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Life Sciences 78 (20

Low dose (�)deprenyl is cytoprotective: It maintains mitochondrial

membrane potential and eliminates oxygen radicals

L. Simon a,*, G. Szilagyi a, Z. Bori a, G. Telek b, K. Magyar c, Z. Nagy a

a National Institute of Psychiatry and Neurology, National Stroke Center, Department of Vascular Neurology, Semmelweis University,

Huvosvolgyi Street 116., Budapest, H-1021 Hungaryb IIIrd Department of Surgery, Semmelweis University, Budapest, Hungary

c Neurochemical Research Unit of the Hungarian Academy of Sciences, Budapest, Hungary

Received 2 February 2005; accepted 12 April 2005

Abstract

Hypoxia leads to a collapse in mitochondrial transmembrane potential (DwM), a fall in the ATP/ADP ratio, and finally cell death. Since

(�)deprenyl directly modulates DwM and production of reactive oxygen species (ROS) by altering the respiratory function of mitochondria, we

were interested in the dose–response relations of these effects. The changes in JC-1 red /green signal ratios {mitochondrial transmembrane

potential}, and the changes in the cerium staining (intracellular ROS) in hypoxic and normoxic PC12 cell cultures were measured following 1 h of

Argon hypoxia and 24 h of re-oxygenation in the absence and in the presence of various concentrations of (�)deprenyl. DwM shifted to lower

values following hypoxia/re-oxygenation and all cells had decreased and uniform DwM levels. The amount of ROS increased. Following 24 h of

treatment with various concentrations of (�)deprenyl during the re-oxygenation period, survival increased, the DwM shift caused by oxygen

deprivation was reversed and the peroxy radical levels decreased except for at 10�3 M.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Mitochondrial transmembrane potential; Reactive oxygen radicals (ROS); (�)Deprenyl; JC-1; Cerium

Introduction

During hypoxia, the impairment of mitochondrial function,

the consequent collapse in mitochondrial transmembrane

potential (DwM), a fall in the ATP /ADP ratio, and the

hypoxia-induced cytoplasmic accumulation of cytochrome c

leads to cell death (Smets et al., 1994; Kroemer et al., 1997;

Yermolaieva et al., 2004; Hardie, 2003; Kim et al., 2003). The

outward pumping of protons across the inner mitochondrial

membrane produces a proton gradient that drives the conversion

of ADP to ATP and is reflected by the DwM (Sherrat, 1991).

Decreased DwM induces opening of the mitochondrial perme-

ability transition pores (PTP), which may lead to the release of

mitochondrial apoptosis initiation factors (AIFs) (Marchetti et

al., 1996). The loss of mitochondrial membrane potential (DwM)

in itself may or may not lead to apoptotic cell death depending

on the model system being used (Salvioli et al., 2000). An

0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.lfs.2005.04.078

* Corresponding author. Tel.: +36 6 70 319 7163; fax: +36 1 391 5440.

E-mail address: simla@freemail.hu (L. Simon).

overall decrease in DwM was reported to occur late in apoptosis,

well after the release of cytochrome c from mitochondria (Yong

et al., 1997). During re-oxygenization after a hypoxic period

high amounts of reactive oxygen species (ROS) are generated

(Yermolaieva et al., 2004). Mitochondria are the major

generators of ROS. ROS production is associated with

excessive oxidative stress. Membrane lipids are the primary

targets of ROS, but proteins, carbohydrates and nucleic acids are

also damaged, leading thus to cellular dysfunction and death.

(�)Deprenyl (phenyl-isopropyl-methyl-propargylamine,

selegiline) is a relatively selective, irreversible inhibitor of

monoamine oxidase-B (Knoll and Magyar, 1972; Birkmayer et

al., 1975; Sowa et al., 2004). Deprenyl is used in the treatment

of Parkinson’s disease (Oerthel and Quinn, 1997). Since

(�)deprenyl directly modulates DwM (Wadia et al., 1998)

and thus the production of reactive oxygen species (ROS) by

altering the respiratory function of mitochondria in a dose-

dependent fashion (Gotz et al., 1995; Thyffault et al., 1997;

Wadia et al., 1998). The aim of the present study was to

investigate the possible cytoprotective mechanisms of

05) 225 – 231

www.e

L. Simon et al. / Life Sciences 78 (2005) 225–231226

(�)deprenyl following hypoxia/re-oxygenization using nerve

growth factor-differentiated PC12 cell culture. The present

study demonstrates that (�)deprenyl improves mitochondrial

function and minimizes damage in PC-12 cells subjected to

hypoxia/re-oxygenation.

It is most probable that this mechanism could be the basis of

the protective effects of (�)deprenyl in middle cerebral artery

occlusion stroke model in rat (Simon et al., 2001; Puurunen et

al., 2001; Maia et al., 2004), as well as in human stroke patients

(Sivenius et al., 2001).

Methods

Culturing and NGF differentiation of PC12 cells

Rat phaechromocytoma (PC-12) cells were maintained in

Dulbecco_s modified Eagle’s medium (DMEM) (Gibco BRL),

supplemented with 10% (vol/vol) calf serum, 2 mM l-

glutamine(Gibco BRL), penicillin (50 international units/ ml),

streptomycin (50 Ag/ml) and PC-12 cells were predifferentiated

on round cover glasses (d =12 mm) covered with a collagen

membrane prepared from acid soluble collagen isolated from

rat tail (Csonka et al., 1980) for 5 days in Dulbecco’s modified

Eagle_s medium (DMEM) (Gibco BRL), with 10% (vol/vol)

calf serum, 5% (vol/vol) horse serum, 2 mM l-glutamine

(Gibco BRL), penicillin (50 international units/ ml), strepto-

mycin (50 Ag/ml) and 50 ng/ml nerve growth factor (NGF) in a

humidified incubator aerated with 5% CO2 at 37 -C. All

treatments of the cells were carried out in 24-well-cluster cell

culture dishes with a diameter of 15 mm per well (Nunclon,

Intermed, Denmark). Each well contained about 1000 cells.

Hypoxia/re-oxygenation

Cells were subjected to hypoxia and re-oxygenation as

follows: hypoxia was produced by placing cultures on the

bottom of an open chamber. Subsequently, it was filled with

Argon gas and then the chamber was closed. A blood gas

analyser (ABL Radiometer, Copenhagen) was used to control

the partial O2 pressure in the cell culture medium. After 1 h of

oxygen deprivation, the cultures were returned to the incubator

(re-oxygenation) for 24 h. Control cultures were maintained in

the incubator under normal conditions (normoxia). In the

oxygen deprivation experiments, the various concentrations of

(�)deprenyl to be tested were added to the culture medium

right after the oxygen deprivation and remained there during

the 24 h re-oxygenation period.

Assessment of cell death in PC12 cell culture with propidium

iodide staining

The extent of cell death was determined by staining the

cultures with 1.5 Ag/ml propidium iodide dissolved in physio-

logical saline for 2 min. The procedure was the following: the

DMEM was removed and 300 Al of 1.5 Ag/ml propidium iodide

solution was added to the cell cultures. The numbers of viable

and dead cells were counted with a fluorescence microscope

using 450–490 nm excitation and 520 nm barrier filters. In

order to avoid problems caused by uneven cell distribution,

cellular death was expressed as a mean percentage of dead cells

in three separate cultures, in twelve samples.

Combined staining procedure (JC-1+cerium)

The changes in DwM were assessed using the lipophilic

cationic membrane potential-sensitive dye JC-1, and the changes

in the amounts of intracellular ROS were assessed using the

cerium method. The staining procedure was the following: the

DMEM was removed and 300 Al of 10 Ag/ml JC-1 (Molecular

Probes) solution (dissolved in physiological) saline was added to

the cultures for 10 min. The staining solution was removed and

the cell cultures were rinsed with physiological saline for 2 min.

Then, 300 Al of 20 mmol/l CeCl3 solution (dissolved in lactated

Ringer) was applied for 2 min. After the CeCl3 solution was

removed the cell cultures were rinsed again with physiological

saline for 2 min and subsequently the cells were fixed in 0.25%

(vol/vol) glutaraldehyde solution for 2 min. The fixed cells on

the round cover glasses were then covered with Vectashield

mounting medium for fluorescence (Vector Laboratories, Inc.

Burlingame, CA) and put on glass slides. Intracellular distribu-

tion of the dye was assessed by confocal microscopy. Fluores-

cence present in the cells was measured at 488-nm excitation/

510- to 625-nm emission. The reaction of cerium ions with ROS

forms stable, insoluble cerium perhydroxide (CeIII [OH]2OOH

or CeIV[OH]3OOH precipitates detectable with reflectance

methods (Robinson and Batten, 1990; Van Norden and

Frederiks, 1993; Halbhuber et al., 1996; Bestwick et al., 1997;

Telek et al., 1999, 2001).

Fluorescent image acquisition and processing

We measured the JC-1 fluorescence signal and the cerium-

ROS precipitate reflectance intensities in fixed samples. Experi-

ments were performed in a darkened room at room temperature.

A BIO-RAD MRC 1024 confocal system (Bio-Rad Corp.,

Hertfordshire, England) was used installed on a Nikon OPTI-

PHOT inverted microscope (Donsanto Corp., Nattick, Massa-

chusetts). Imaging of JC-1-labeled cells was performed using

multichannel detection in fluorescence mode excitation with 488

line of a Krypton–Argon laser; standard filter set: T1, T2A).

Imaging of cerium-labeled cells was performed using a single

channel detection in reflectance mode, excitation with the 488

line of a Krypton–Argon laser; standard filter set (T1, T2A).

Exposures, laser intensities and acquisition parameters were set

the same during all acquisitions (across the groups being

compared). Routine observations were carried out by simulta-

neously generating a green and a red fluorescent image (at 510 to

625 nm emission). For quantitative analysis high-resolution

(100�) images were taken followed by a digital superposition.

Data analysis and statistics

The quantitative analysis of fluorescent JC-1 monomer

(green fluorescence), J-aggregate (red fluorescence) and cerium

Changes in JC1 red/green signal ratios in hypoxic

and normoxic PC12 cells following (-)deprenyl

treatment (mean+/-SEM)

Red

/gre

en s

igna

l int

ensi

ty r

atio

3

2

1

0N H H+10-12M H+10-8M H+10-3M

**

******

***

Fig. 2. Right after 1 h of hypoxia and 24 h of re-oxygenation the cells had

decreased and relatively uniform JC-1 red /green ratios 0.681T0.013 (n =50)

versus 1.07T0.18 (n =50) ( p <0.01). The average JC-1 red /green signal

intensity ratio (the representative of mitochondrial activity) of oxygen

deprived/re-oxygenated cultures was elevated by (�)deprenyl treatment in a

concentration dependent fashion, the 10�12 M being the most potent membrane

potential booster and the 10�3 M being the least potent. The average JC-1 red /

green signal intensity ratio was 0.68T0.013 (n =50) in hypoxic control cultures,

2.83T0.1 (n =50) ( p <0.001) at 10�12 M, 2.33T0.074 (n =50) ( p <0.001) at

10�8 M and 1.68T0.095 (n =50) ( p <0.001) at 10�3 M. The data were

statistically evaluated using ANOVA and a post hoc Duncan test (*=p <0.05;

** = p < 0.01; *** = p < 0.001). (N = normoxia; H = hypoxia; H + 10�12

M=hypoxia + 10�12 M (�)deprenyl; H + 10�8 M=hypoxia + 10�8 M

(�)deprenyl; H +10�3M=hypoxia+10�3 M (�)deprenyl).

Cerium signal intensities in hypoxic and normoxic

L. Simon et al. / Life Sciences 78 (2005) 225–231 227

reflectance present in the cells was performed using a built in

evaluation software LaserSharp Processing (Bio-Rad Corp.,

Hertfordshire, England. The fluorescence (red and green) (or

reflectance) intensity was averaged within the selected cells.

The ratio of red and green fluorescence was captured. In case of

the cerium reflectance method the average intensity data were

processed. The data were statistically evaluated using ANOVA

and a post hoc Duncan test. The results were considered to be

statistically significant if p <0,05. The postcapture analysis was

the same in all cases. Data are shown as meanTSEM.

Results

There was no visually discernible difference in images of JC-

1-stained partially neuronally differentiated PC12 cells before

and after fixation with 0,25% (vol/vol) glutaraldehyde solution

for 2 min. However, the postfixation JC-1 red /green pixel

intensity ratios are approximately twice as big as the prefixation

intensity ratios [1.57T0.19 (n =15) versus 0.66T0.06 (n=15)].

The partial O2 pressure in the cell culture medium was

154.65T1.35 mm Hg (n =6) in normoxic conditions and it was

131.96T2.29 mm Hg (n =6) (meanTSEM) following the

hypoxic period. Right after 1 h of hypoxia and 24 h of re-

oxygenation the percentage of propidium iodide-positive (PI+)

cells increased as compared to the normoxic control

(36.66T3.25%) (n =12) versus (PI+ : 19.5T2.18% (n =12)

( p <0.001). The JC-1 red /green ratios of the cells decreased

and were relatively uniform (0.681T0.013 (n =50) versus

1.07T0.18 (n =50) ( p <0.01)). They also showed an increased

cytoplasmic levels of peroxyl radicals (28.99T0.65 (n =100)

versus 23.78T0.78 (n =100) ( p <0.001)) as measured with the

cerium reflectance method.

Treatment with (�)deprenyl decreased the percentage of PI+

cells in oxygen-deprived and reperfused PC12 cell cultures at

The extent of cell death following 1 h argonhypoxia and 24 h re-oxygenization in PC12 cellsfollowing (-)deprenyl treatment (mean+/-SEM)

Per

cent

age

of p

ropi

dium

iodi

de-p

ositi

ve c

ells

60

50

40

30

20

10

0

******

**

N H H+10-12M H+10-8M H+10-3M

Fig. 1. Right after 1 h of hypoxia and 24 h of re-oxygenation the percentage of

propidium iodide-positive (PI+) cells increased as compared to the normoxic

control ( 36.66T3.25%) (n =12) versus (PI+: 19.5T2.18% (n =12) ( p <0.001).

Treatment with (�)deprenyl decreased the percentage of PI+ cells in oxygen-

deprived and reperfused PC12 cell cultures at 10�12 (21.41T3.97%) (n =12)

( p <0.01) and 10�8 M (21.54T2.33%) (n =12) ( p <0.001) significantly while

10�3 M (�)deprenyl, the highest concentration, increased the percentage of

PI+ cells (44.25T5.76%) (n =12) ( p >0.05) as compared to the hypoxic control

(36.66T3.25%) (n =12). The data were statistically evaluated using ANOVA

and a post hoc Duncan test (*=p <0.05; **=p <0.01; ***=p <0.001).

(N =normoxia; H =hypoxia; H +10�12M=hypoxia+10�12 M (�)deprenyl;

H +10�8 M=hypoxia+10�8 M (�)deprenyl; H +10�3 M=hypoxia+10�3

M (�)deprenyl).

10�12 M (21.41T3.97%) (n =12) ( p <0.01) and 10�8 M

(21.54T2.33%) (n =12) ( p <0.001) significantly. 10�3 M

(�)deprenyl, the highest concentration, increased the percentage

of PI+ cells (44.25T5.76% ) (n =12) ( p >0.05) as compared to

the hypoxic control (36.66T3.25%) ( p <0.01) (n =12) (Fig. 1).

The average JC-1 red /green signal intensity ratio (the

representative of mitochondrial activity) of oxygen deprived/

PC12 cells following (-)deprenyl treatment(mean+/-SEM)

Mea

n si

gnal

inte

nsity

N H H+10-12M H+10-8M H+10-3M

30

20

10

0

*** ***

**

Fig. 3. Right after 1 h of hypoxia and 24 h of re-oxygenation the cells showed an

increased cytoplasmic levels of peroxyl radicals 28.99T0.65 (n =100) versus

23.78T0.78 (n =100) ( p <0.001) measured with the cerium reflectance method.

The cerium signal intensity in oxygen deprived/re-oxygenated PC12 cultures

was changing concentration dependently. At 10�12 M (20.84T0.58) (n =100)( p <0.001) (�)deprenyl treatment decreased the cerium signal level in hypoxic/

re-oxygenated PC12 cultures below that of the normoxic control (23.78T0.78)

(n =100), at 10�8 M (28.86T0.78) (n =100) (�)deprenyl did not affect it, and at

10�3 M (32.15+0.75) (n =100) ( p <0.01) (�)deprenyl considerably increased

it as compared to the hypoxic control (28.99+0.65) (n =100). The data were

statistically evaluated using ANOVA and a post hoc Duncan test (*=p <0.05;

** = p < 0.01; *** = p < 0.001). (N = normoxia; H = hypoxia; H + 10�12

M=hypoxia + 10�12 M (�)deprenyl; H +10�8 M=hypoxia + 10�8 M

(�)deprenyl; H +10�3M=hypoxia+10�3 M (�)deprenyl).

Fig. 4. Partially neuronally differentiated, normoxic PC12 cells stained with JC1. Red color denotes active mitochondria, green color denotes cytoplasm. Imaging of

JC-1-labeled cells was performed using multichannel detection in fluorescence mode excitation with 488 line of a Krypton–Argon laser; standard filter set: T1,

T2A); 40� lens; image acquisition mode: simultaneous double label+accumulate to peak. Bar equals 5 Am.

L. Simon et al. / Life Sciences 78 (2005) 225–231228

re-oxygenated cultures was elevated by (�)deprenyl treatment

in a concentration dependent fashion. The concentration of

10�12 M was the most potent membrane potential booster and

the 10�3 M was the least potent. The average JC-1 red /green

signal intensity ratio was 0.68T0.013 (n =50) in hypoxic

control cultures, 2.83T0.1 (n =50) ( p <0.001) at 10�12 M,

2.33T0.074 (n=50) ( p <0.001) at 10�8 M and 1.68T0.095(n =50) ( p <0.001) at 10�3 M (Figs. 2, 4, and 5).

The cerium signal intensity in oxygen deprived/re-oxygen-

ated PC12 cultures also changed in a concentration dependent

fashion. (�)Deprenyl at 10�12 M (20.84T0.58) (n =100)

( p <0.001) decreased the cerium signal level in hypoxic/re-

oxygenated PC12 cultures below that of the normoxic control

(23.78T0.78) (n =100). On the other hand, (�)deprenyl at

10�8 M (28.86T0.78) (n =100) did not affect the level of

cerium signal, while at 10�3 M (32.15T0.75) (n =100)

( p <0.01) it considerably increased cerium reflectance intensi-

ties as compared to the hypoxic control (28.99T0.65) (n =100)(Fig. 3).

In the normoxic control cells treated with (�)deprenyl the

percentage of PI+ cells was not affected at 10�8 M

(19.67T2.82%) (n =12). The concentration of 10�12 M,

however, considerably decreased the percentage of PI+ cells

(10.18T0.79% (n =12) and slightly decreased it at 10�3 M

(14.83T1.06%) ( p >0.05) (n =12) as compared to the nor-

moxic control (19.5T2.18%) (n =12). JC-1 red /green pixel

intensity ratio was significantly increased in (�)deprenyl-

treated normoxic control PC12 cultures at all (�)deprenyl

concentrations. It was 1.07T0.025 (n =50) in normoxic control

Fig. 5. Picture A: normoxic control (Mitochondrial polarization is indicated by an

picture B: hypoxia/re-oxygenation control (Mitochondrial depolarization is indicate

red.); picture C: hypoxia/re-oxygenization+10�12 M (�)deprenyl. Red: active mit

present within the cell. Imaging of JC-1-labeled cells was performed using multich

Argon laser; standard filter set: T1, T2A); 100� lens; image acquisition mode:sim

cultures, 2.15 T0.088 (n =50) ( p <0.001) at 10�12 M,

2.17T0.077 (n =50) ( p <0.001) at 10�8 M and 2.83T0.18(n =50) ( p <0.001) at 10�3 M.

The cerium signal level, however, in the same (�)deprenyl-

treated normoxic PC12 cultures was the lowest at 10�8 M

(16.76T1.11) (n =100) ( p <0.001). At 10�3 M of (�)deprenyl

concentration it was slightly increased (26.86T1.05) (n =100)( p <0.05), while at 10�12 M (24.63T0.93) (n =100) it was atthe level of normoxic absolute control (23.78T0.78) (n =100).Data are summarized in Table 1.

Discussion

In the present study we demonstrated that the combination

of the methods propidium iodide staining (for the assessment

of cell death), modified JC-1 staining method (for the

characterization of mitochondrial membrane potential) and

cerium staining (for the in situ quantitative measurement of

free radical production) provide us with a useful data set to

evaluate the cytoprotective features of a selected drug. In

(�)deprenyl-treated hypoxic and normoxic PC12 cell cultures

evaluated by these assays a dose dependent protective effect

has been found. The sensitivity of this combination of assays

was such that significant effects could be detected with doses

as low as 10�12 M.

The present study using this combination of stainings has

also demonstrated that (�)deprenyl protects cells from

hypoxia/re-oxygenization, maintains mitochondrial membrane

potential and prevents increases in the amounts of ROS

increase in the red /green fluorescence intensity ratio. Red overrides green.);

d by a decrease in the red /green fluorescence intensity ratio. Green overrides

ochondria are present within the cell. Green: non-functional mitochondria are

annel detection in fluorescence mode excitation with 488 line of a Krypton–

ultaneous double label. Bar equals 5 Am.

Table 1

Data summary of treated and control PC12 cell cultures

Treatment group/deprenyl

concentration

Normoxia Hypoxia

Percentage of

PI+cells

JC-1 R /G

ratio

Cerium reflectance

intensity

Percentage

of PI+cells

JC-1 R /G

ratio

Cerium reflectance

intensity

Control 19.5T2.18 1.07T0.18 23.78T0.78 36.66T3.25 0.681T0.01 28.99T0.65

10–12 M 10.18T0.79 2.15T0.088 24.63T0.93 21.41T3.97 2.83T0.1 20.84T0.5810–8 M 19.67T2.82 2.17T0.077 16.76T1.11 21.54T2.33 2.33T0.074 28.86T0.78

10–3 M 14.83T1.06 2.83T0.18 26.86T1.05 44.25T5.76 1.68T0.095 32.15T0.75

L. Simon et al. / Life Sciences 78 (2005) 225–231 229

induced by hypoxia/re-oxygenization in a dose-dependent

manner.

In PC12 cell cultures DwM progressively shifted across

mitochondrial populations to lower values following hypoxia/

re-oxygenation and as a consequence of this shift all the cells

had decreased and relatively uniform DwM levels. The same

phenomenon was observed in heart-derived H9c2 cells

following hypoxia (Kim et al., 2003), in hypothalamic neurons

following hypoxia (Wu et al., 2004), and in primary rat

hippocampal cultures following oxygen–glucose deprivation

(Iijima et al., 2003). The re-oxygenation considerably increased

the amount of intracellular ROS generated in PC12 cell

cultures as observed by other authors as well (Yermolaieva et

al., 2004). Following 24 h of treatment with various

concentrations of (�)deprenyl during the re-oxygenation

period the percentage of PI+ cells was decreased, the overall

decrease in DwM caused by an oxygen deprivation was

reversed and the peroxy radical levels were decreased except

for the concentration of 10�3 M, revealing a U-shaped curve.

Normoxic PC12 cell cultures treated with various concentra-

tions of (�)deprenyl showed the presence of very low

percentages of PI+ cells. The mitochondrial DwM was found

to be increased at all (�)deprenyl concentrations, and the free

radical level was decreased at 10�12 M.

Our data are consistent with an earlier work by Wadia and

her colleagues (Wadia et al., 1998), who found that (�)deprenyl

at 10�9 M concentration maintains mitochondrial membrane

potential and reduces apoptosis by inducing new protein

synthesis.

Since changes in mitochondrial and cellular function as a

result of hypoxia/re-oxygenization have been implicated in the

cascades leading to cell death, changes in DwM were monitored

Table 2

(�)Deprenyl studies in animal stroke models

Study Species Stroke model Dose range S

Jolkkonen et al., 2000 Rat 72 h of transient cerebral

artery occlusion

0.3 mg/kg S

a

Semkova et al., 1996 Rat Permanent middle cerebral

artery occlusion

15 mg/kg S

d

Simon et al., 2001 Rat Permanent middle cerebral

artery occlusion

0.2 mg/kg/day 0

i

Unal et al., 2001 Mice 30 min middle cerebral

artery occlusion and

72 h reperfusion

10 mg/kg D

Erdo et al., 2000 Gerbil Transient global cerebral

ischemia

0.0001 mg/kg

0.001 mg/kg

0.01 mg/kg

i

t

by using the potentiometric, fluorescent dye JC-1. The

mitochondrial membrane potential (DwM) controls not only

ATP synthesis, mitochondrial Ca2+ accumulation, and redox

poise but also ROS generation.

Oxidative stress/ROS generation is a common detrimental

factor in many different forms of neurodegenerative disease,

or in hypoxia/reperfusion conditions (Yermolaieva et al.,

2004). The present cerium staining used for the in situ

detection of intracellular ROS is the adaptation of previous

methods used by Robinson and Batten (1990), Van Norden

and Frederiks (1993), Halbhuber et al. (1996), Bestwick et al.

(1997) and Telek et al. (1999, 2001). Its limitation, however,

similarly to all the available assays, that it is not rigorously

quantitative, since the dye competes with other molecules for

the interaction with ROS. The rate of oxidation of such

probes shows the steady-state concentration, not the rate of

production.

On the basis of their observations, Thyffault and his

colleagues (1997) deducted that (�)deprenyl enhances O2.

formation by altering the rate of electron transfer within the

respiratory chain leading to increases in SOD activities. The

double nature of this drug involves the induction of an increase

in mitochondrial transmembrane potential as well as elimina-

tion of free radicals at low concentrations, but at extremely

high concentrations it elevates mitochondrial transmembrane

potential to a harmful level resulting in the overproduction of

ROS (Thyffault et al., 1997). This result, however, is only

partially supported by our observations because in our

experiments (�)deprenyl elevated mitochondrial transmem-

brane potential maximally at the concentration of 10�12 M, and

the maximal intracellular ROS production was found at the

concentration of 10�3 M.

tudy design Results

ingle dose following operation,

s a post-treatment

Non-significant decrease

in infarct size

ingle daily doses for 8 consecutive

ays as a pretreatment

Significant decrease

in infarct size

.2 mg/kg/day continuously, for 3 days using an

ntraperitoneal minipump as a post-treatment

Significant decrease

in infarct size

aily doses for 10–14 days as a pretreatment Significant decrease

in infarct size

.p. 6 times in 4 days following

ermination of ischemia

Significant, dose dependent

effect

L. Simon et al. / Life Sciences 78 (2005) 225–231230

(�)Deprenyl prevents ROS production in various in vitro

and in vivo model systems (Wu et al., 1993, 1996; Khaldy et

al., 2000. In our model, this unique antioxidant nature of

(�)deprenyl was associated with maintenance/boost of mito-

chondrial membrane potential. High DwM increases the

generation of ROS and maintains the reduced environment

(Nicholls and Budd, 2000). (�)Deprenyl, however, at 10�12 M

increases DwM and decreases ROS production at the same

time. (�)Deprenyl balances the positive side of increased DwM

(increased mitochondrial activity) against the negative side of

increased DwM (increased ROS production).

The increased DwM associated with a decrease in ROS

levels is the possible basis of (�)deprenyl_s anti-apoptotic

(Magyar and Szende, 2000) and pro-longevity effect (Milgram

et al., 1991; Knoll, 1988). (�)Deprenyl may create the

energetical basis and a comparatively peroxy radical free

environment for penumbral plasticity processes following

middle cerebral artery occlusion in rats (Puurunen et al.,

2001; Simon et al., 2001), and following a stroke event in

humans (Sivenius et al., 2001). (�)Deprenyl may also exert a

significant protective effect against memory deficits and lipid

hyperperoxidation observed after cerebral ischemia (Maia et

al., 2004).

However, previous studies on animal models indicate that

(�)deprenyl does not necessarily reduce the lesion size in

occlusion models of brain ischemia (Jolkkonen et al., 2000;

Semkova et al., 1996; Simon et al., 2001; Unal et al., 2001;

Erdo et al., 2000) (Table 2). The results of these studies suggest

a need for further experiments.

If we look at the overall picture, cytoprotection was

achieved at 10�12 and 10�8 M concentrations of (�) deprenyl,

mitochondrial membrane protection was increased over control

levels at 10�12, 10�8 and 10�3 M concentrations and

reduction of ROS was achieved only at 10�12 M. In other

words there seems to be no correlation between these three

parameters. (�)Deprenyl caused a significant inhibition of

apoptosis in a concentration range of 10�7 to 10�13 M in A-

2058 human melanoma cell culture induced by serum

deprivation, while treatment with 10�3 M (�)deprenyl resulted

in 50% apoptosis 72 h after treatment. This biphasic effect of

(�)deprenyl on cell death was in parallel with changes in

caspase-3 activity. When (�)deprenyl was used in a high

concentration (10�3 M) without serum deprivation, the

caspase-3 activity was raised by 5 to 6 times, while low

concentrations (10�7 to 10�9 M) of the drug inhibited caspase-

3 activity induced by serum deprivation (Magyar and Szende,

2004). (�)Deprenyl seems to be cytoprotective in the dose

range not activating caspase-3.

Conclusion

Our study demonstrates a persistent respiratory defect of

PC12 cell cultures during re-oxygenation after hypoxia. The

defect is associated with lack or heavy delay of recovery of

DwM and reflected mostly by mitochondria progressing

probably to MPT or cytochrome c release (cell death) and

by increased free radical production. This mitochondrial

lesion is prevented and is totally reversed by supplementation

with (�)deprenyl decreasing intracellular ROS levels and

mitochondrial susceptibility to damage at 10�12 M, while at

10�3 M promoting cell death and ROS production, showing

thus a characteristic biphasic action. Because mitochondrial

defects generated by hypoxia/reperfusion can cause irrevers-

ible cell injury, (�)deprenyl treatment merits serious recon-

sideration for rescuing tissues injured by ischemia/reperfusion

and related insults.

Acknowledgements

This study was supported by the Hungarian National

Science Foundation OTKA 2001 T-037887 and ETT 096/

2003 grants.

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