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Neurobiology of Disease 18 (2005) 450–458

Caspase-3-mediated cleavage of PHF-1 tau during apoptosis

irrespective of excitotoxicity and oxidative stress:

an implication to Alzheimer’s disease

Hyo Jung Kang,a,b Won Joo Yoon,a Gyeong Joon Moon,a Doo Yeon Kim,c Seonghyang Sohn,d

Hyuk Jae Kwon,d and Byoung Joo Gwagb,e,*

aDepartment of Neuroscience, Ajou University School of Medicine, Suwon, Kyungkido, KoreabDepartment of Pharmacology, Ajou University School of Medicine, Suwon, Kyungkido, KoreacNeurobiology of Disease Laboratory, Genetics and Aging Research Unit, Department of Neurology and Mass General Institute for Neurodegenerative

Diseases, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 01219, USAdLaboratory of Cell Biology, Ajou University School of Medicine, Suwon, Kyungkido, KoreaeCenter for the Interventional Therapy of Stroke and Alzheimer’s Disease, Ajou University School of Medicine, Suwon, Kyungkido, Korea

Received 9 March 2004; revised 13 November 2004; accepted 10 December 2004

Available online 25 January 2005

Excitotoxicity, oxidative stress, and apoptosis have been recognized as

routes to neuronal death in various neurological diseases. We examined

the possibility that PHF-1 tau, a substrate for various proteases, would

be selectively cleaved depending upon routes of neuronal death.

Cleavage form of PHF-1 tau was not observed in cortical cell cultures

exposed to excitotoxins or oxidative stress that cause neuronal cell

necrosis. PHF-1 tau was cleaved within 8 h following exposure of

cortical cell cultures to apoptosis-inducing agents. This cleavage was

blocked by inclusion of zDEVD-fmk, an inhibitor of caspase-3, and

accompanied by activation of caspase-3. Levels and cleavage of PHF-1

tau were markedly increased in AD brain compared with control.

Moreover, PHF-1 tau and active caspase-3 were colocalized mostly in

tangle-bearing neurons. The current findings suggest that PHF-1 tau is

cleaved by caspase-3 during apoptosis and neurodegenerative process

in AD.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Alzheimer’s disease; Neurofibrillary tangles; Caspase-3;

Apoptosis; Tau; Glutamate

Introduction

Accumulating evidence suggests that oxidative stress, excitotox-

icity, and apoptosis comprise the major routes of neuronal death in

0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.nbd.2004.12.004

* Corresponding author. Department of Pharmacology, Ajou University

School of Medicine, San 5, Woncheon, 442-749, Suwon, Kyungkido,

Korea. Fax: +82 31 219 5069.

E-mail address: bjgwag@ajou.ac.kr (B.J. Gwag).

Available online on ScienceDirect (www.sciencedirect.com).

Alzheimer’s disease (AD). Redox-active iron is accumulated in

extracellular senile plaques and intracellular neurofibrillary tangles

(NFTs) in AD (Smith et al., 1997). Reduced energy metabolism,

advanced glycation end products, and beta amyloid can produce

oxidative stress in AD (Castellani et al., 2001). A great deal of work

reports increase in oxidation products of lipid, DNA, and protein in

AD (Ando et al., 1998; Smith et al., 1991). Glutamate transporters

are deficient in AD, which can cause accumulation of glutamate at

the synaptic cleft (Keller et al., 1997; Masliah et al., 1996). Cortical

neurons treated with beta amyloid are highly vulnerable to

excitotoxicity (Koh et al., 1990; Mattson et al., 1992). This implies

that oxidative stress and excitotoxicity may contribute to neuro-

degeneration in AD.

Recently, apoptosis has been proposed as additional type of death

in AD based upon prominent DNA damage positive to terminal

transferase-mediated dUTP-biotin nick end labeling (TUNEL) and

activation of caspases, a family of cysteine proteases mediating

apoptosis in various types of cells (Smale et al., 1995; Su et al.,

2000). However, TUNEL-positive cells and activated caspases have

been reported following exposure to H2O2 and hypoxic ischemic

injury that can cause necrosis as well as apoptosis (Cole and Perez-

Polo, 2002; Moroni et al., 2001). Thus, it remains to be resolved if

apoptosis contributes to neuronal death in AD.

Evidence has been accumulated suggesting that excitotox-

icity, oxidative stress, and apoptosis are propagated through

distinctive signaling pathways (Gwag et al., 2002). Recently, we

have observed that PHF-1-positive phosphorylated tau (PHF-1

tau), a major constituent of paired helical filaments (PHF) in

AD, is cleaved by caspase-3 in the process of calyculin A-

induced neuronal cell apoptosis (Ko et al., 2000). The present

study was aimed to examine the possibility that PHF-1 tau

would be cleaved selectively in the process of neuronal cell

H.J. Kang et al. / Neurobiology of Disease 18 (2005) 450–458 451

apoptosis. We additionally studied cleavage of PHF-1 tau and

activation of caspase-3 in AD to determine if apoptosis would

contribute to neuronal death in AD as additional route besides

excitotoxicity and oxidative stress.

Materials and methods

Human brain tissue

Autopsy brain tissue from the cortex of five neuropatholog-

ically confirmed AD cases and three age-matched cases with no

AD changes or any other neurodegenerative condition, e.g.,

Pick’s disease, Parkinsons disease, Dementia with Lewy Bodies,

Progressive Supranuclear Palsy (Table 1). Each brain underwent

exhaustive neuropathological analysis. Human brain tissues used

in this study were provided by Boston University Alzheimer’s

Disease Center (Dr. Ann C. Mckee).

Cell culture

Cortical cells were prepared from fetal ICR mice (E15) and

mechanically triturated. Dissociated cells were plated on 24 well

plates (5 hemispheres/plate, approximately 105 cells/well) in a

plating medium consisting of Eagle’s minimal essential media

(MEM, Earle’s salts, 11090-081) supplemented with 5% horse

serum, 5% fetal bovine serum, 2 mM glutamine and 21 mM

glucose. Proliferation of non-neuronal cells was halted by adding

cytosine arabinoside (final concentration 10 AM) at 7–9 days in

vitro (DIV 7–9) when astrocytes became confluent. Cultures were

then fed twice a week with plating medium lacking fetal serum.

Cultures were maintained at 378C in a humidified 5% CO2

atmosphere.

Transmission electron microscopic study

Cultures were fixed in Karnovsky’s fixative solution (1%

paraformaldehyde, 2% glutaraldehyde, 2 mM calcium chloride,

100 mM cacodylate buffer, pH7.4) for 2 h, washed with cacodylate

buffer, and postfixed in 1% osmium tetroxide and 1.5% potassium

ferrocyanide for 1 h. Cells were then stained en bloc in 0.5% uranyl

acetate, dehydrated through a graded ethanol series and embedded

in Poly/Bed 812 resin (Pelco, CA, USA). Cells were sectioned

using Reichert Jung Ultracut S (Leica, Cambridge, UK). After

staining cells with uranyl acetate and lead citrate, cells were

observed and photographed under Zeiss EM 902 A.

Table 1

Case demographics

Case Group Age/sex

(years)

PMI

(h)

Neur

diagn

1 Control 66/M 8.5 Norm

2 Control 85/M 19.0 Norm

3 Control 73/M 17.0 Norm

4 AD 80/M 8.5 AD

5 AD 80/M 7.0 AD

6 AD 70/M 3.0 AD

7 AD 79/M 7.0 AD

8 AD 83/M 12.0 AD

Note. PMI, postmortem interval.

Western blot analysis

Cultured cells were lysed in a lysis buffer containing 50 mM

Tris–HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5%

deoxycholic acid, 0.1% SDS, 1 mM PMSF, and 100 Ag/ml

leupeptin. Brain tissues were lysed in a lysis buffer containing

10 mM Tris–HCl pH 7.5, 50 mM NaCl, 1% Triton X-100, 30 mM

sodium pyrophosphate, 50 mM NaF, 5 AM ZnCl2, 2 mM PMSF,

and 100 Ag/ml leupeptin, 10 Ag/ml pepstatin A, and 1 mM DTT.

Lysates were centrifuged at 13,000 � g for 10 min, the

supernatants collected, subjected to electrophoresis on a 12%

SDS-polyacrylamide gel, and transferred to a nitrocellulose

membrane. The blot was incubated in 5% nonfat dry milk for

30 min, reacted with primary antibodies for overnight at 48C, andthen incubated with a biotinylated anti-mouse or rabbit secondary

antibody (1:1000) for 2 h. For internal controls, polyclonal

antibody for actin (Sigma, St. Louis, MO, USA, 1:1000) was

probed on the same blot. Signals were detected using the

VECTASTAIN ABC kit (Vector Lab., Burlingame, CA, USA)

and luminol as an enhanced chemiluminescence substrate (Amer-

sham, Buckinghamshire, UK), and then analyzed using Kodak X-

Omat film or an image analyzer LAS1000 (Fuji Photo Film Co.,

Ltd.). The PHF-1 tau monoclonal antibody recognizes tau

phosphorylated at Ser-396 and Ser-404 (a generous gift from

Dr. P. Davis, Department of Pathology, Albert Einstein College of

Medicine, Bronx, NY, USA, 1:1000). The Tau-5 monoclonal

antibody recognizes the tau at 200–233 amino acids independent

on phosphorylation state (a generous gift from Dr. Lester I Binder,

Northwestern University School of Medicine, Chicago, IL, USA,

1:5000). The rabbit polyclonal antibody for caspase-3 recognizes

active caspase-3 of 17–20 kDa (Cell Signaling, Beverly, MA,

USA, 1:1000) or pro caspase-3 of 32 kDa (Sigma, St. Louis, MO,

USA, 1:1000).

Immunohistochemistry

Brain sections were incubated in 10% normal horse serum

for 1 h, reacted with mouse monoclonal antibody recognizing

PHF-1 (1:200), rabbit polyclonal antibody recognizing active

caspase-3 (Cell Signaling, Beverly, MA, USA, 1:100) overnight,

and then reacted with FITC- or Texas-red-labeled secondary

antibodies (Vector, Burlingame, CA, USA, 1:200). To stain

nuclei, Hoechst (Molecular Probes, Eugene, OR, USA) was

applied for 10 min at RT. Finally, sections were washed with

distilled water, air dried, and mounted with Vectashield (Vector,

Burlingame, CA, USA). All images were collected and analyzed

opathological

osis

Braak and

Braak stage

Cause of death

al 0 Sepsis

al 0 Sepsis

al 0 Unknown

VI Pneumonia

VI Congestive heart failure

VI Urosepsis

VI Carcinoma of stomach

VI Pneumonia

H.J. Kang et al. / Neurobiology of Disease 18 (2005) 450–458452

with a fluorescence microscopy (Zeiss, Germany) equipped with

the REAL-14k precision digital camera (Apogee Instruments,

Tucson, AZ, USA) and ImagePro Plus Plug-in.

Results

Selective cleavage of PHF-1 tau during apoptosis

We first examined if PHF-1 tau would be cleaved in the

process of apoptosis by staurosporine, a non-specific protein

kinase inhibitor known to cause apoptosis in various types of

cells as well as calyculin A. Cortical cell cultures exposed to 100

nM staurosporine or 10 nM calyculin A revealed apoptotic

features of degenerating neurons within 8 h that was evident by

cell body shrinkage, aggregated condensation of nuclear chro-

matin, and collapse of nuclear membrane prior to plasma

membrane (Figs. 1A–1C). As previously reported (Ko et al.,

2000), PHF-1 tau was cleaved within 8 h following exposure of

cortical cell cultures to calyculin A. PHF-1 tau was also cleaved

in cortical cell cultures exposed to staurosporine for 8–12 h

(Fig. 1D). We also observed cleavage of PHF-1 tau in the

process of neuronal cell apoptosis induced by BAPTA-AM, an

intracellular calcium chelator, or serum deprivation (data not

shown). This implies that PHF-1 tau is cleaved in the process of

neuronal cell apoptosis. Size of PHF-1 tau cleaved by

staurosporine or other apoptosis-inducing agents was similar to

that of caspase-3-mediated cleavage of PHF-1 tau by calyculin,

suggesting that treatment with pro-apoptotic agents induces

proteolytic cleavage of PHF-1 tau through activation of

Fig. 1. Cleavage of PHF-1 tau during neuronal apoptosis by staurosporine or ca

following sham control (A) or exposure of cortical cell cultures (DIV 11) to 100 n

evident by cell body shrinkage and aggregated condensation of nuclear chroma

mitochondria. Scale bar denotes 2.5 Am. (D) Western blot analysis of PHF-1 tau in c

calyculin A for indicated points of time.

caspase-3. This was supported by blockade of PHF-1 tau

cleavage in the presence of 100 AM zDEVD, an inhibitor of

caspase-3 (Fig. 2A). Caspase-3-mediated cleavage of tau was

observed using tau-5 antibody, a monoclonal antibody recogniz-

ing 45–68 kDa tau, in cortical cell cultures exposed to

staurosporine (Fig. 2B). Furthermore, the active form of

caspase-3 was observed within 4–8 h after exposure of cortical

cell cultures to staurosporine or calyculin A (Fig. 2C).

We next examined if neuronal death by excitotoxicity or

oxidative stress would be accompanied by cleavage of PHF-1 tau.

As previously reported (Gwag et al., 1995), cortical cell cultures

underwent widespread neuronal cell necrosis within 2 h following

exposure to low doses of excitotoxins, 30 AM NMDA or 50 AMkainite, that was evident by marked swelling of cell body and

mitochondria, scattering condensation of the nuclear chromatin,

and early fenestration of plasma membrane (Figs. 3A–3C). PHF-1

tau was not cleaved in cortical cell cultures exposed to excitotoxins

up to 2 h (Fig. 3D). As most cortical neurons treated with

excitotoxins for 2 h revealed lysis of plasma membrane, exposure

time longer than 2 h was avoided.

Oxidative stress was induced by exposing cortical cell cultures

to Fe2+ or menadione that produces hydroxyl radical or superoxide,

respectively. Most cortical neurons revealed marked cell body

swelling within 8 h after exposure to Fe2+ or menadione. The

degenerating neurons were accompanied by ultrastructural changes

reminiscent of necrosis that was observed in neurons undergoing

excitotoxic degeneration (Figs. 4A–4C). Cleavage of PHF-1 tau

was not observed in the process of neuronal necrosis following

exposure of cortical cell cultures to Fe2+ or menadione for up to 8 h

(Fig. 4D).

lyculin A. (A–C) Electron photomicrographs of cortical neurons taken 8 h

M staurosporine (B) or 10 nM calyculin A (C). Note the apoptotic neurons

tin. Abbreviations: PM, plasma membrane; NM, nuclear membrane; MI,

ortical cell cultures (DIV 10–12) exposed to 100 nM staurosporine or 10 nM

Fig. 2. Caspase-3-mediated cleavage of PHF-1 tau and tau-5 during apoptosis. (A) Western blot analysis of PHF-1 tau in cortical cell cultures (DIV 11) exposed

to sham control (BL) or 100 nM staurosporine (left panel) or 10 nM Calyculin A (right panel) for 12 h, alone or with 100 AM zDEVD. (B) Western blot

analysis of tau-5 in cortical cell cultures (DIV 11) exposed to sham control (BL) or 100 nM staurosporine for indicated points of time, alone or with 100 AMzDEVD. (C) Western blot analysis of caspase-3 and actin in cortical cell cultures (DIV 11) exposed to 100 nM staurosporine (left panel) or 10 nM Calyculin A

(right panel) for different points of time.

H.J. Kang et al. / Neurobiology of Disease 18 (2005) 450–458 453

Cleavage of PHF-1 tau and activation of caspase-3 in AD brain

To determine if excitotoxicity, oxidative stress, or apoptosis

would be involved in the process of neuronal death in AD

brain, cleavage patterns of PHF-1 tau in AD brain were

analyzed. There are the six abnormally phosphorylated brain

tau isoforms in AD brains (Schmidt et al., 2001). Western blot

analysis showed that levels of PHF-1 tau were prominently

increased in the temporal cortex of the AD brain compared to

the control brain (Fig. 5A). Small fragments of PHF-1 tau were

also observed in the AD brain, suggesting that caspase-3 might

cleave PHF-1 tau accumulated in the AD brain. This was

supported by increase in levels of the active caspase-3 and the

procaspase-3 (CPP32) in the AD brain that were slightly

detectable in the control brain (Figs. 5B, 5C). Finally, double

immunohistochemistry was performed to determine if active

caspase-3 and its substrate PHF-1 tau would be colocalized in

cortical neurons in the AD brain. PHF-1 tau and active caspase-

3 were barely detectable in cortical cells in the age-matched

control brain (Figs. 6A–6C). The immunofluorescence signal of

active caspase-3 was markedly increased in the cytoplasm of

cortical cells in the AD brain (Figs. 6D–6F). Pre-adsorption of

the caspase-3 antibody with active caspase 3 markedly reduced

caspase-3-immunoreactive signal in the AD brain (Figs. 6G–6I).

Double immunohistochemical analysis revealed that approxi-

mately 70% of neurons immunoreactive to active caspase 3

were colocalized with PHF-1 tau-positive neurons. The similar

patterns were observed in the other AD brains. This implies that

caspase-3 is activated primarily in PHF-1 tau-positive and thus

tangle-bearing cortical neurons in the AD brain.

Fig. 3. PHF-1 tau is not cleaved during excitotoxicity. (A–C) Electron photomicrographs of cortical neurons taken 2 h following sham control (A) or exposure

of cortical cell cultures (DIV 12) to 30 AM NMDA (B) or 50 AM Kinate (C). Note the necrotic neurons evident by cell body swelling and scattering

condensation of nuclear chromatin. Abbreviations: PM, plasma membrane; NM, nuclear membrane; MI, mitochondria. Scale bar denotes 2.5 Am. (D) Western

blot analysis of PHF-1 tau in cortical cell cultures (DIV 10–12) exposed to 30 AM NMDA or 50 AM Kinate for indicated points of time.

H.J. Kang et al. / Neurobiology of Disease 18 (2005) 450–458454

The present study suggests that caspase-3-mediated cleavage of

PHF-1 tau is selective in the process of neuronal apoptosis and

possibly contributes to apoptotic degeneration of tangle-bearing

neurons in AD.

Fig. 4. PHF-1 tau is not cleaved during free radical neurotoxicity. (A–C) Electron p

exposure of cortical cell cultures (DIV 12) to 50 AM Fe2+ (B) or 20 AM menadione

condensation of nuclear chromatin. Abbreviations: PM, plasma membrane; NM, n

blot analysis of PHF-1 tau in cortical cell cultures (DIV 10–12) to 50 AM Fe2+ o

Discussion

We demonstrate that PHF-1 tau is cleaved in neurons selectively

undergoing apoptosis induced by staurosporine, calyculin A, or

hotomicrographs of cortical neurons taken 8 h following sham control (A) or

(C). Note the necrotic neurons evident by cell body swelling and scattering

uclear membrane; MI, mitochondria. Scale bar denotes 2.5 Am. (D) Western

r 20 AM menadione for indicated points of time.

Fig. 5. Cleavage of PHF-1 tau and activation of caspase-3 in Alzheimer’s disease brain. (A) Western blot analysis of PHF-1 tau (top panel) and tau-5 (bottom

panel) in the temporal cortex obtained from the postmortem brain of 3 age-matched controls (CT) and 5 Alzheimer’s disease (AD) patients. (B–D) Western blot

analysis of active caspase-3 (B), procaspase-3(C), and actin (D) in the temporal cortex of 3 control group (CT) and 5 Alzheimer’s disease (AD) patients.

H.J. Kang et al. / Neurobiology of Disease 18 (2005) 450–458 455

serum deprivation, but not necrosis induced by excitotoxicity or

oxidative stress in cultured cortical neurons. Cleavage of PHF-1 tau

depends upon activation of caspase-3. Cleaved PHF-1 tau and

activated caspase-3 were observed in the temporal cortex of the AD

brain and appeared to be colocalized to a great extent, suggesting

that activation of caspase-3 may contribute to cleavage of PHF-1 tau

and neuronal apoptosis in AD brain through mechanisms irrespec-

tive of excitotoxicity and oxidative stress.

The hyperphoshorylated forms of tau in the paired helical

filaments constitute the major component of neurofibrillary tangles

in AD brain (Goedert, 1993; Trojanowski and Lee, 1995). The

monoclonal antibody PHF-1 tau recognizes the phosphorylated

forms of tau at Ser396 or Ser404 that is sensitive to calpain or

caspase-3 (Han et al., 2001; Ko et al., 2000; Yang and Ksiezak-

Reding, 1995). Asp-Met-Val-Asp at the carboxy-terminal region of

PHF-1 tau is cleaved by caspase-3 during apoptosis induced by

calyculin A, a selective inhibitor of Ser/Thr phosphatase I and IIA

(Chung et al., 2001; Ko et al., 2000). We also found that PHF-1 tau

was cleaved in the course of neuronal apoptosis following exposure

to staurosporine as well as serum deprivation. Pro-apoptotic

cleavage of PHF-1 tau was accompanied by activation of caspase-

3 and blocked by a selective inhibitor of caspase-3. This suggests

that neuronal apoptosis is accompanied by caspase-3-mediated

cleavage of PHF-1 tau.

Excess activation of ionotropic glutamate receptors appears to

cause neuronal cell death exclusively through necrosis (Dessi et al.,

1993; Gwag et al., 1995). The ultrastructural features of degenerat-

ing cortical neurons following exposures to low doses of NMDA or

kainate confirmed necrotic degeneration evident by cell body

swelling, scattering condensation of nuclear chromatin, and early

fenestration of plasma membrane. While tau proteins were sensitive

to the calcium-dependent protease calpain that was shown to be

activated by administration of the excitotoxins (Johnson et al., 1989;

Yang and Ksiezak-Reding, 1995), PHF-1 tau was not cleaved in

cortical cell cultures undergoing excitotoxic necrosis after exposure

to NMDA or kainate. This is consistent with previous findings that

phosphorylated forms of tau including PHF-1 tau are extremely

resistant to proteolytic cleavage by calpain (Litersky and Johnson,

1995; Yang and Ksiezak-Reding, 1995).

Oxidative stress has been reported to cause neuronal cell death

through apoptosis or necrosis. Ultrastructural analysis of degenerat-

ing cortical neurons demonstrates that necrosis is a predominant

pattern of neuronal cell death in cortical cell cultures exposed to

prooxidants, Fe2+ or menadione as previously reported (Ryu et al.,

1999; Won et al., 2000). PHF-1 tau was not cleaved in the course of

free radical-induced neuronal cell necrosis. Thus, PHF-1 tau appears

to be cleaved by caspase-3 selectively during neuronal cell

apoptosis.

Occurrence of apoptosis in AD has been supported by beta

amyloid-induced apoptosis in cultured neurons, DNA fragmenta-

tion and TUNEL-positive neurons in AD brain, and induction of

apoptosis in neurons overexpressing mutant presenilin 1 (Chui et

al., 1999; Guo et al., 1997; Kim et al., 1997; Loo et al., 1993;

Weihl et al., 1999). In the present study, we have found that PHF-1

tau abundantly accumulated in the AD brain is present as cleaved

fragments as well as full isoforms. While further study will be

Fig. 6. Colocalization of active caspase-3 and PHF-1 tau in AD. Fluorescence photomicrographs of control (top panel) and AD (middle and bottom panels)

brain sections after double immunolabeling with antibodies for PHF-1 tau (A, D and G, red) and active caspase-3 (B and E, green). In bottom panel, active

caspase-3 antibody was pre-absorbed with active caspase-3 prior to immunolabeling (H). The merged fluorescence photomicrographs showed that pre-

adsorption of the caspase-3 antibody markedly reduced caspase-3-immunoreactive signal in colocalization of PHF-1 tau in AD brain (F and I). Double-labeled

sections were stained with Hoechst dye labeling (blue) (C). Arrow indicates a PHF-1 tau and active caspase-3 -positive neuron (yellow).

H.J. Kang et al. / Neurobiology of Disease 18 (2005) 450–458456

needed to determine whether and where human PHF-1 tau in AD is

cleaved by caspase-3, apoptosis-specific and caspase-3-dependent

cleavage of PHF-1 tau in murine cortical cell cultures suggests that

cleaved PHF-1 tau in AD is associated with activation of caspase-3

during apoptosis. In support of this, levels and activation of

caspase-3 are also increased in the AD brain (Shimohama et al.,

1999). Moreover, activated caspase-3 is observed in most of PHF-1

tau-positive neurons in the AD brain. Caspase-3-dependent

cleavage form of fodrin has been detected in neurofibillary

tangle-bearing neurons in AD (Rohn et al., 2001).

Hyperphophorylated forms of tau are accumulated in the AD

brain due to activation of protein kinases such as glycogen

synthase kinase-3h (GSK-3h) and cyclin-dependent kinase-5

(cdk5) or reduced activity of protein phosphatase 2A- and protein

phosphatase 1 (Geschwind, 2003; Godemann et al., 1999; Iqbal et

al., 1998; Pei et al., 1998; Yamaguchi et al., 1996). Tau-positive

neurofibrillary tangles are also observed in various dementing

diseases including progressive supranuclear palsy (PSP), cortico-

basal degeneration, Pick’s disease, and familial frontotemporal

dementia and Parkinsonism linked to chromosome 17 (FTDP-17)

(Spillantini and Goedert, 1998). Overexpression of mutant tau

derived from FTDP-17 causes accumulation of hyperphosphory-

lated tau and neuronal degeneration (Tanemura et al., 2001;

Tatebayashi et al., 2002; Vogelsberg-Ragaglia et al., 2000). Beta

amyloid fibrils that induce accumulation of PHF-1 tau cause

neuronal death in the presence of tau (Busciglio et al., 1995;

Rapoport et al., 2002). Blockade of caspase 3-mediated cleavage

of tau attenuates apoptotic cell death induced by staurosporine

and okadaic acid (Chung et al., 2001). Taken together, the

phosphorylation and caspase-3-mediated cleavage of tau likely

play a role in the formation of neurofibrillary tangles and

neurodegeneration.

The present study supports that apoptosis may contribute to

neurondegeneration in AD possibly through mechanisms involv-

ing activation of caspase-3 and cleavage of PHF-1 tau. The

apoptosis appears to be different from excitotoxicity and oxidative

stress that cause neuronal death primarily through necrosis and do

not cleave PHF-1 tau. Thus, apoptosis as well as excitotoxic and

oxidative necrosis may constitute additional route of neuronal

death in AD.

Acknowledgments

We thank Dr. Peter Davis (Albert Einstein College of Medicine)

for the PHF-1 antibody, Dr. Lester I Binder (Northwestern

University School of Medicine) for the Tau-5 antibody and Boston

H.J. Kang et al. / Neurobiology of Disease 18 (2005) 450–458 457

University Alzheimer’s Disease Center (Dr. Ann C. Mckee) for

human brain tissues. This work was supported by a National

Research Laboratory grant and the 21st Century Frontier Research

Program (01610) (BJG).

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