Biochemical and Biophysical Research Communications 308 (2003) 660–667

www.elsevier.com/locate/ybbrc

BBRC

Impairment of superoxide dismutase activation by N-terminallytruncated prion protein (PrP) in PrP-deficient neuronal cell line

Akikazu Sakudo,a,1 Deug-chan Lee,a,1 Keiichi Saeki,a Yuko Nakamura,a Keiichi Inoue,b

Yoshitsugu Matsumoto,a Shigeyoshi Itohara,b and Takashi Onoderaa,*

a Department of Molecular Immunology, School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japanb Laboratory for Behavioral Genetics, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan

Received 12 July 2003

Abstract

Previous studies have reported a neuroprotective role for cellular prion protein (PrPC) against apoptosis induced by serum

deprivation in an immortalized prion protein gene (Prnp)-deficient neuronal cell line, but the mechanisms remain unclear. In this

study, to investigate the mechanisms by which PrPC prevents apoptosis, the authors compared apoptosis of Prnp�=� cells with that

of Prnp�=� cells expressing the wild-type PrPC or PrPC lacking N-terminal octapeptide repeat region under serum-free conditions.

Re-introduction of Prnp rescued cells from apoptosis, upregulated superoxide dismutase (SOD) activity, enhanced superoxide anion

elimination, and inhibited caspase-3/9 activation. On the other hand, N-terminally truncated PrPC enhanced apoptosis accompanied

by potentiation of superoxide production and caspase-3/9 activation due to inhibition of SOD. These results suggest that PrPC

protects Prnp�=� cells from apoptosis via superoxide- and caspase-3/9-dependent pathways by upregulating SOD activity. Fur-

thermore, the octapeptide repeat region of PrPC plays an essential role in regulating apoptosis and SOD activity.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Antioxidant; Apoptosis; Caspase; Oxidative stress; Prion disease; Prion protein; PrP-deficient cell line; Superoxide dismutase

Prion diseases such as Creutzfeldt–Jakob disease(CJD), scrapie, and bovine spongiform encephalopathy

are disorders characterized by the intracerebral accu-

mulation of abnormal prion proteins (PrPSc) [1]. Cellu-

lar prion proteins (PrPC), which are constitutively

expressed on the cell surface of neural and nonneural

cells, are identical in amino acid sequence to PrPSc [2].

Mice lacking the expression of PrPC cannot be infected

with prion agents [3,4] and neurons lacking PrPC ex-pression cannot be killed by the toxicity of PrPSc [5,6].

Therefore, the expression of PrPC is essential for the

induction of prion diseases, and the conversion of PrPC

to PrPSc is closely associated with the main features of

prion diseases. Six lines of prion protein (PrP) gene

(Prnp) knockout mice, designated as Zur I [3], Zur II [7],

Npu [8], Ngsk [9], Rcm0 [10], and Rikn [11], have been

generated. Some lines do not display any anatomical or

* Corresponding author. Fax. +81-3-5841-8020.

E-mail address: aonoder@mail.ecc.u-tokyo.ac.jp (T. Onodera).1 These two authors contributed equally to the work.

0006-291X/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/S0006-291X(03)01459-1

developmental defects [3]. Although altered circadianrhythms [12] and electrophysiological abnormality [13]

have been reported, the latter remains controversial

[14,15]. Other lines of PrP-null mice exhibit a late-onset

ataxia, but this is possibly due to abnormal splicing by

the destruction of splicing acceptor [7,10]. Therefore,

little is known about the function of PrPC.

Recently, a line of PrP-deficient immortalized hippo-

campal neuronal cells derived from Rikn Prnp�=� micehas been established [16]. Moreover, it has been reported

that removal of serum from the cell culture causes ap-

optosis in Prnp�=� cells, but not in Prnpþ=þ cells.

Transfection of PrP gene suppresses apoptosis of

Prnp�=� cells under serum-free conditions [16]. However,

the mechanisms by which PrPC prevents apoptosis re-

main unclear. These recent studies suggest that PrPC may

itself work as an apoptosis suppressor and/or regulatorof apoptosis suppressors and that the loss of functions

causes neuronal cell death. Several apoptotic pathways

are activated by various stimuli. One of the pathways is

regulated by expression of the proto-oncogene bcl-2

A. Sakudo et al. / Biochemical and Biophysical Research Communications 308 (2003) 660–667 661

family, whose members include bcl-2 and bcl-x [17]. Afamily of cysteine proteases, termed caspases, which have

been shown to cleave a number of intracellular targets

including poly(ADP-ribose) polymerase (PARP), are

also involved in the initiation/execution of apoptosis [18].

Several reports concerning the relationship between

PrPC and superoxide dismutase (SOD) using brain or

primary cultures of neuron have provided inconsistent

results [19–22]. Additionally, the regulation of SOD ac-tivity by PrPC may not be a mechanism in an indepen-

dent cell but rather reflect a systemic process of

heterogeneous cell population in the brain. Recently,

rabbit kidney epithelial RK13 cells or human neuro-

blastoma SH-SY5Y cells overexpressing PrPC have been

resistant to hydrogen peroxide, 3-morpholinosydnoni-

mine (SIN-1) or copper induced toxicity possibly due to

increase of antioxidant enzyme activity [23,24]. However,since these cell lines have endogenous Prnp, the effect of

endogenous PrPC could not be excluded from the ex-

planation. Therefore, the antioxidative property of PrPC

was analyzed by investigating the mechanisms of apop-

tosis in the presence or absence of PrPC in Prnp�=� cells.

In this study,weassessed roles ofPrPC andPrPC lacking

an octapeptide repeat region in the apoptosis of Prnp�=�

cells. The results suggested that antioxidative property ofPrPC was the mechanism for preventing apoptosis, and

that the octapeptide repeat region of PrPC essentially

contributed to regulation of SOD activity, superoxide

elimination, and inhibition of caspase-3/9 activation.

Materials and methods

Cell cultures and animals. Murine PrP-deficient cell line (HpL3-4)

[16] was maintained in Dulbecco’s modified Eagle’s medium (DMEM)

(Sigma, St. Louis, MO) supplemented with 10% fetal calf serum (FCS)

at 37 �C in a humidified 5% CO2 incubator. Occasionally, cells were

serum-deprived. For serum deprivation, cells were plated on 60-mm

culture dishes (5� 105 cells per dish) in a complete medium. Two dayslater, cells were washed twice with a serum-free medium and then

placed in a fresh serum-free medium for the indicated time. The male

Rikn Prnp�=� mice [11] and C57BL/6CrSlc (WT) mice purchased from

Nippon SLC were analyzed at 8 weeks of age.

Plasmid construction and gene transfer. In the gene transfer of Prnp,

the coding region of Prnp cDNA was amplified by polymerase chain

reaction (PCR) using the following primers:

Prnp:

50-GCGAATTCCGCCACCATGGCGAACCTTGGCTACTGGCTG-30

50-CACGAATTCCACCTCAATTGAAAGAGCTACAGGTGG-30

The EcoRI linker is underlined and the Kozak consensus sequence

(GCCACCATGG) [25] is boxed. The PCR products were cloned

into pT7Blue vector and subcloned into the multicloning site of

pMSCVpuro (Clontech, Palo Alto, CA). The resulting construct

pMSCVpuro-PrP was transiently transfected into the packaging cell

line PT67 by the Lipofectamine-mediated method (Life Technologies,

Gaithersburg, MD). Viral supernatants were harvested for the trans-

duction of HpL3-4 cells. These cells were selected for more than 10

days in a complete medium containing 20 lg/ml puromycin (WAKO,Osaka, Japan). Ten individual colonies were further selected. Of 10

stable Prnp-transfected lines, two clones (HpL3-4-PrPNo3 and HpL3-

4-PrPNo7) with abundant PrPC were used for this study. Additionally,

a plasmid containing a Prnp cDNA rendering an N-terminally trun-

cated PrP lacking the octapeptide repeat region [PrP(D53–94, Q52H)]was constructed using the restriction enzymes Eco81I and KpnI and a

DNA blunting kit (Takara Bio, Otsu, Japan). The Prnp and Prnp

deletion cDNAs were subcloned into the multicloning site of

pMSCVpuro-EGFP, which was constructed by insertion of the BglII–

HpaI fragment of pIRES2-EGFP (Clontech) into pMSCVpuro,

resulting in pMSCVpuro-PrP-EGFP and pMSCVpuro-PrP(D53–94,Q52H)-EGFP. HpL3-4 cells were transduced with pMSCVpuro-

EGFP, pMSCVpuro-PrP-EGFP or pMSCVpuro-PrP(D53–94, Q52H)-EGFP by a retrovirus-mediated method as described above. The

resulting stable transfectants were named HpL3-4-EM, HpL3-4-PrP,

and HpL3-4-D#1, respectively.SOD activity assay. A SOD assay kit-WST (Dojindo, Kumamoto,

Japan) was used for the quantification of SOD activity. This method is

a xanthine-based spectrophotometric assay. Each protein extract

(20 lg) was assayed and compared with 1U of bovine erythrocyte Cu/Zn-SOD (Sigma S2515) activity. The SOD activity was estimated using

the standard curve of SOD activity versus absorbance.

Western blot assay. To fractionate tissue samples, 10% (w/v) ho-

mogenate was prepared in PBS by sonication. After centrifugation at

600g for 15min at 4 �C, supernatants were ultracentrifuged at 200,000gfor 1 h, at 4 �C. The pellet was solubilized by 27-gauge syringe. Eachfraction was solubilized in radio-immunoprecipitation assay (RIPA)

buffer composed of 10mM Tris–HCl (pH 7.4) containing 1% sodium

deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS),

and 0.15M sodium chloride supplemented with 2mM phenylmethyl-

sulfonyl fluoride (PMSF).

For detection of Bcl-2, Bcl-xL, and Cu/Zn-SOD, tissue homogen-

ates were directly lysed in RIPA buffer, while the membrane fraction of

tissue samples was used for detection of PrPC.

Cells were pelleted by centrifugation and washed with PBS before

lysis with RIPA buffer. After centrifugation at 10,000g for 10min at

4 �C, the protein concentration of the supernatant was measured usingthe Bio-Rad DC assay, and an equal quantity of protein (60lg forPrPC, 50lg for Bcl-2, 30 lg for Bcl-x, and 20 lg for Cu/Zn-SOD,cleaved caspase-3, cleaved caspase-9, and cleaved PARP) was dena-

tured in 2� SDS gel-loading buffer [90mM Tris–HCl (pH 6.8), 10%

mercaptoethanol, 2% SDS, 0.02% bromophenol blue, and 20% glyc-

erol] and separated on SDS/12% polyacrylamide gel before electrical

transfer onto polyvinyl difluoride (PVDF) membrane (Hybond-P;

Amersham–Pharmacia Biotech, Piscataway, NJ) for 60min at 15V.

Blots were treated by BLOCK-ACE (Dainippon Pharmaceutical,

Osaka, Japan) at 4 �C overnight and then incubated in PBS containing0.1% Tween 20 (PBS–T) and 10% BLOCK-ACE for 1 h at room

temperature with one of the following: 3F11 monoclonal anti-Bcl-2

(Pharmingen, San Diego, CA), polyclonal anti-Bcl-x (Pharmingen),

polyclonal anti-Cu/Zn-SOD (Stressgen, Victoria, BC), polyclonal

anti-cleaved caspase-3 (Cell Signaling Technology, Beverly, MA),

polyclonal anti-caspase-9 (Cell Signaling Technology), polyclonal anti-

cleaved PARP (Cell Signaling Technology), P8 polyclonal anti-PrP,

which recognizes amino acids 92–109 of mouse PrP (GGTHNQWN

KPSKPKTNLK), or 6H4 monoclonal anti-PrP, which recognizes

residues 144–152 of mouse PrP. After washing thrice with PBS–T, the

membrane was incubated in horseradish peroxidase-conjugated sec-

ondary antibody for 1 h at room temperature. After washing twice

with PBS–T, the probed proteins were detected using an enhanced

chemiluminescence detection kit (Amersham–Pharmacia Biotech).

Apoptosis assays. Internucleosomal DNA fragmentation was de-

tected after 24-h incubation in a serum-free medium by DNA lad-

dering assay [16]. Cells were lysed with lysis buffer [10mM Tris–HCl

(pH 7.4), 10mM EDTA (pH 8.0), and 0.5% Triton X-100], incubated

for 20min on ice, and centrifuged at 12,000g for 30min at 4 �C. Theaqueous phase was incubated with 400lg/ml DNase-free RNase A for1 h at 37 �C. Proteins were digested with 400lg/ml of proteinase K for1 h at 37 �C and then DNA was precipitated in a solution of 0.4M

Fig. 1. Re-introduction of Prnp did not change the expression of Bcl-2,

Bcl-xL, and Cu/Zn-SOD. Western blot analysis of protein extracts

using anti-PrP (P8), Bcl-2, Bcl-x, and Cu/Zn-SOD antibodies. The

expressions of Bcl-2, Bcl-xL, and Cu/Zn-SOD were not significantly

different between Prnp transfected HpL3-4 cells [HpL3-4-PrPNo3 (lane

2) and HpL3-4-PrPNo7 (lane 3)] and the parental HpL3-4 cell lines

(lane 1). Bcl-2, Bcl-xL, and Cu/Zn-SOD were similarly expressed in WT

and Prnp�=� (KO) mouse brain.

662 A. Sakudo et al. / Biochemical and Biophysical Research Communications 308 (2003) 660–667

NaCl in 50% isopropanol overnight at )20 �C. Precipitated DNAsamples were resuspended in TE buffer [10mM Tris–HCl (pH 7.4),

1mM EDTA (pH 8.0)] and electrophoresed through 2.0% agarose gel.

The gel was stained with ethidium bromide (EB) (0.5mg/ml) for 10min

and destained with milli-Q for 10min. DNA bands were visualized by

UV light-transilluminator and photographed.

Reactive oxygen species probes. Intracellular H2O2 and superoxide

anion levels were determined based on methods adapted from those

previously described [26,27] using 20,70-dichlorofluorescein diacetate

(DCFH-DA) (Lambda Fluoreszenztechnologie, Graz, Austria) and

Dihydroethidium (DH) (Molecular Probes, Eugene, OR), respectively.

DCFH-DA is incorporated into cells and hydrolyzed by intracellular

esterase, generating the nonfluorescent molecule DCFH. H2O2 oxi-

dizes DCFH to the fluorescent molecule DCF. DH permeates to viable

cells and is directly oxidized to EB by superoxide anion intracellularly

to yield fluorescence for measurement.

DCFH-DA or DH was added to cell cultures to a final concen-

tration of 10lM and incubated for 30min at 37 �C. The cells werecollected, washed twice, and then analyzed with flow cytometry.

Fluorescence was collected using an excitation of 488 nm and a 530/

30-nm band pass for DCF and a 585/42-nm band pass for EB.

Caspase activity assay. Equal quantity of protein (20lg for caspase-3/7 and 60lg for caspase-9) from cells before and after serum depri-

vation for the indicated time was incubated in reaction buffer [20mM

Hepes (pH 7.5); 10% glycerol; and 2mM DTT] containing 100lMAc-LEHD-methyl-coumaryl-7-amide (MCA) (specific substrate for

caspase-9) or Ac-DEVD-MCA (specific substrate for caspase-3/7)

obtained from Peptide Institute (Osaka, Japan). LEHD and DEVD

cleavage was assessed by monitoring fluorescence with excitation and

emission wavelengths of 355 and 460 nm using a Fluoroscan fluo-

rometer (Labsystems Oy, Helsinki, Finland).

Statistical analysis. Quantification of SOD activity, superoxide

anion production, and caspase activity was tested statistically by the

unpaired Student’s t test. p Values smaller than 0.05 were considered tobe statistically significant.

Results

Influence of PrP expression on the protein levels of Bcl-2,

Bcl-xL, and Cu/Zn-SOD

Elucidation of the mechanisms by which PrPC inhibits

apoptosis was approached by analysis of Prnp-trans-

fectedPrnp�=� cells byWestern blotting. Re-introductionof Prnp was confirmed by Western blotting (Fig. 1) and

flow cytometry (data not shown). There were no differ-

ences in the expression level of Bcl-2, Bcl-xL, and Cu/Zn-

SOD protein among HpL3-4, HpL3-4-PrPNo3, and

HpL3-4-PrPNo7 (Fig. 1). In a similar tendency, equal

expressions of Bcl-2, Bcl-xL, and Cu/Zn-SOD protein

were observed betweenwild-type (WT) andPrnp�=� (KO)

murine brain (Fig. 1). In the brain samples, Bcl-xL wasdetected as a doublet with a molecular mass of approxi-

mately 28 kDa. The higher band of this doublet may be a

phosphorylated form of the Bcl-xL protein [28].

Correlation of PrPC production and SOD activity using

tissue and cell lines

For simple analysis of PrPC distribution, the precip-

itate after ultracentrifugation (200,000g for 1 h at 4 �C)

of the tissue homogenate (considered to be the mem-

brane fraction) and the supernatant (the soluble frac-

tion) were employed. PrPC was detected only in the

membrane fraction of WT mouse brain, whereas Cu/Zn-SOD was detected in the soluble fraction of WT and KO

mouse tissues (Fig. 2A). The SOD activity in the above-

mentioned fraction was then measured. The membrane

fraction of WT mouse brain, where PrPC is abundant,

showed elevated SOD activity as compared with the

membrane fraction of other tissues (Fig. 2B). The SOD

activity of the soluble fraction of Prnp�=� mouse brain

and testis indicated a slight but significant decreasewhen compared to WT mouse brain and testis, respec-

tively (Fig. 2C).

To further examine the relationship between expres-

sion of PrPC and SOD activity, PrP-deficient neuronal

cells were analyzed. The lysates of HpL3-4-PrPNo3 and

HpL3-4-PrPNo7 cells, which expressed PrPC (Fig. 1),

revealed enhanced SOD activity as compared with

HpL3-4 cells (data not shown). Flow cytometry analysisusing ROS probes demonstrated that HpL3-4PrPNo3

cells eliminated superoxide anion generation and en-

hanced H2O2 production as compared with HpL3-4 cells

under serum deprivation for 6 h (Figs. 3A and B), sug-

gesting that augmented SOD activity by PrPC effectively

converted superoxide anion into H2O2.

Role of the N-terminal octapeptide repeat region of

PrPC for inhibition of apoptosis and superoxide anion

elimination

Because the N-terminal octapeptide repeat region of

PrPC has been shown to bind to copper and is involvedin the enzymatic function [29], it was of interest to de-

termine whether the octapeptide repeat region of PrPC

affected the suppression of apoptotic neuronal cell death

under serum deprivation. This was examined in PrP-

negative cells (HpL3-4-EM), PrP-positive cells (HpL3-4-

Fig. 3. Re-introduction of Prnp eliminated superoxide anion and in-

creased hydrogen peroxide. Flow cytometric analysis of ROS in serum-

deprived cells. (A) The amount of intracellular superoxide anion was

determined by DH. (B) Hydrogen peroxide was detected by the

staining of DCFH-DA. HpL3-4 and HpL3-4PrPNo3 (PrPNo3) cells

were stained with the fluorescent probes as described in Materials and

methods. The histograms of cells under serum-deprivation for 6 h and

control (0 h) are indicated by filled and open areas, respectively.

Fig. 2. SOD activity in the wild-type and Prnp�=� mouse tissues. Brain

and testis homogenates from Prnp�=� and WT mice were fractionated

by ultracentrifugation. (A) PrPC and Cu/Zn-SOD in precipitate (Ppt)

and supernatant (Sup) were examined by Western blotting. PrPC was

detected in the wild-type (WT) mouse brain but not in Prnp�=� (KO)

mouse brain. PrPC was detected only in the membrane fraction of WT

mouse brain, while Cu/Zn-SOD was detected in the soluble fraction of

WT and KO mouse tissues. (B–C) SOD activity of the above-men-

tioned fraction of tissue was determined. SOD activity correlates with

the expression level of PrPC in the membrane fraction (B). In the

soluble fraction, KO mouse tissue showed reduced SOD activity as

compared with WT mouse tissue (C). Differences with p < 0:05 (*)

between WT and KO tissue and differences with p < 0:01 (**) versus

other fraction of tissue were significant.

A. Sakudo et al. / Biochemical and Biophysical Research Communications 308 (2003) 660–667 663

PrP), and cells transfected with a plasmid expressingPrnp cDNA rendering an N-terminally truncated PrP

lacking the octapeptide repeat region (HpL3-4-D#1).Western blotting showed that HpL3-4-PrP and HpL3-4-

D#1 expressed PrPC at the equivalent level on WT

mouse brain (Fig. 4A). When compared with empty

vector transfected cells (HpL3-4-EM), the cells ex-

pressing PrPC (HpL3-4-PrP) were markedly resistant to

apoptosis following serum deprivation for 24 h (Fig. 4B).The cells expressing N-terminally truncated PrPC

(HpL3-4-D#1), which lacks an octapeptide repeat re-gion, showed enhanced apoptosis when compared with

HpL3-4-EM (Fig. 4B), thus illustrating the importance

of this region for survival in serum-derived medium.

To examine whether the increased susceptibility of

HpL3-4-D#1 cells to serum deprivation was due to ab-errant activity of SOD, SOD activity and superoxide

anion production in the presence and absence of serum

were analyzed. HpL3-4-D#1 cells demonstrated signifi-cantly low activity of SOD and a significant increase in

superoxide anion production compared with HpL3-4-

EM cells under serum deprivation for 6 h (Figs. 4C and

D). However, HpL3-4-PrP cells showed significantly

high activity of SOD and a significant decrease in su-peroxide anion production compared with HpL3-4-EM

cells (Figs. 4C and D).

Serum deprivation induced caspase-3/9 activation which

was inhibited by PrPC and enhanced by N-terminally

truncated PrPC lacking octapeptide repeat region

To further characterize the effects of PrPC and N-

terminally truncated PrPC lacking the octapeptide repeat

region on apoptosis, levels and activities of apoptosis

related proteins including caspase-3, caspase-9, and

PARP in HpL3-4-EM, HpL3-4-PrP, and HpL3-4-D#1cells were examined by Western blotting and caspaseactivity assay. Western blot analysis demonstrated that

active cleaved forms of caspase-3 (17 kDa) and caspase-

9 (37 and 39 kDa) were observed in HpL3-4-EM and

Fig. 4. N-terminal octapeptide repeat region of PrPC plays an essential

role in regulating apoptosis and SOD activity. (A) Western blot

analysis with anti-PrP 6H4 of equal quantity of protein fromWT brain

(WT) and HpL3-4 cells expressing wild-type PrP (PrP: HpL3-4-PrP),

PrP(D53–94, Q52H) (#1: HpL3-4-D#1) or the empty vector only (EM:HpL3-4-EM). (B) DNA fragmentation assay showed that HpL3-4-EM

cells exhibited more apoptosis than HpL3-4-PrP cells after serum de-

privation for 24 h. HpL3-4-D#1 cells (#1) elicited increased ratio ofapoptosis compared with HpL3-4-PrP cells (PrP) or HpL3-4-EM cells

(EM). (C–D) Effect of PrPC on SOD activity (C) and superoxide anion

production (D). HpL3-4-EM (EM), HpL3-4-PrP (PrP), and HpL3-4-

D#1 (#1) cells were serum-deprived for 0 h (white bars) or 6 h (blackbars). SOD activity was determined by xanthine-based spectrophoto-

metric assay as described in Materials and methods. Results are ex-

pressed as units/mg protein. The oxidation of DH (a measure of

superoxide anion production) was determined as the increase in the

fluorescence of EB. Results are expressed as superoxide anion level

(relative mean fluorescence intensity of EB). Superoxide anion level of

HpL3-4-EM cells under serum-deprivation for 0 h was taken as 100%.

One asterisk (*) and two asterisks (**) denote a statistically significant

difference compared to HpL3-4-EM cells serum-deprived for 6 h

[p < 0:05 (*) and p < 0:01 (**)]. A plus (+) denotes a significant dif-

ference compared to HpL3-4-EM cells serum-deprived for 0 h

[p < 0:01 (+)].

Fig. 5. Caspase-3/9 activation and PARP cleavage were inhibited by

PrPC and enhanced by N-terminally truncated PrPC. (A) Levels of

cleaved caspase-3, cleaved caspase-9, and cleaved PARP in HpL3-4-

EM (EM), HpL3-4-PrP (PrP), and HpL3-4-D#1 (#1) cells serum-de-prived for the indicated time were analyzed by Western blotting. Note

that cleaved caspase-3, cleaved caspase-9, and cleaved PARP were

detected in HpL3-4-EM and HpL3-4-D#1 cells at 12 and 24 h. Thelevels of the cleaved proteins were decreased in HpL3-4-PrP cells and

increased in HpL3-4-D#1 cells compared with HpL3-4-EM cells. (B–

C) LEHD (B) and DEVD (C) cleavage activity in cell lysates obtained

from serum-deprived cultures of HpL3-4-EM, HpL3-4-PrP, and

HpL3-4-D#1 cells. DRFU is equivalent to the change in relative fluo-rescent units (RFU) per minute (100�). One asterisk (*) and two as-terisks (**) denote a statistically significant difference compared to

HpL3-4-EM cells serum-deprived for 24 h [p < 0:05 (*) and p < 0:01

(**)].

664 A. Sakudo et al. / Biochemical and Biophysical Research Communications 308 (2003) 660–667

HpL3-4-D#1 cells following serum deprivation for 12 h

and 24 h (Fig. 5A). HpL3-4-D#1 cells showed enhancedcleavage of caspase-3 and caspase-9 when compared

with HpL3-4-EM cells (Fig. 5A). In contrast, active

cleaved forms of caspase-3 and caspase-9 were not ob-

served in HpL3-4-PrP cells until 24 h (Fig. 5A). Con-firming these observations, PARP fragment (89 kDa)

cleaved by active caspase-3 was detected in HpL3-4-EM

and HpL3-4-D#1 cells at 12 and 24 h, while increasedlevel of cleaved PARP was observed in HpL3-4-D#1cells compared with HpL3-4-EM cells at 12 and 24 h

(Fig. 5A). On the other hand, reduced level of cleaved

PARP fragment was observed in HpL3-4-PrP cells

compared with HpL3-4-EM cells at 12 and 24 h(Fig. 5A). Next, to further confirm the relationship be-

tween caspase-3/9 activity and full-length or N-termi-

nally truncated PrPC, caspase activity assay was

performed. HpL3-4-D#1 cells showed significantly highactivity of DEVD cleavage activity, which represents

caspase-3/7 activity [30], and LEHD cleavage activity,

which represents caspase-9 activity [30], compared with

HpL3-4-EM cells under serum deprivation for 24 h

(Figs. 5B and C). On the other hand, HpL3-4-PrP cells

showed significantly low activity of DEVD and LEHD

cleavage activity compared with HpL3-4-EM cells (Figs.

5B and C).

Discussion

Balancing between the function of ROS and the anti-

oxidant defense mechanism is thought to be essential for

the regulation and prevention of apoptosis. One efficient

A. Sakudo et al. / Biochemical and Biophysical Research Communications 308 (2003) 660–667 665

anti-oxidant enzyme in neurons is SOD. This enzymeconverts superoxide anion radicals into H2O2. Accord-

ing to Brown et al. [19], the brains of Prnp�=� mice have

reduced the activity of cytosolic Cu/Zn-SOD. Further-

more, PrPC per se elicits SOD activities. The N-terminal

octapeptide repeat region has been shown to bind to

copper and is involved in the enzymatic function of PrPC

[29]. These studies suggest that PrPC plays a role in

cellular resistance to oxidative stress. However, a re-cently conflicting report has demonstrated that the

amount of brain copper and the activity of cuproen-

zymes, such as SOD and cytochrome C oxidase, do not

differ with the PrPC expression level [20]. More recent

papers demonstrated that PrPC overexpression increased

resistance to hydrogen peroxide, SIN-1 or copper in

induced toxicity in RK13 or SH-SY5Y cell lines possibly

due to increase of antioxidant enzyme activity [23,24].However, since RK13 and SH-SY5Y cells have endog-

enous Prnp, the effect of endogenous PrPC could not be

excluded from the explanation. Therefore, further

studies using PrP-deficient immortalized neuronal cells

are warranted to fully elucidate the relationship between

PrPC and SOD activity. The present study analyzed this

relationship using PrP-deficient neuronal cells; Prnp�=�

neuronal cells were apoptotic after serum deprivation,and re-introduction of Prnp upregulated SOD activity,

suggesting that PrPC inhibited apoptosis by upregula-

tion of SOD activity. Moreover, PrPC did not change

Bcl-2, Bcl-xL, and Cu/Zn SOD expression levels, sug-

gesting that PrPC inhibits apoptosis regardless of Bcl-2,

Bcl-xL and Cu/Zn SOD expression levels. Furthermore,

as transfection of SOD-1, which encodes Cu/Zn SOD,

but not treatment of Cu/Zn SOD inhibited apoptosis inPrnp�=� cells (data not shown), superoxide anion gen-

erated within an individual cells and acting inside cells is

an important event in apoptosis. PrPC expression in

Prnp�=� cells resulted in effective elimination of super-

oxide anion and H2O2 accumulation under serum de-

privation, suggesting that SOD activated by PrPC

efficiently converts superoxide anion into H2O2. These

data support the notion that PrPC acts to regulate SODactivity and superoxide anion elimination in an inde-

pendent cell.

The membrane fraction of WT mouse brain, where

PrPC is highly expressed, manifested a higher level of

SOD activity than those of other membrane fractions of

tissues such as WT and Prnp�=� mouse testis and

Prnp�=� mouse brain, suggesting indeed that PrPC per se

elicits SOD activity. In addition, the soluble fraction ofWT mouse brain and testis, where cytosolic Cu/Zn-SOD

exists, showed higher SOD activity compared with

Prnp�=� mouse brain and Prnp�=� mouse testis, respec-

tively, suggesting that low levels of PrPC are necessary

for the activation of cytosolic Cu/Zn-SOD because ex-

pression of PrPC in testis is low but sufficient for stabi-

lizing SOD activity. Furthermore, re-introduction of

Prnp upregulated SOD activity without changing Cu/Zn-SOD expression levels. It has been reported that copper

binding promotes PrPC endocytosis [31]. N-terminal

deletion mutants of chicken PrPC show diminished en-

docytosis [32]. These suggest that PrPC upregulates SOD

activity by incorporation of copper into Cu/Zn-SOD via

the N-terminal domain of PrPC. Zeng et al. [24] recently

reported that substitution of signal peptide sequence of

PrPC for the uncleaved signal sequence and transmem-brane sequence compromises cellular SOD activity. In-

terestingly, our studies indicated that PrPC lacking an

octapeptide repeat region accelerates apoptosis and in-

hibits SOD activity. As the N-terminal deletion mutant

of PrPC enhanced caspase-3/9-mediated pathway, it is

suggested that the toxicity of the deletion mutant of PrPC

functions in the upstream of caspase-3/9 cascade. It is

consistent with the recent report that overexpression ofPrPC suppresses cell death induced by Bax-overexpres-

sion, but PrPC lacking an octapeptide repeat region does

not enhance Bax-induced cell death in human primary

culture neurons [33]. Taken together with our studies, the

toxicity of PrPC lacking an octapeptide repeat region

functions in the upstream of caspase-3/9 cascade at least

in part by inhibition of SOD activity. As the N-termi-

nally truncated PrPC decreased SOD activity, these re-sults raise the possibility that some factors, which

interact with PrPC, may mediate copper transfer from

PrPC to Cu/Zn-SOD, and the existence of the octapep-

tide repeat region of PrPC may be indispensable for

copper transfer and regulation of SOD activity. Stress-

inducible protein 1 (STI1) is a candidate for involvement

in such an interaction with PrPC [34]. STI1, which me-

diates PrPC dependent neuroprotective signals, interactswith amino acids 113–128 of PrPC [34]. However, to test

the possibility, further study to investigate the functional

interaction of PrPC and copper with STI1 or unknown

factors would be necessary. Overall, the present study

supports the notion that PrPC aids the elimination of

superoxide anion in an independent cell and N-terminal

octapeptide repeat region of PrPC is essential for its

regulation of apoptosis and SOD activity.The present study showed that serum deprivation

caused apoptosis of Prnp�=� cells via intracellular su-

peroxide production and caspase-3/9 activation, and

PrPC suppressed apoptosis, at least in part, by upregu-

lation of SOD activity and inhibition of caspase-3/9

activation via N-terminal octapeptide repeat region of

PrPC. These results advocate the fact that superoxide

anion production and caspase-3/9 activation are criticalevents in the apoptosis of Prnp�=� cells under serum

deprivation, and the octapeptide repeat region of PrPC

essentially contributes to regulation of SOD activity,

superoxide elimination, and inhibition of caspase-3/9

activation. When animals are infected with prions, PrPSc

is converted from PrPC and accumulates. The conver-

sion of PrPC to PrPSc leads to PrPC deficiency [11].

666 A. Sakudo et al. / Biochemical and Biophysical Research Communications 308 (2003) 660–667

Our studies suggest that neuronal cells are susceptible tooxidative stress due to reduced SOD activity and cas-

pase-3/9 activation induced by PrPC deficiency. These

findings suggest that not only accumulation of PrPSc but

also deficiency of PrPC are crucial events in the

neurodegeneration of prion diseases.

Acknowledgments

The authors are grateful to Dr. Yoshio Yamakawa (Department of

Biochemistry and Cell Biology, National Institute of Infectious Dis-

eases) for the gift of anti-prion protein P8. Thanks are due to Dr. An-

thony Foong for reading the manuscript. This work was supported by

the Sasakawa Scientific Research Grant from The Japan Science Soci-

ety (to A.S.) and aGrant-in-Aid for Science Research from theMinistry

of Education, Science, Culture and Technology of Japan (to T.O.).

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