Zinc-Superoxide Dismutase Inhibits the

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MOLECULAR

for Pharmacology

ACCELERATED

and

Experimental

Therapeutics

reserved. (1995).

47:1095-1100

PHARMACOLOGY,

COMMUNICATION

Expression of Human Copper/Zinc-Superoxide Inhibits the Death of Rat Sympathetic Neurons Withdrawal of Nerve Growth Factor JOAQUIN RICHARD

JORDAN,1 J. MILLER

Departments

GHANASHYAM

of Pharmacological

and

Chicago,

Chicago,

Illinois

Received

February

8, 1 995; Accepted

D. GHADGE,1

Physiological

JOCHEN

Sciences

(J.J.,

H. M.

J.H.M.P.,

PREHN,

P.T.T.,

PETER

R.J.M.)

and

Dismutase Caused by

T. TOTH,

Neurology

RAYMOND

(G.D.G.,

P. ROOS,

R.P.R.),

The

and

University

of

60637 March

1 0, 1995

SUMMARY Rat superior

cervical ganglion neurons require the presence of factor (NGF) to develop and survive in culture. If NGF is removed from the culture medium, then the neurons die of programmed cell death. We investigated the potential role of

sion of the Ca2-binding protein calbindin D28k or the enzyme -galactosidase did not. We also observed that treatment of the cells with the cytokine transforming growth factor-31 , which has been shown to protect neurons against oxidative injury,

Ca2

delayed

nerve growth and

reactive

oxygen

species

in this

process.

We

found

that overexpression of human wild-type copper/zinc-superoxide dismutase in cultured superior cervical ganglion neurons, using an adenovirus-based vector, substantially protected the cells

from

the

effects

Elucidation tion

of NGF

of the

is of critical

development

withdrawal,

molecular

importance

and

basis

disease.

Recently,

have

general under

for understanding conditions. For

citotoxicity useful The

directed

involved

in the normal

now

well

way

might

important

and

that

activated

inappropriate

under neural

dismutase;

PCD,

programmed

BHK, baby hamster

zineethanesulfonic

kidney;

cell death;

produced

widely the

types

by

or die of ex-

to be extremely

NGF

withdrawal.

These

data

types

common

features

roles

for

a program is

of

species

a process

been

system is same path-

pathological

effects

con-

degeneration,

that

has

NGF,

TGF, transforming

nerve

growth

growth

intracellular

in

the

amyotrophic

factor;

that

include

role

of the enzyme

i.e.,

in culture

(8, 9) and

by the

cervical

PBS, phosphate-buffered

the

removal and

ganglion;

the

discovery

induces

apoptotic

linked

,

to

present model

of NGF investigated

SOD-i

reducing

in humans,

directly

(12),

recently protein

or

SOD

and

oxygen

antiapoptotic

Moreover,

are

Ca24

of reactive

formation

enzyme (10).

some

possible

has

the

radicals

also

including

The

the

oxygen

system,

superior

to

different as being

are

neurodegeneration idea

neurons

neurons SCG,

6, 7).

inhibiting

same

These

lateral sclerosis (11). In the we have used a well established

nervous

in

discussed there

mediators,

the

down-regulation

experiments

factor;

by

of reactive

tions

important

necrosis

usually

considered.

and by

act

clearly

process,

(1,

apoptosis

of cultured

thetic

be

species

death

the

to

several

may

is and

are

of degenerative

highlighted

Bcl-2

necrosis

and

oxygen in

It

of PCD

degeneration.

PCD

different

(5).

roles

of neuronal

reactive this

discussed

relative

Although

This work was supported by a postdoctoral fellowship from NATO to J.J., by a grant from the German Research Foundation to J.H.M.P., by United States Public Health Service Grants DA02575, DA02121, and MH40165 to R.J.M., and by United States Public Health Service Grant N521442 and the ALS Association to R.P.R. 1 JJ and G.D.G. contributed equally to this study. ABBREVIATIONS:

been

consider

possible

neurons live the concepts

proven

now

areas

some

the problem. die by initiating

suicide

be inadvertently to

example,

degeneraof neuronal

development of the nervous (4). The possibility that the

established leading

cell

us with why

(3) have

about can

cell

several

to provide

ofPCD

of thinking that neurons

genetically

ditions,

started

(1, 2) and ways idea

of nerve

death

overexpres-

to an understanding

ofinvestigation principles different

although

cell

suggest a role for reactive oxygen species in triggering programmed cell death of rat sympathetic neurons upon growth factor withdrawal.

mutafamilial

series ofPCD

of in

from

sympa-

the

possible

copper/zinc-superoxide

saline; HEPES, 4-(2-hydroxyethyl)-i

-pipera-

acid.

1095

I 096 roles

of

some

degeneration. based system in

et aL

Jordan

of

sympathetic

oxygen

these

To do for the

factors

this, we efficient

neurons.

species

may

in

Our

play

the

resulting

have used expression results

a key

role

nerve

a novel of genes indicate

in

this

that type

cell

adenovirusof interest reactive

of

neuronal

death.

Materials

and

Methods

em

Cell culture. SCGs were isolated from 1-5-day-old Holzman rats (13). Rat pups were killed by a cut made into the thorax and through the heart, under anesthesia. This procedure reduced the bleeding from the carotid artery during dissection of the ganglia. After dissection, ganglia were incubated for 40 mm, at 37#{176}, in Ca2and Mg-free

Hanks’

solution

containing

0.2%

trypsin

(Worthington,

Freehold, NJ) and were then dissociated into single cells by gentle trituration. Dissociated cells were washed twice with L-15 culture medium containing 0.1% (w/v) bovine serum albumin (CalbiochemNovabiochem, La Jolla, CA) and were plated on poly-L-lysineand laminin-treated

glass

coverslips.

Glass

coverslips

were

prepared

by

incubation for a minimum of 15 mm with 0.5 mg/ml poly-L-lysine (Sigma Chemical Co., St. Louis, MO) dissolved in borate buffer. The coverslips

were

then

rinsed

three

times

with

water

and

covered

with

0.02 mg/ml laminin in L-15 medium, for a minimum of 1 hr, before the cells were replated. Cells were maintained in L-15 culture medium supplemented with 1 p.g/ml NGF (United States Biochemicals, Cleveland, OH), 5% rat serum (GIBCO, Grand Island, NY), glucose, bicarbonate, penicillin-streptomycin, and vitamins, as described (14). Construction of recombinant, replication-defective adenovirus containing wild-type copper/zinc-SOD (SOD-i). A SOD-i cDNA clone was obtained from the American Type Culture Collection (15). A blunt-ended PstI-NheI fragment was inserted into the EcoRV site of the transfer vector pAdKN downstream of the elongation factor i-a promotor and upstream of the cellular heavy chain enhancer (4F2) and the bovine growth hormone polyadenylation site (pA+), to generate pAdKN.SODWT plasmid. The pAd.KN plasmid also contains 0-i and 9-16 map units ofDNA sequence of adenovirus type 5. To construct a replication-defective adenovirus (AdEF.SODWT) containing SOD-i cDNA, NheI-digested pAdKN.SODWT and XbaIand Clal-digested adenovirus type 5 sub36O were co-transfected into human embryonic kidney 293 cells using a calcium phosphate-DNA precipitation method. The recombinant AdEF.SODWT was plaque purified three times to isolate a homogeneous population ofthe virus. The virus used in the studies was further purified by CsC1 isopycmc ultracentrifugation

and

line (10 mM HEPES, 10% glycerol. Purified titered

by

plaque

was

dialyzed

against

HEPES-buffered

sa-

mM NaCl, 2 mM MgCl2, pH 7.5) containing virus was stored at -70#{176} in small aliquots and

140 assay.

Adenoviruses

carrying

plicity of infection of i500 or were not infected. Four days after infection, neurons were washed with cold PBS and scraped. Neurons were resuspended in 15 l ofH10K10D1 buffer (iO mM HEPES, iO m’vi KC1, 1 mM dithiothreitol, pH 7.4) and lysed on ice with 1% Nonidet P-40 for 15 mm. Cytoplasmic protein (10 j.g), obtained after centrifugation at rpm for iO mm at 4#{176}, was analyzed for the presence of i9-kDa SOD-i protein by Western blotting using horseradish peroxidase-conjugated anti-SOD-i polyclonal antibody (catalogue no. PP077; Binding Site) and an enhanced chemiluminescence West-

the

genes

for

cal-

bindin D28k (AdCABP-i) and f3-galactosidase (AdBAc.LacZ) have been described (i6, i7). Infection protocoL After 3 days in vitro the cultures were infected with the respective virus at a multiplicity ofinfection of i500. For this purpose, aliquots of the high titer viruses were added to the culture medium and distributed by gentle agitation. After 2 hr, the infection medium was exchanged with regular, NGF-supplemented, culture medium. After an additional 3 days, cultures either were subjected to NGF deprivation (see NGF withdrawal) or were fixed, and expression of the respective proteins was verified by immunocytochemistry. No nonspecific toxic effects ofthe viruses were apparent under the conditions used in these studies. The percentage of neurons showing positive staining for each antigen was >95% in every experiment and was usually not significantly different from 100%. In contrast, no neurons were stained for the antigens under noninfected conditions (see Fig. 2). Western blot analysis. Sympathetic neurons were infected with either AdBAc.LacZ, AdCABP-i, or AdEF.SODWT virus at a multi-

blotting

SOD

detection

system

activity

gels.

AdEF.SODWT

or not

(Amersham).

BHK-2i infected,

cells and

were

96

hr

either

after

infected

infection

with

cytoplasmic

extracts were prepared in H10K10D1 buffer containing 0.5% Nonidet P-40. One milligram of protein was electrophoresed on a 7.5% nondenaturing polyacrylamide gel at 4#{176}, and SOD enzyme activity was determined by using the nitroblue tetrazolium method (i8). Immunohistochemistry. In each experiment successful expression

of

proteins

was

verified

by

immunohistochemistry.

For

this

purpose cultures were fixed with 4% paraformaldehyde in culture medium at 37#{176} for iS mm. After cells were washed three times with 0.i M PBS (0.1 M Na2 HPO4, 0.i M NaH2 P04, 0.2 M NaCl), pH 7.4, they

were

permeabilized

with

0.i%

Triton

X-100

(Eastman

Kodak,

Rochester, NY) in PBS for 2.5 mm. The coverslips were then incubated in blocking medium [0.i% Tween-20 (Sigma) and 4% bovine serum albumin (Sigma) in 0.1 M PBS] for i hr at room temperature. Incubations with primary antibodies were performed overnight at 4#{176}, using murine monoclonal anti-human SOD-i (1/300), anti-calbinthn D28k (1/1000), and anti-3-galactosidase (111000) antibodies (Sigma), diluted in blocking medium. Monoclonal antibodies were detected using

anti-mouse

Grove,

IgG

(Jackson

ImmunoResearch

diluted 112000 in blocking ABC kit (Vector Laboratories, Burlingame, visualized using 3,3’-diaminobenzidine substrate.

Coverslips

were

mounted

Laboratories,

solution, CA). (Sigma)

PA),

in

90%

West

and a Vectastain The peroxidase was as the chromogenic glycerol

with

0.1

phosphate buffer containing 0.01% NaN3. TGF-f11 treatment. Twenty-four hours before NGF cultures received 10 ng/ml TGF-131 (recombinant human

withdrawal,

Systems,

solution

Minneapolis,

saline

containing

MN) i mg/ml

from

a

1000

ovalbumin

and

ng/ml 4 mri

form;

stock HC1).

M

R & D (in

Controls

were

treated with an equal volume of the vehicle; TGF-J31 or vehicle was also present during the period of NGF withdrawal. NGF withdrawal. For induction of PCD, the original culture medium was exchanged for fresh culture medium with (control conditions) or without (NGF withdrawal) the NGF supplement. Neuronal degeneration was analyzed by phase-contrast microscopy after 24, 48, and 72 hr. Neurons exhibiting a rough appearance with irregularly

shaped

cell

bodies,

blebs,

and

vacuoles,

followed

by

cell

shrinkage, neurite fragmentation, and loss ofphase-brightness, were considered to be damaged. Frequently, nuclear fragmentation could be observed by phase-contrast microscopy. Healthy neurons were identified as having round to oval, smooth, phase-bright cell bodies and intact neurites. A total of iOO-200 neurons were examined in three to five randomized subfields ofthe coverslips. Similar numbers of neurons were examined under all experimental conditions. Each condition was represented by six coverslips. All experiments were performed

in quadruplicate.

Results It has survival dent culture factor

been widely demonstrated of rat sympathetic neurons

on the

form

In

is thought

medium, anism

presence

medium.

that of PCD

of the

appropriate

growth

most

paradigms

the

to be NGF.

neurons shows (12).

die

over

many We

that the development in cell culture are

When

NGF

a period of the

readily

hallmarks confirmed

factors

major days, of the this

in the

important

is removed

of a few

and depen-

from

the

by a mech“apoptotic”

to be

the

case.

SOD Neurons

in our

withdrawal criteria and

cultures

(Fig. suggestive

membrane

drawal,

died

1) and

over

many

of apoptosis, blebbing.

neurons

also

a 3-day

exhibited

incuding

Forty-eight stained

D28k,

on

neuronal

the

human

positively

death

observed

chosen

because

they

iological

ways

of perturbing

cell

D28k) (i7, 19) and reactive anions (copper/zinc-SOD-i).

oxygen

of the protein expression chemistry increased

neurons 3 days

species

made to infection

of each antigen was 24 hr after infection. over

the

next

24-48

after

(calbindin

was

then

appropriate Evidence

in

of NGF

was

using

the

rus)

(Figs.

i and

of

time

D28k

we

not

or

A

reasons.

First,

us

(24

that

to

and

Western

blots

control

48 hr)

de-

in

the

conditions vi-

virus of cells

perform neurons in these clearly

protected

as well

expression, died

any

overexpression in SCG activity

The

expression

of SOD-i with number

copper/zinctime. neurons

-galactosidase

allow

think

the that

from

in

to the control died by 72 hr

f3-galactosidase-expressing

points

treated small

man SOD-i protein increase in enzyme 2).

of by

2). Copper/zinc-SOD-i

not the

did

iO% died

the

ofSOD-i

In contrast neurons had

different D28k

to the effects

calbindin

assays,

not

over

ofoverexpression

had

1097

PCD

observed

SOD-i-overexpressing

calbindin

at earlier

In contrast

cultures

(Fig.

death

control cells Although

immunohistoof staining

stabilized

was

only cells

(i.e.,

ing

efficient expression we used adenovirusapproximately 100%

express the (Fig. 2) (17).

effect

withdrawal,

of cell

neurons

as superoxide

expression

intensity

a striking

NGF

presence

phys-

staining

SOD-1-overexpressing gree

These

such

in

these neurons (Figs. 1 and 2). situation, in which >90% of the

cal-

relatively

obtained by The intensity

hr and

with-

removal. of Ca24

13-Galactosidase

could be after viral

somata NGF

diminution

next 7 days. We observed

f3-galactosidase

NGF

handling

No

double-stranded

represent

used as an additional control. To achieve of these proteins in cultured neurons, based vectors (i7). Using these vectors,

NGF

procedure. of the proteins and

after

were

after

for

copper/zinc-SOD-i,

proteins

after

morphological

shrunken

hours

DNA breaks, using the TUNEL staining We examined the effects of expression bindin

period of the

and

(Fig.

1).

cells expressas

rapidly

(Fig. i). in the SCG conventional

as

did

neuron enzyme

of immunoreactive

hu-

was associated cells, for the

with an following

demonstrated

the

ex-

B S

I

I

1

I S..

23

I Turn. 1k)

C

caubundun

Time

(ii)

TIm.

(3,)

D

928K

I

I

I

I #{149}5#{149}

24

TIrne(h)

E

24 1mw

(3,)

FIg. I . Histograms representing the time course of survival of rat SCG neurons grown in the presence (U) or absence (Li) of NGF. A, Control conditions, without any treatment; B, cells overexpressing p-galactosidase; C, cells overexpressing calbindin D28k; D, cells overexpressing human SOD-i ; F, treatment with TGF-p1 24 hr before the start of NGF withdrawal (four experiments in each case; see Materials and Methods). *, p