Apoptotic signaling cascades

Paula C. Ashe*, Mark D. Berry

ALviva Biopharmaceuticals Inc., 218-111 Research Drive, Saskatoon, Saskatchewan, Canada S7N 3R2

Accepted 8 January 2003

Abstract

Apoptosis is a form of programmed cell death that results in the orderly and efficient removal of damaged or unnecessary cells, such as

those resulting from DNA damage or during development. There are many factors that contribute to this process, each demonstrating

specificity of function, regulation, and pathway involvement. The aim of this brief overview is to provide an introduction to a number of

these factors as well as the various apoptotic pathways that have been identified.

D 2003 Elsevier Science Inc. All rights reserved.

Keywords: Apoptosis; Bcl-2 proteins; Caspases; Death receptor; Mitochondria; Signal transduction

1. Introduction

Programmed cell death is recognized as a critical element

in the removal of cells following exposure to toxic com-

pounds as well as during development and in degenerative

disorders. This is a complex area that is continually being

expanded to incorporate newly defined modes of cell death

such as anoikis and abortosis. In general, programmed cell

death represents a continuum of cell death ranging from

classical apoptosis to necrosis at its two poles (Fig. 1).

In the last decade, much progress has been made towards

elucidating the various signal transduction pathways that

can ultimately lead to a cell’s demise. Based on this

information, many apoptotic cascades have been described,

such as intrinsic and extrinsic, mitochondrial and death

receptor (DR), p531-dependent and -independent, and cas-

pase-dependent and -independent pathways in association

with initiation, commitment, and execution phases. While

such categorization is undoubtedly of value, it has become

increasingly apparent that apoptosis is not a series of clearly

defined pathways, but rather, a multitude of highly regu-

lated, interconnected pathways (Fig. 1). While trying to

view any of these pathways in isolation is clearly an over-

simplification, the sheer magnitude of possible events

makes it a necessity. For the purposes of this review, the

broad-based intrinsic and extrinsic classifications of cell

death will be used as a platform from which to describe

apoptotic signal cascades.

The intrinsic cell death pathway involves the initiation of

apoptosis as a result of a disturbance of intracellular homeo-

stasis. In this pathway, mitochondria are critical for the

execution of cell death, and so this pathway has also been

referred to as the mitochondrial cell death pathway. The

extrinsic pathway involves the initiation of apoptosis

through ligation of plasma membrane DRs, and so this

pathway is also referred to as the DR pathway. While the

initiation mechanisms of these pathways are different, both

0278-5846/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved.

doi:10.1016/S0278-5846(03)00016-2

Abbreviations: AIF, apoptosis-inducing factor; Apaf-1, apoptotic

protease-activating factor-1; ASK1, apoptosis signal-regulating kinase-1;

BH, Bcl-2 homology; BIR, baculovirus IAP repeat; CARD, caspase

recruitment domain; CRADD, caspase and RIP adaptor with death domain;

CRD, cysteine-rich domain; DcR, decoy receptor; DD, death domain; DED,

death effector domain; DIABLO, direct IAP-binding protein with low pI;

DISC, death-inducing signaling complex; DR, death receptor; FADD, Fas-

associated death domain protein; FLIP, FLICE inhibitory protein; I-kB,inhibitor of NF-kB; IKK, I-kB kinase complex; JNK, c-Jun amino terminal

kinase; MAP, mitogen-activated protein; NEMO, NF-kB essential modu-

lator; NF-kB, nuclear factor kB; NIK, NF-kB-inducing kinase; pI,

isoelectric point; PLAD, preligand-binding assembly domain; RAIDD,

RIP-associated ICH-1/Ced-3-homologous protein with death domain; RIP,

receptor-interacting protein; Smac, second mitochondrial activator of

caspases; SODD, silencer of death domains; TNF, tumor necrosis factor;

TNFR, tumor necrosis factor receptor; TRADD, tumor necrosis factor-

associated death domain protein; TRAF, tumor necrosis factor receptor-

associated factor; TRAIL, tumor necrosis factor a-related apoptosis-

inducing ligand; XIAP, X-linked inhibitor of apoptosis protein.

* Corresponding author. Tel.: +1-306-956-6883; fax: +1-306-956-

6877.

E-mail address: paula.ashe@nrc-cnrc.ga.ca (P.C. Ashe).

www.elsevier.com/locate/pnpbp

1 Tumor suppressor gene.

Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 199–214

pathways converge to result ultimately in cellular morpho-

logical and biochemical alterations characteristic of apopto-

sis. In addition to the ultimate convergence, it is also

apparent that considerable cross talk between the pathways

occurs upstream of the convergence point, and that indi-

vidual cells possess a considerable degree of redundancy in

their apoptotic pathways. There is an extensive literature

describing apoptotic pathways, much of it arising from the

use of various cancerous cell lineages. It is necessary,

therefore, to be cautious when extrapolating these data to

normal cells. By definition, cancer involves a failure in

apoptotic mechanisms. Therefore, it can be assumed that

while apoptosis induced in these cells utilizes much of the

machinery involved in the death of normal cells, some

components may be altered or bypassed altogether. Further-

more, as discussed below, even within normal cells, mech-

anisms that are present within one cell type, may not be

relevant to other cell types since cells of different origins

may utilize different suicide pathways. As such, with respect

to neurodegenerative disorders, for example, it is of par-

ticular importance that apoptotic cascades described in

nonneuronal cells be confirmed as active in neuronal cells.

Because of the limited nature of this overview, it is useful

to refer to several excellent, recent reviews (Table 1), as well

as the separate chapters in this issue that discuss specific

apoptotic cascades in relation to individual disease states

(see Love, 2003; Raina et al., 2003; Hickey and Chesselet,

2003; Lev et al., 2003; Waldmeier, 2003, this issue).

2. Caspases

Caspases (cysteine aspartate-specific proteases) are a

family of intracellular proteins involved in the initiation

Fig. 1. A schematic representation of the continuum of programmed cell death cascades. Cell death can be regarded as a continuum that contains, at its two

poles, classical apoptosis (as defined by Kerr et al., 1972) and necrosis. Between these two extremes exist a multitude of pathways that show varying degrees of

apoptotic and necrotic characteristics. At any point within the continuum of cell death, different pathways exist, which can be subdivided according to various

classification systems (see text for further details) such as the extrinsic and intrinsic pathways, p53-dependent and -independent pathways, and physiologic and

pathologic cell death. In this schema, we regard cell death pathways as being analogous to a tree (background). As such, there are a vast number of

interconnected initiation pathways (the branches) that eventually filter down to a common commitment phase (the trunk). Beyond this common commitment

point, there is a degree of divergence once more with a number of different execution pathways (the roots). In this scheme, an inappropriately functioning

pathway (analogous to a diseased branch) can be removed without affecting the system as a whole (the tree). As such, in this scheme, treatment with

sufficiently selective antiapoptotic compounds would be feasible, without necessarily affecting ongoing, physiological, homeostatic, cell death cascades.

Table 1

Recent reviews on apoptotic cascade components

Component References

Bcl-2 family

proteins

Tsujimoto and Shimizu, 2000;

Cory and Adams, 2002

Caspases Nicholson, 1999;

Wolf and Green, 1999;

Yakovlev and Faden, 2001;

Shi, 2002;

Troy and Salvesen, 2002

DRs Sartorius et al., 2001;

Chen and Goeddel, 2002;

Wajant, 2002

Mitochondria Halestrap et al., 2000;

Nicholls and Budd, 2000;

Ravagnan et al., 2002

General Kaufmann and Hengartner, 2001;

Zimmermann et al., 2001;

Green and Evan, 2002

P.C. Ashe, M.D. Berry / Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 199–214200

and execution of apoptosis. Activation of the execution

caspases is often referred to as the apoptotic commitment

point; i.e., the point in the signaling cascade where the cell

commits to die. To date, at least 14 mammalian caspases

have been identified; active human homologs for all family

members have not been identified, however (see Table 2

for a brief overview). Further, not all these family mem-

bers have been well characterized with respect to their

physiological roles and targets, although, it is known that

certain distinct caspases play roles in apoptosis and

inflammation.

Caspases are synthesized as procaspases that are then

proteolytically processed, at critical aspartate residues, to

their active forms. All procaspases contain a highly homo-

logous protease domain as well as an NH2 terminal prodo-

main. The protease domain contains two subunits of

approximately 20 and 10 kDa, respectively, that associate

to form a heterodimer following proteolytic processing. Two

heterodimers then associate to form a tetramer, which is the

active form of caspases (Walker et al., 1994). The NH2

terminal domain is of variable length depending on the

functional category of the caspase. Initiator and inflammat-

ory caspases possess long prodomains (>100 amino acids),

whereas effector caspases have short prodomains ( < 30

amino acids). Long prodomains contain specific motifs

essential for caspase activity. These motifs may be either

death effector domains (DEDs) as in caspases 8 and 10, or

caspase recruitment domains (CARDs) as in caspases 1, 2,

4, 5, 9, 11, 12, 13, and 14. As discussed below, these

domains mediate interactions between caspases and a vari-

ety of adaptor molecules involved in cell signaling. Both

DED-containing caspases are initiator caspases, whereas

CARD-containing caspases may be either initiator caspases

(caspases 2 and 9) or inflammatory caspases (1, 4, 5, 11, 12,

13, 14). For a detailed discussion of inflammatory caspases,

please refer to Cohen (1997) and Chang and Yang (2000).

Caspase 12 is generally categorized as an inflammatory

caspase; however, it also plays a role in endoplasmic

reticulum-stress-induced apoptosis (Nakagawa et al.,

2000). With the exception of caspase 12, active forms of

all of the apoptosis-related caspases have been identified in

humans. The gene for caspase 12 has been identified in

humans; however, loss of function mutations prohibit the

proteolytic activity of any protein that may be translated

(Fischer et al., 2002). This suggests that caspase 12 may not

be involved in human apoptotic cascades. Human homologs

of the murine inflammatory caspases 11, 13, and 14 have

not yet been identified.

The induction of apoptosis through extrinsic or intrinsic

death mechanisms results in the activation of initiator

caspases. DRs, through adaptor molecules, recruit initiator

caspases 2, 8, or 10, while intrinsic death signals result in

the activation of caspase 9. Activation of initiator caspases

is the first step of a highly regulated, irreversible, self-

amplifying proteolytic pathway. Initiator caspases are able

to cleave procaspases, and thus, are able to activate effector

caspases (caspases 3, 6, and 7) or are able to amplify the

caspase cascade by increased activation of initiator caspases.

Effector caspases are common to both the extrinsic and

intrinsic death pathways, and therefore, the ultimate mor-

phological and biochemical hallmarks of apoptosis are

relatively independent of the apoptotic inducer.

A large number of apoptosis-related caspase targets have

been identified. Please refer to Stroh and Schulze-Osthoff

(1998), Chan and Mattson (1999), and Chang and Yang

(2000), for further details. In brief, through proteolytic

processing, caspases activate or inhibit target proteins that

exhibit roles in the maintenance of cell morphology (Taka-

hashi et al., 1996; Kothakota et al., 1997; Sahara et al.,

1999), cell death (Cheng et al., 1997; Clem et al., 1998),

DNA metabolism (Lazebnik et al., 1994; Enari et al., 1998;

Sakahira et al., 1998), cell cycle regulation (Levkau et al.,

1998; Zhou et al., 1998), or signal transduction (Cardone et

al., 1997; Rudel and Bokoch, 1997; Widmann et al., 1998).

The specificity of caspases for these targets results in a

highly controlled and efficient removal of damaged or

unwanted cells.

3. Bcl-2 family

Members of the Bcl-2 family of intracellular proteins are

essential mediators of cell survival and apoptosis. Both anti-

and proapoptotic family members have been characterized

and their classification is related to the presence or absence

of Bcl-2 homology (BH) domains. Four BH domains have

been described: BH1, BH2, BH3, and BH4. Bcl-2 and Bcl-

XL, both containing all four BH domains, possess estab-

lished roles in the inhibition of apoptosis, although their

exact mechanism remains elusive. It has been proposed that

antiapoptotic Bcl-2 family members inhibit apoptosis by

antagonizing the actions of proapoptotic family members.

This antagonism is proposed to occur upstream of apopto-

Table 2

The caspases

Caspase Alternate name(s) Physiological role Human

homolog

1 ICE cytokine activation yes

2 Nedd2, ICH-1 apoptosis initiator yes

3 CPP32, Yama, apopain apoptosis effector yes

4 ICErelII, TX, ICH-2 cytokine activation yes

5 ICErelIII, TY cytokine activation yes

6 Mch2 apoptosis effector yes

7 Mch3, ICE-LAP3, CMH-1 apoptosis effector yes

8 Mch5, MACH, FLICE apoptosis initiator yes

9 ICE-LAP6, Mch6 apoptosis initiator yes

10 Mch4 apoptosis initiator yes

11 ICH-3 cytokine activation no

12 cytokine activation no

13 ERICE cytokine activation no

14 MICE cytokine activation no

P.C. Ashe, M.D. Berry / Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 199–214 201

sis-related mitochondrial alterations. Two subfamilies of

proapoptotic Bcl-2 family members have been identified;

the bax family (Bax, Bok, and Bak), containing BH1, BH2,

and BH3, and the BH3-only family (Bid, Bim, Bik, Bad,

Bmf, Hrk, Noxa, and PUMA). Similar to the antiapoptotic

family members, the exact mechanism of action of proa-

poptotic Bcl-2 family members is uncertain. In a number of

systems, these proteins have been demonstrated to be

essential for the completion of apoptotic programs (Lindsten

et al., 2000; Wei et al., 2001; Yin et al., 2002). However,

Bax-independent apoptosis has also been described (D’Sa-

Eipper et al., 2001). The BH3-only proteins are proposed to

function upstream of Bax family proteins because they do

not appear to induce apoptosis in the absence of Bax and

Bak (Zong et al., 2001); however, their mechanism of action

remains largely unknown. Regulation of Bcl-2 family pro-

teins is diverse both between and within the subfamilies and

includes transcriptional control, posttranslational control,

protein translocation, and protein–protein interactions. This

complexity of Bcl-2 family proteins underlies the uncer-

tainty of their specific roles in various apoptotic systems,

but what is indisputable is their critical role in cell death

execution.

4. Intrinsic cell death signaling

Intrinsic cell death pathways follow from proapoptotic

signals resulting in a disruption of intracellular homeostasis,

i.e., signals for cell suicide originate within the cell. The

mitochondria are the primary intracellular initiation sites

although the endoplasmic reticulum has also been impli-

cated (see Bratton and Cohen, 2001; Ferri and Kroemer,

2001, for reviews). Mitochondria play a dominant role in

cellular metabolism; however, they also play an important

role in apoptosis. A number of apoptotic proteins are

compartmentalized within functional mitochondria. Follow-

ing an apoptotic insult, these proteins are released from the

mitochondria, placing them in close proximity to their

apoptotic sites of action (Fig. 2).

Cytochrome c, normally localized in the intermembrane

space of the mitochondria, is released into the cytosol where

Fig. 2. A schematic representation of the intrinsic pathway of apoptosis signaling (adapted from Zuo et al., 1999).

P.C. Ashe, M.D. Berry / Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 199–214202

it interacts with apoptotic protease-activating factor-1

(Apaf-1), ATP/dATP, and caspase 9 to form the apoptosome

(Liu et al., 1996a; Li et al., 1997). Apaf-1 contains a CARD,

which mediates its interaction with caspase 9, as well as a

WD-40 repeat domain that is proposed to maintain protein

inactivity in the absence of cytochrome c (Hu et al., 1998).

In the presence of cytochrome c and ATP/dATP, Apaf-1

undergoes a conformational change that permits self-

aggregation (Zou et al., 1999). This exposes the CARD,

thus, inducing recruitment of procaspase 9 and its sub-

sequent transproteolytic activation. Caspase 9 can then

directly activate caspases 3 and 7, which results in the

orderly death of the cell through controlled proteolytic

processing of various downstream targets. Smac/DIABLO

(second mitochondrial activator of caspases/direct IAP-

binding protein of low isoelectric point [pI]) is also released

from the mitochondria into the cytosol in response to

apoptotic stimuli (Du et al., 2000; Verhagen et al., 2000).

Smac/DIABLO binds to baculovirus IAP repeat (BIR)

domains within inhibitor of apoptosis proteins (IAPs) to

remove their inhibitory effect on caspase activity (Sriniva-

sula et al., 2000, 2001; LeBlanc, this issue). A third

mitochondrial factor implicated in apoptosis is apoptosis-

inducing factor (AIF) (Susin et al., 1996). AIF is a mito-

chondrial flavoprotein that translocates to the nucleus fol-

lowing apoptotic stimuli (Susin et al., 1999; Lorenzo et al.,

1999). In the nucleus, AIF induces partial DNA fragmenta-

tion and chromatin condensation. AIF appears to promote

apoptosis independently of caspases although it likely acts

in a cooperative manner with other factors to promote

nuclear apoptosis.

A number of mechanisms have been proposed to be

responsible for the release of these proapoptotic proteins.

One proposes that the opening of the mitochondrial per-

meability transition pore results in a loss of mitochondrial

membrane potential. Since the permeability transition pore

does not allow passage of molecules greater than 1500 Da, it

seems unlikely this could be the mechanism allowing the

release of proapoptotic proteins. Associated with pore

opening and membrane potential disruption, however, is

an influx of fluid into the mitochondria. This is proposed to

result in the rupture of the outer mitochondrial membrane

and the release of proapoptotic proteins (Zamzami et al.,

1995, 1996; Vander Heiden et al., 1997). This hypothesis is

supported by several reports (Wadia et al., 1998; Heiskanen

et al., 1999); however, it has also been demonstrated that

cytochrome c release occurs prior to the loss of mitochon-

drial membrane potential (Kluck et al., 1997; Yang et al.,

1997a; Krohn et al., 1999). A second hypothesis to explain

mitochondrial protein release during apoptosis is based on

the demonstration that antiapoptotic members of the Bcl-2

family can prevent cytochrome c release (Kluck et al., 1997;

Yang et al., 1997a; Johnson et al., 2000; Sun et al., 2002). In

this case, it is proposed that these antiapoptotic proteins

interact with the proapoptotic family members to inhibit

their induction of mitochondrial protein release. This hypo-

thesis is based both on their structure as well as their

translocation from the cytosol to the mitochondria during

apoptosis (Wolter et al., 1997; Putcha et al., 1999). Bcl-2

family members are structurally similar to the pore-forming

diphtheria toxin (Muchmore et al., 1996), and so it has been

proposed that proapoptotic Bax family members oligomer-

ize to form mitochondrial channels through which proapop-

totic proteins can pass (Antonsson et al., 2000; Saito et al.,

2000). A novel, Bax-dependent channel activity does exist

in mitochondria during apoptosis, but it has not yet been

fully characterized (Pavlov et al., 2001). In further support

of this hypothesis, it has been shown that Bcl-2 can prevent

the oligomerization of Bax in the outer mitochondrial

membrane (Mikhailov et al., 2001).

In addition to the numerous regulatory points that exist in

the pathway described above, the tumor suppressor gene,

p53, has also been demonstrated to play an important role in

the regulation of cell death in many systems. A number of

insults which induce DNA damage result in the activation

of p53 and its subsequent involvement in apoptosis (Sakhi

et al., 1994; Wood and Youle, 1995; Morrison et al., 1996).

p53 promotes apoptosis through the direct activation of

Bax, although overexpression of p53 can induce apoptosis

independently of Bax (Miyashita and Reed, 1995; Xiang et

al., 1998). In addition, p53 has been demonstrated to induce

the BH3 family members, Noxa and PUMA; thus, dem-

onstrating multiple points of apoptosis regulation within the

Bcl-2 family (Oda et al., 2000; Nakano and Vousden,

2001). Recently, Apaf-1 has been identified as a direct

target of p53; thus, revealing yet another mechanism by

which p53 can promote apoptosis (Fortin et al., 2001). In

addition to these targets, Polyak et al. (1997) have identified

a number of genes regulated by p53 that are implicated in

apoptosis. p53, however, is not only involved in intrinsic

cell death pathways since it has also been shown to be

involved in the regulation of DRs.

5. Extrinsic cell death signaling

5.1. Death receptors

Cell surface DRs belong to the tumor necrosis factor

receptor (TNFR) superfamily. They transmit their apoptotic

signals following binding of death ligands. These receptor–

ligand complexes initiate apoptotic cascades within seconds

of ligand binding and can result in apoptotic cell death

within hours. The best-characterized family members

include Fas (also known as Apo1 or CD95) and TNFR1

(Ashkenazi and Dixit, 1998, 1999; Chen and Goeddel,

2002; Wajant, 2002). Additional members of the TNFR

superfamily include DR3 (also known as Apo3, WSL-1,

TRAMP, or LARD) (Chinnaiyan et al., 1996a; Marsters et

al., 1996), DR4 (Pan et al., 1997b), DR5 (also known as

Apo2, TRAIL-R2, TRICK2, or KILLER) (Pan et al., 1997a;

Sheridan et al., 1997), and DR6 (Pan et al., 1998). These

P.C. Ashe, M.D. Berry / Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 199–214 203

receptors are Type I transmembrane receptors characterized

by extracellular cysteine-rich domains (CRD) and intra-

cellular death domains (DDs). The extracellular CRD are

responsible for receptor self-association (CRD1) as well as

receptor–ligand interactions (CRD2 and CRD3) (Siegel et

al., 2000). Members of the TNF receptor superfamily

require self-association prior to ligand binding; therefore,

the CRD1 domain has also been termed the preligand-

binding assembly domain (PLAD) (Chan et al., 2000).

The cytoplasmic tail contains a DD, an approximately 80-

amino acid domain that signals programmed cell death

(Tartaglia et al., 1993a). The DD consists of six antiparallel,

amphipathic a-helices folded in a configuration that results

in the surface exposure of a number of charged residues

(Huang et al., 1996). Following receptor–ligand binding,

receptor DDs interact, presumably via electrostatic interac-

tions. Subsequent to this activation of DRs, other DD-

containing proteins are recruited and function as adaptor

proteins in the signal transduction cascade (see below).

These adaptor proteins interact with a variety of other

proteins to complete the DR signaling pathways. The

previously described CARDs and DEDs, a specific example

of the more global CARD, mediate these subsequent inter-

actions. NMR spectroscopy has demonstrated that the DED

shares a similar tertiary structure with the DD (Eberstadt et

al., 1998). In contrast to the surface-charged DD, however,

the DED contains two hydrophobic regions that appear to be

important for apoptotic activity.

6. DR-mediated apoptosis

6.1. FasL signaling

Fas is expressed in various tissues, but is most abundant

in the heart, thymus, liver, and kidney (Nagata, 1997). The

preferred ligand for the Fas receptor is FasL, a Type II

transmembrane protein almost exclusively expressed in

activated T cells (Suda et al., 1993). FasL contains an

extracellular self-assembly domain necessary for the oligo-

merization of the ligand (Orlinick et al., 1997). The FasL

gene is generally transcriptionally inactive; therefore, Fas/

FasL-mediated events are regulated by activation of this

gene (Pinkoski and Green, 1999). The Fas/FasL system is

mainly responsible for three types of cell killing: (i) activa-

tion-induced cell death (AICD) of T cells, (ii) cytotoxic T

lymphocyte-mediated killing of target cells, and (iii) killing

of inflammatory cells in immune privilege sites and killing

of cytotoxic T lymphocytes by tumor cells (Nagata, 1997).

More recently, however, it has been demonstrated that the

Fas/FasL system is also involved in neuronal apoptosis

following traumatic brain injury (Beer et al., 2000; Qiu et

al., 2002), cerebral ischaemia (Martin-Villalba et al., 1999;

Rosenbaum et al., 2000), and in apoptosis during neuro-

development (Felderhoff-Mueser et al., 2000; Raoul et al.,

2000).

Ligation of Fas by FasL promotes the recruitment of the

cytosolic adaptor protein Fas-associated death domain pro-

tein (FADD; also known as MORT1) (Chinnaiyan et al.,

1996b). In addition to its DD, FADD also contains an N-

terminal DED responsible for association with other DED-

containing proteins such as caspases 8 and 10 (Boldin et al.,

1996; Muzio et al., 1996; Medema et al., 1997; Kischkel et

al., 2001). The complex involving Fas, FasL, FADD, and

either caspase 8 or 10 is referred to as the death-inducing

signaling complex (DISC) (Kischkel et al., 1995; Wajant,

2002). The oligomerization-induced proteolytic activation

of these initiator caspases results in the execution of the

apoptotic program by cleavage of downstream targets.

Despite the commonality of DISC association, two types

of cells using distinct Fas signaling pathways have been

identified (Scaffidi et al., 1998). Type I cells require the

activation of caspase 8 that is closely followed by the

activation of caspase 3. In Type II cells, limited activation

of caspase 8 results in an amplification loop mediated by

mitochondrial activation (Scaffidi et al., 1999). In other

words, the extrinsic apoptotic pathway recruits an intrinsic

apoptotic pathway. To date, a limited number of cell types

have been classified as Type I or II. Peripheral T cells and

thymocytes have been demonstrated to be Type I cells and

liver cells appear to be typical Type II cells (Algeciras-

Schimnich et al., 2002). Neurons may represent Type II cells

(Plesnila et al., 2001; Yin et al., 2002). In Type I cells, the

activation of caspase 8 can directly activate downstream

caspases including caspases 3, 6, and 7 (Srinivasula et al.,

1996; Muzio et al., 1997) (Fig. 3). Apoptosis of Type I cells

is associated with mitochondrial disruption and cytochrome

c release; however, death is not inhibited by antiapoptotic

members of the Bcl-2 family (Scaffidi et al., 1998).

The mitochondrial amplification loop in Type II cells has

been demonstrated to involve the caspase 8-mediated cleav-

age of the BH-3 only protein, Bid (Li et al., 1998; Luo et al.,

1998) (Fig. 3). Truncated Bid (tBID) is critical for caspase

8-induced release of cytochrome c from mitochondria

(Gross et al., 1999). In addition to the release of cytochrome

c, tBid has also been demonstrated to promote the release of

Smac/DIABLO from mitochondria (Madesh et al., 2002). It

should be noted that Smac/DIABLO might play a role in

both Types I and II cells. This protein can circumvent the

antiapoptotic effect of Bcl-XL, thereby negating the protect-

ive effect of Bcl-XL on Type II cells and in effect, convert-

ing them to Type I cells (Srinivasula et al., 2000).

As stated above, regulation of Fas-mediated apoptosis is

dependent on the expression of FasL; however, additional

regulatory mechanisms have also been identified. It has

been demonstrated that p53 activation mediates cell surface

redistribution of Fas, thereby sensitizing cells to Fas-medi-

ated apoptosis (Bennett et al., 1998). Apoptosis following

p53 activation is not inhibited by actinomycin D nor by

cycloheximide, indicating that under these circumstances,

apoptosis is independent of transcription and/or translation.

Subsequent studies have also demonstrated a transcription-

P.C. Ashe, M.D. Berry / Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 199–214204

dependent role for p53 in Fas-mediated apoptosis. A p53-

responsive element has been identified in the human and the

mouse Fas gene (Muller et al., 1998; Munsch et al., 2000).

This suggests that following its activation, p53 can directly

up-regulate Fas at the level of transcription. In support of

this, Fas up-regulation in response to a variety of chemo-

therapeutic agents was demonstrated to be dependent on the

presence of wild-type p53 (Muller et al., 1998). In addition,

differential regulation of Fas has been observed in tissues

from wild-type and p53 knockout mice (Lin et al., 2002).

Fas expression in the liver and spleen was dependent on the

presence of p53, whereas expression in other tissues,

including the brain, was independent of p53 status, suggest-

ing that the requirement of p53 for Fas-mediated apoptosis

is tissue specific (Fuchs et al., 1997). Tissue specificity may

not be absolute; however, in contrast to the findings of Lin

et al. (2002), Tan et al. (2001) demonstrated that Fas

induction correlated with the presence of p53 and TUNEL

in neurons following kainate-induced seizures. It remains to

be determined whether the mechanism of cellular injury acts

in concert with the cell type to determine the requirement for

p53.

Regulation of Fas-mediated apoptosis can also occur

through the association of FasL with the decoy receptor 3

(DcR3), a secreted extracellular DR (Pitti et al., 1998). DcRs

function to inhibit apoptotic signal transduction by compet-

ing death ligands away from their respective DRs. A

number of tumor cells express amplified levels of DcR3, a

Fig. 3. A schematic representation of Fas-mediated apoptosis signaling.

P.C. Ashe, M.D. Berry / Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 199–214 205

finding that may account for their relative resistance to

immune–cytotoxic attack. Downstream of the receptor,

regulation of Fas-mediated signaling can occur at the level

of caspase 8 interaction. The FLICE inhibitory protein

(FLIP) exists in two forms, FLIPS and FLIPL. Both of these

forms have been demonstrated to associate with FADD via

DED interactions (Irmler et al., 1997). FLIPL is also able to

interact directly with procaspase 8 via DEDs and the

proteolytically inactive C-terminal caspase-like region of

FLIPL. This interaction inhibits apoptotic signal transduc-

tion through direct interference with the FADD–caspase 8

interaction and through inhibition of procaspase 8 process-

ing. FLIP up-regulation has been demonstrated in FasL-

resistant melanoma tumor cell lines; therefore, similar to the

proposed function of DcR3, FLIP expression may also

mediate tumor cell resistance to apoptosis.

In addition to the already complex signaling pathway

mediated by Fas/FasL, it has been demonstrated that an

alternate proapoptotic, FADD-independent, cell type-spe-

cific pathway exists. Daxx, a Fas-binding protein, binds

specifically to the Fas DD and activates the Jun NH2-

terminal kinase (JNK) pathway (Yang et al., 1997b). Spe-

cifically, Daxx may function as a bridge between Fas and

apoptosis signal-regulating kinase-1 (ASK1), a mitogen

activated protein (MAP) kinase kinase kinase able to activ-

ate apoptosis (Chang et al., 1998). ASK1-mediated apopto-

sis is dependent on the activation of caspase 9 (Hatai et al.,

2000), which is, in turn, dependent on the release of

cytochrome c from the mitochondria. Activation of the

JNK pathway has been proposed to enhance apoptosis

through phosphorylative inactivation of Bcl-2 (Yamamoto

et al., 1999) as well as phosphorylation-mediated stabiliza-

tion of c-myc (Noguchi et al., 2001). c-Myc has been

demonstrated to result in a Bax-dependent destabilization

of mitochondrial integrity; thus, promoting the release of

proapoptotic factors (Juin et al., 2002). The Fas–Daxx–

ASK1 apoptotic pathway is reported to be independent of

the activation of caspase 8. In contrast to the proposed

FADD- and caspase 8-independent JNK-mediated death

pathway, a separate report indicates that JNK can also result

in FADD- and caspase 8-dependent apoptosis. Cortical

neurons treated with b-amyloid demonstrate activation of

JNK and the subsequent induction of the transcription

factor, c-Jun (Morishima et al., 2001). c-Jun directly up-

regulates the expression of FasL, which can induce FADD-

and caspase 8-dependent apoptosis. The exact role that the

JNK pathway plays in Fas-mediated apoptosis is unclear;

however, it appears that the timing of signaling events and

the cell type involved may be critical elements in JNK-

mediated apoptosis.

6.2. Tumor necrosis factor signaling

Tumor necrosis factor (TNF)-mediated cell signaling

constitutes a second major DR pathway. TNF is predom-

inantly expressed in activated macrophages, T cells, and

some epithelial tumor cell lines (Decker et al., 1987;

Turner et al., 1987; Spriggs et al., 1988). Signal trans-

duction is mediated through two cell surface receptors,

TNF-R1 and TNF-R2. These two receptors transduce

distinct cellular responses; TNF-R1 mediates cytotoxicity,

whereas TNF-R2 mediates T cell proliferation (Tartaglia et

al., 1991). Membrane-bound TNF preferentially binds

TNF-R2; however, ligand passing from TNF-R2 to TNF-

R1 has been demonstrated to occur (Tartaglia et al.,

1993b). This is presumed to be mediated by the fast off-

rate of TNF from TNF-R2 that creates a local high

concentration of TNF; thus, facilitating binding to TNF-

R1, a receptor with a slow dissociation rate. Ligation of

TNF-R1 by TNF results in the recruitment of the adaptor

protein, TNF receptor-associated DD (TRADD), an adaptor

protein (Hsu et al., 1995). The interaction of TNF with TNF-

R1 promotes the dissociation of silencer of death domains

(SODD) from the cytoplasmic tail of TNF-R1; thus, per-

mitting the recruitment of TRADD (Jiang et al., 1999). The

association between SODD and TNF-R1 is presumed to

prevent the spontaneous activation of TNF-R1-mediated

signaling. Following its association, TRADD can interact

with FADD, TNF receptor-associated factor-2 (TRAF2),

and receptor-interacting protein (RIP), a serine/threonine

kinase (Hsu et al., 1996a,b). The interaction with FADD

induces apoptosis similar to that described for Fas (see

above). TNF can also induce apoptosis via the DD-medi-

ated interaction between RIP and RIP-associated ICH-1/

Ced-3-homologous protein with DD/caspase and RIP

adaptor with death domain (RAIDD/CRADD) (Fig. 4).

RAIDD contains a CARD through which it can interact

with caspase 2 to induce apoptosis (Ahmad et al., 1997;

Duan and Dixit, 1997; Shearwin-Whyatt et al., 2000).

Caspase 2, similar to caspase 8 in Type II cells, induces

the release of the mitochondrial proapoptotic factors, cyto-

chrome c, Smac/DIABLO, and AIF (Guo et al., 2002). This

is accomplished through the direct cleavage of Bid by

caspase 2; however, an alternate Bid-independent pathway

also appears to exist.

Recruitment of TRAF2 by TRADD can result in the

activation of nuclear factor kB (NF-kB) and JNK (Fig. 4).

TRAF2 can also activate NF-kB and JNK via a TRADD-

independent interaction with TNF-R2 (Rothe et al., 1995;

Reinhard et al., 1997). In contrast to the Fas-mediated

activation of JNK, TNF-R-mediated activation of JNK does

not appear to be associated with the induction of apoptosis

(Liu et al., 1996b; Natoli et al., 1997a). However, this

finding is controversial as TRAF2 has been demonstrated

to interact with and activate ASK1 resulting in subsequent

JNK activation and apoptosis (Ichijo et al., 1997; Hoeflich

et al., 1999). The induction of apoptosis through this

pathway, in contrast to the Fas-mediated pathway, is

dependent on the generation of reactive oxygen species

(Liu et al., 2000; Tobiume et al., 2001). It seems likely that

the role of JNK activation in TNF-induced apoptosis is

dependent on the cell type and circumstances surrounding

P.C. Ashe, M.D. Berry / Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 199–214206

cell death, such as the presence or absence of free radical

scavengers. In contrast to the potential dual role of JNK

activation, activation of NF-kB appears to protect cells

from TNF-induced apoptosis. NF-kB, but not JNK activa-

tion, has been demonstrated to be dependent on NF-kB-inducing kinase (NIK) (Natoli et al., 1997b; Song et al.,

1997). It is, therefore, concluded that NF-kB and JNK

activation pathways bifurcate directly at the level of

TRAF2. Activation of NF-kB requires the interaction

between RIP and TRAF2. Specifically, RIP interacts with

NF-kB essential modulator (NEMO; IKKg), a component

of the inhibitor of kB kinase complex (IKK) (Zhang et al.,

2000). The IKK complex also contains IKKa and IKKbsubunits, which are required for the interaction between

IKK and TRAF-2 (Devin et al., 2001). The RIP-TRAF2

complex activates NIK, a member of the MAP kinase

kinase kinase family (Malinin et al., 1997), which, in turn,

activates IKK via phosphorylation. Subsequent phosphor-

ylation of inhibitor of NF-kB (I-kB) by IKK targets this

protein for ubiquitination and proteosome degradation

(DiDonato et al., 1996, 1997). Removal of I-kB unmasks

the nuclear localization signal of NF-kB and allows its

translocation to the nucleus (Beg et al., 1992). NF-kB can

then activate transcriptional events mediating cell survival

(Li and Stark, 2002). NF-kB has been demonstrated to

control the expression of TRAF1, TRAF2, cIAP1, and

cIAP2 (Wang et al., 1998). It is proposed that TRAF-

mediated recruitment of cIAP1 and cIAP2 to the TNF

Fig. 4. A schematic representation of TNF-R1-mediated apoptosis signaling.

P.C. Ashe, M.D. Berry / Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 199–214 207

receptor complex results in the inhibition of caspase 8;

thus, protecting cells from TNF-induced apoptosis. TNF-

mediated NF-kB activation also appears to inhibit sustained

JNK activation, a further mechanism by which NF-kB may

exert its antiapoptotic action (Tang et al., 2001). Increased

expression of X-linked inhibitor of apoptosis protein

(XIAP), a proposed NF-kB responsive gene, is presumed

responsible for inhibition of the JNK pathway. These

complex interactions between various arms of the TNF

signal transduction cascade may represent a mechanism of

tight regulation for determination of a cell’s ultimate fate.

While pieces of the TNF puzzle are slowly being unveiled,

a great deal of work remains to decipher all of the

intricacies of TNF signaling.

6.3. Apo3L signaling

The Fas and TNF pathways are the best-characterized

DR pathways; however, a number of other pathways also

contribute to DR signaling. DR3 (Apo3) and its ligand,

Apo3L, share considerable similarities to TNF-R1 and

TNF, respectively. Signaling by DR3 has been demonstra-

ted to induce apoptosis as well as the activation of NF-kB(Chinnaiyan et al., 1996a; Marsters et al., 1996, 1998).

Similar to TNF signaling, apoptosis induced by DR3 is

mediated via interactions with TRADD, FADD, and cas-

pase 8, while NF-kB activation is mediated by TRADD,

TRAF2, and RIP. In contrast to the TNF system, however,

Apo3L displays a relatively wide tissue distribution,

whereas DR3 demonstrates a more focused tissue distri-

bution. This suggests that these DR systems likely have

distinct biological roles despite their similarities in cell

signaling. Signaling mediated by the DR6 receptor also

shares a number of similarities with TNF signaling. The

ligand and the exact physiological functions of this recep-

tor are unknown; however, it has been demonstrated that

DR6 activation can induce apoptosis via an interaction

with TRADD (Pan et al., 1998). Similar to TNF-R1, DR6

activation can also induce the activation of NF-kB and

JNK. In addition, it appears that DR6 is regulated by NF-

kB and that treatment with TNFa increases the expression

of DR6, thereby providing additional regulatory mecha-

nisms in an already complex and highly regulated system

(Kasof et al., 2001).

6.4. TRAIL signaling

Tumor necrosis factor a-related apoptosis-inducing

ligand (TRAIL; Apo2L) is a Type II transmembrane death

ligand expressed in the majority of human tissues (Wiley et

al., 1995; Pitti et al., 1996). Four DRs have been shown to

bind specifically to TRAIL: DR4, DR5, DcR1, and DcR2

(Chaudhary et al., 1997; Marsters et al., 1997; Pan et al.,

1997a,b; Sheridan et al., 1997). Like TRAIL, these recep-

tors are expressed in a wide variety of tissues. Despite the

wide variety of tissues expressing the elements necessary for

TRAIL-induced apoptosis, it has been reported that TRAIL

demonstrates selective toxicity to cancer cells. This select-

ivity may be a function of the tissue expression profiles of

DcR1 and DcR2, DcRs that inhibit the apoptotic signaling

of TRAIL. Selectivity may also be conferred by p53-

mediated induction of DR5 (Kim et al., 2001). These

authors reported that cancer cells, but not normal cells,

demonstrate enhanced sensitivity to TRAIL following p53

expression; an effect correlated with DR5 expression. In

accordance with this explanation, DR4 is also up-regulated

by p53; however, the antiapoptotic DcRs are also induced

by p53; thus, the specific role of p53 regulation of TRAIL

receptors is questionable (Meng et al., 2000; Guan et al.,

2001). One further role for p53 in TRAIL-mediated apop-

tosis may be through its removal of the FLIP-mediated

inhibition of caspase 8. p53 has been demonstrated to

suppress the expression of FLIP, an effect apparently medi-

ated through enhancement of the ubiquitin-proteasome

degradation of FLIP (Fukazawa et al., 2001). FLIP expres-

sion may also be related to resistance to TRAIL-mediated

apoptosis as a number of resistant cell types display elevated

expression of FLIP, whereas a large proportion of TRAIL-

sensitive cells have lower expression levels (Kim et al.,

2000). Despite the reported cancer cell selectivity of

TRAIL, it has been demonstrated that TRAIL can induce

apoptosis in normal primary cortical neurons and may play a

role in ischaemia-induced neuronal apoptosis (Martin-Vil-

lalba et al., 1999).

A number of earlier studies reported that FADD is not

required for TRAIL-induced apoptosis (Chaudhary et al.,

1997; Pan et al., 1997b); however, more recent reports

indicate that DR4 and DR5, similar to Fas, both signal

apoptosis through an interaction with FADD and caspase 8

(Kischkel et al., 2000; Kuang et al., 2000; Sprick et al.,

2000). Caspase 10 is also recruited to the TRAIL DISC

through its association with FADD, although its function in

apoptosis appears to be separate from that of caspase 8

(Sprick et al., 2002). The activation of caspase 8 following

DR4 or DR5 ligation by TRAIL has been demonstrated to

recruit the intrinsic mitochondrial cell death pathway similar

to that following Fas ligation by FasL (Deng et al., 2002;

Werner et al., 2002). It appears that, although cytochrome c

is released, it is the mitochondrial release of Smac/DIABLO

that is critical for the induction of apoptosis following

TRAIL signaling. In the absence of Smac/DIABLO, XIAP

is able to inhibit the activation of caspase 3 even in the

presence of active caspase 9.

In addition to apoptosis, TRAIL ligation of DR4 and

DR5 can activate NF-kB as well as the JNK pathway

(Chaudhary et al., 1997; Schneider et al., 1997; Muhlenbeck

et al., 1998). Similar to TNF signaling, RIP and TRAF2 are

involved in the activation of JNK and NF-kB. RIP is

essential for both the TRAIL-induced activation of JNK

and NF-kB, whereas TRAF2 is necessary for the activation

of JNK but does not appear to effect NF-kB activation (Lin

et al., 2000). While TRADD is proposed to be the adaptor

P.C. Ashe, M.D. Berry / Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 199–214208

molecule that recruits RIP and TRAF2 to DR4 and DR5

receptors, the identity of the adaptor molecule is not con-

firmed. NF-kB activation, by analogy to TNF signaling, is

proposed to protect cells from TRAIL-induced apoptosis.

The role of JNK activation, however, is unknown, as

TRAF2 knockout cells are as sensitive to TRAIL-induced

death as wild-type TRAF2 cells. Similar to other DR path-

ways, this is a highly regulated, complex system, and many

questions regarding the physiological role of TRAIL signal-

ing as well as components and regulatory mechanisms

remain unanswered.

7. Conclusions

In summary, apoptosis is a complex area involving

many protein families, subcellular compartments, and

signal transduction cascades. While much is known, more

work is necessary on the multiple pathways in multiple

cell types. At present, the precise pathways executed

under specific circumstances are unconfirmed and many

details of the complex regulatory mechanisms are

unknown. It does seem likely, however, that cell type

and mode of induction are critical factors with respect to

which apoptotic pathway is recruited. With the increas-

ingly demonstrated role of apoptosis in various degener-

ative conditions, it is becoming urgent to obtain a clear

understanding of this phenomenon. As our understanding

improves and more apoptotic cascade components are

identified, the opportunity to develop target specific drugs

for clinical intervention becomes increasingly realistic.

Already, many companies are focusing on the devel-

opment of antiapoptotic compounds (Larner, 2000) as a

way to tackle the immense field of acute and chronic

neurodegeneration.

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