan plc. Printed in Great Britain

A Rapid Publication

CYCLOSPORIN A, AN IMMUNESUPPRESSOR, ENHANCEDNEUROTOXICITYOF N-METHYL-4-PHENYL-1,2,3,6-TETRAHYDROPYRIDINE (MPTP) TO MICE

Masako Hagihara I , Kennichiro, Fujishiro 2, Akira Takahashi 2, Makoto Naoi I

and Toshiharu Nagatsu I

1 Department of Biochemistry

and 2 Neurology,

Nagoya University School of Medicine, Nagoya, Japan

(Received 15 June 1989; accepted 20 June 1989)

SUMMARY

N-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine administrated into C57BL/6N mice for 8 days.

(MPTP)

was

systemically

Tyrosine hydroxylase

activity

and dopamine content in striatum and hypothalamus were reduced markedly, the reduction was more manifest in the striatum.

and

Cyclosporin A, an immune

suppresser, enhanced the neurotoxicity of MPTP, when it was injected to mice in combination with MPTP.

Tyrosine hydroxylase activity in the striatum

was

reduced to 109 ± 20 from 350 Z 46 pmol/min/mg protein of control, when mice were injected by MPTP alone.

The enzyme activity was further reduced to 68.4

12.2 pmol/min/mg protein by injection of MPTP in combination to cyclosporin A.

In addition,

dopamine and biopterin contents in the striatum decreased

quite in parallel to the reduction of tyrosine hydroxylase activity.

On the

other hand, cyclosporin A itself did not effect tyrosine hydroxylase activity and dopamine and biopterin contents in the striatum and hypothalamus.

These

data suggest cumulative effect of cyclosporin A to the neurotoxicity of MPTP.

N-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine

(MPTP)

is

a

well-known

neurotoxin, which elicits symptoms very similar to those with parkinsonism in humans

(Davis et aZ., 1979; Langston et aZ., 1983) and which causes specific

neurodegeneration 1983)

and rodents

in

the nigro-striatal

(Del Zompo

system

and Boccetta,

in primates 1984).

(Burns

Molecular

et

aZ.,

basis

of

neurotoxicity of MPTP has been extensively studied (Chiba et aZ., 1984, Javitz

Address correspondence

and reprint requests

to Dr

Toshiharu

Nagatsu

at

Department of Biochemistry, Nagoya University School of Medicine, 65 Tsurumaicho, Showa-ku, Nagoya 466, Japan. 249

250

MASAKO HAGIHARAet al. et aZ.,

1985).

Recently

we

reported

[tyrosine,

tetrahydropteridine

1.14.16.2,

TH]

and

markedly

: oxygen

in

aromatic L-aminoacid

the

striatum

of

dopamine

content

cerebrospinal

fluid

microglobulin

were

suggests

that disorder

parkinsonism. pathological

changes

systematically suppresser,

to reduce

in immune

To d e m o n s t r a t e of

mice

patients

system

may

the

dopaminergic

administrated

into

8

system

contents et a~.,

mice,

of

in

and

days'

hand,

in

of b e t a

2-

1989),

which

in pathogenesis

immune

system

parkinsonism,

the

systemic

the other

be involved

the c o n t r i b u t i o n

Lwere

marked reduction of

On

(Mogi

EC

(aromatic

EC 4.1.i.28)

after

In addition,

markedly

hydroxylase

(5-hydroxylating),

decarboxylase

in the striatum.

from p a r k i n s o n i a n found

tyrosine

carboxy-lyase,

C57BL/6N

of MPTP (Mogi et a~., 1988a). was observed

of

oxidoreductase

administration (DA)

activity

L-5,4-dihydroxyphenylalanine

aminoacid decarboxylase, reduced

that

effect

of

of

to

the

MPTP

was

an

immune

cyclosporin A (CsA), on the toxicity of MPTP was examined.

EXPERIMENTAL PROCEDURES MPTP

was

dissolved

purchased

from

Aldrich

in phosphate-buffered

Chemical

saline

(Milwaukee,

(PBS) and MPTP

day) injected into C57BL/6N mice (male, 30 week-old, kindly

donated

intravenous

by

injection.

mice

(qmg/kg/day,

After

8-days'

immediately

with

0.52

produced

sucrose.

chemical

quantitatively amounts

on

ice,

as

TH

activity

was

were 1980).

using bovine

detection

(ECD,

assayed by HPLC with ECD determined

for into

by HPLC with

of

combination mice

were

then killed.

by

(Oka et aZ.,

fluorescence

198q).

detection

(Hirata

of

and

DOPA

(HPLC) with

DA contents

were

Total biopterin (Fukushima

Protein content was determined by the method of Bradford T-globulin

were

and homogenized

measurement

1979a).

MPTP.

without

The brains

liquid chromatography

et a L ,

with left

previously

were dissected

determined

Nagatsu

ml

CsA was

with PBS and injected

reported

The striatum and hypothalamus

and

solvent)

without

or CsA for 8 days and placed

g/5

of ~LPTP and CsA,

from L-tyrosine by high-performance

electro

Nixon,

and

or

U.S.A.)

20-25 g weight).

(0.25

was diluted with

administration

removed

M

a solution

The solution

of MPTP

1986).

as

subcutaneously),

daily

administration

Nagatsu,

Sandoz,

WI,

(30 mg /kg body weight/

and

(1976)

as standard.

RESULTS As summarized reduction

in Table

i, after

of TH activity was observed

injected with saline alone. CsA injected

together with MPTP.

and also reduction

by CsA in combination markedly

administration

in the striatum,

of MPTP,

compared

marked

with control

Reduction of TH activity by MPTP was enhanced by On the other hand, CsA alone did not affect

the activity of TH in the striatum. by MPTP,

8 days'

DA contents

in the striatum were reduced

of DA content was more dominant

with MPTP.

In the hypothalamus,

reduced by MPTP alone as in the striatum,

in mice injected

TH activity was not so

as summarized

in Table

i.

251

Cyclosporin A enhanced neurotoxicity of MPTP to mice Table i.

Effects of MPTP and CsA on TH activity and DA contents in the

striatum and hypothalamus

TH activity

Dopamine content (pmol/mg protein)

(pmol/min/ mg protein)

Strlatum Control

4

350

+ 46

461

+ 21

CsA

4

323

± 50

459

Z 59

MPTP

4

109

d 20*

i01

d 19

MPTP + CsA

4

68.4 + 12.2"

78

Control

4

77.5 Z 21

21.1Z

4.9

CsA

4

54.5 Z 15

27.1 ±

5.9

MPTP

4

54.5 i

9.0

18.8 ±

4.7

MPTP + CsA

4

23.5 Z

3.8*

8.2 Z

+ 14.9"

HypothaZomu8

1.6"

Each value represents mean and SD of duplicate measurements of each sample (n : number).

* Significant difference from control, p< 0.001, by Student's t test.

Control mice were injected with saline, and the amounts of CsA and MPTP administered daily were 4 mg/kg and B0 mg/kg per day for 8 days, respectively. All animals were sacrificed 8 days after the last injection.

In contrast,

TH activity

was reduced significantly

in mice administered by

MPTP in combination with CsA: 30.3 % of the control value.

On the other hand,

TH activity was not reduced by injection of CsA alone (Table I). in the h y p o t h a l a m u s combined with CsA.

was reduced s i g n i f i c a n t l y

in m i c e

DA content

t r e a t e d w i t h MPTP

252

MASAKO HAG1HARA el al. Biopterin contents were

measured

contents

and

(a co-factor

the

results

in the striatum

are

of TH) in the striatum shown

in Table

and hypothalamus

were

2.

and hypothalamus

The

reduced

total

biopterin

by administration

of

MPTP alone and the reduction was enhanced by combination with CsA.

Table 2.

Effects of MPTP and CsA on biopterin contents in the striatum and

hypothalamus.

Biopterin content

(pmol/mg protein)

Striatum

HypothaZo2m~8

Control

4

3.79 i 0.36

2.47

± 0.18

CsA

4

B.81 ± 0.81

2.28

± 0.49

MPTP

4

2.01 * 0.26*

1.20

+ 0.16"

MPTP + CsA

4

1.5B i 0.ii*

0.917 ± 0.359*

Each value represents mean and SD of duplicate measurements

of each sample

(n:

number).

*Significant

Control

mice

administered

difference

were

from control: p< 0.001, by Student's

injected

with

saline,

and

the amounts

t test.

of CsA and MPTP

daily were 4 mg/kg and 30 mg/kg per day for 8 days, respectively.

All animals were sacrificed 8 days after the last injection.

DISCUSSION

As r e p o r t e d

in

r e d u c e d TH a c t i v i t y DA. as

our previous and t o t a l

R e d u c t i o n o f TH a c t i v i t y shown by

degeneration

an

enzyme

Severe

toxicity

of

is

(Mogi e t contents

ascribed

immunoassay

in the striatum.

m i g h t be due t o i n c r e a s e d

paper

biopterin

to reduction

(Mogi

et

1987),

which

to

the

cyclosporin

(Berden

CsA was not detected

central

nervous

and c o n v u l s i o n ,

et al.,

1985;

content, suggests

o f MPTP by CsA

cyclosporin

treated though,

oZ.,

MPTP

and d e p l e t e d

o f TH p r o t e i n

of dopaminergic neurons.

in patients Even

and h e r e ,

degeneration

symptoms s u c h a s t r e m o r , n e u r a l g i a ,

1985).

1988)

Enhancement o f the n e u r o t o x i c i t y

neurological

with

aZ.,

in the striatum

system

Wilczek

in the cerebrospinal

with

were o b s e r v e d et al.,

fluid and it

Cyclosporin A enhanced neurotoxicity of MPTP to mice

253

has been considered not to be transported into the brain through blood-brain barrier in man (Palestine et aZ., 1985) or mouse (Fazakerley and Webb, 1985). On the other hand,

an experimental chronic viral infection of the central

nervous system of mouse with the mouse hepatitis virus 3 (MHVS), CsA was found to increase the rate of acute death and viral titer in the brain (Boespflug et al.,

1989),

when cyclosporin treatment was started at the same time as the

virus infection.

On the other hand, CsA inhibited expression of a chronic

MHV~-induced infection of the central nervous system, when CsA treatment was started two weeks after virus infection.

The discrepant effects of CsA

treatment on the infection of a neurotropic virus, MHV3, suggest that CsA may increase acute cytopathogenic lesions in the brain by virus infection, that it may have a beneficial effect on chronic immune response. effect

was

also

shown

by

its

inhibitory

effect

experimental allergic encephalitis (Armending,

of

1982).

the

and

The latter

expression

of

The cumulative effect

of CsA on the MPTP-induced lesion in the dopaminergic neurons may be ascribed to its effect on the immune system or may be dependent on its effect directly on the toxicity of MPTP.

X"ne molecular basis of the enhanced toxicity of MPTP

by CsA awaits further study.

ACKNOWIEDGMENT This work was supported by Grant-in-Aid

for Scientific

Research on

Priority Areas, Ministry of Education, Science and Culture, Japan.

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