Caspase cleavage of members of the amyloid precursor family of proteins

Caspase cleavage of members of the amyloid precursor family

of proteins

Veronica Galvan,* Sylvia Chen,* Daniel Lu,� Anna Logvinova,* Paul Goldsmith,*Edward H. Koo� and Dale E. Bredesen*

*Buck Institute for Age Research, Novato, California, USA

�Department of Neurosciences, University of California, San Diego, La Jolla, California, USA

Abstract

The synapse loss and neuronal cell death characteristic of

Alzheimer’s disease (AD) are believed to result in large part

from the neurotoxic effects of b-amyloid peptide (Ab), a 40–42

amino acid peptide(s) derived proteolytically from b-amyloid

precursor protein (APP). However, APP is also cleaved

intracellularly to generate a second cytotoxic peptide, C31,

and this cleavage event occurs in vivo as well as in vitro and

preferentially in the brains of AD patients (Lu et al. 2000).

Here we show that APPC31 is toxic to neurons in primary

culture, and that like APP, the APP family members APLP1

and possibly APLP2 are cleaved by caspases at their C-ter-

mini. The carboxy-terminal peptide derived from caspase

cleavage of APLP1 shows a degree of neurotoxicity compar-

able to APPC31. Our results suggest that even though APLP1

and APLP2 cannot generate Ab, they may potentially con-

tribute to the pathology of AD by generating peptide fragments

whose toxicity is comparable to that of APPC31.

Keywords: Alzheimer’s disease, b-amyloid precursor pro-

tein (APP), cytotoxic peptides, neurodegeneration, neuronal

cell death, neurotoxicity.

J. Neurochem. (2002) 82, 283–294.

Cell death in the CNS occurs extensively in development,

during normal aging and in some pathological states

associated with degeneration of specific subsets of neurons.

The majority of cell deaths in the developing nervous system

occur by the activation of programmed cell death, and neural

death in at least some disease states may involve pro-

grammed cell death (Bredesen 1995; Sperandio et al. 2000;

Yuan and Yankner 2000). Elucidating the molecular mech-

anisms that initiate and control pathological cell death in the

CNS should help in the development of interventions that

may prevent or ameliorate degenerative CNS diseases.

The loss of hippocampal neurons is one of the prominent

features of Alzheimer’s disease (AD). The pathological

hallmark of AD is the formation of senile plaques and

neurofibrillary tangles in the brain, accompanied by substan-

tial neuronal and synaptic loss in the neocortex. APP is a

ubiquitously expressed membrane-spanning glycoprotein

that is cleaved during its normal metabolism to generate

the amyloid-b protein (Ab), a 40–42 amino acid peptide thatis the main constituent of senile plaques. The deposition of

Ab may account for the enhanced susceptibility of hippo-

campal and cortical neurons to premature death, since

exposure of cultured human neuronal and non-neuronal cells

to amyloidogenic Ab peptide induces the activation of

apoptotic cell death pathways (Cotman and Anderson 1995;

La Ferla et al. 1995).

Work from this and other laboratories has shown that, in

addition to the cleavages that result in the formation of Ab,APP can be cleaved at its C-terminus by caspases, a family of

cysteine proteases central to the execution of apoptosis

(Gervais et al. 1999; LeBlanc et al. 1999; Pellegrini et al.

1999; Lu et al. 2000). In addition, work from several

laboratories (Gervais et al. 1999; LeBlanc et al. 1999) has

raised the possibility that the cleavage of APP precedes and

may favor the intramembrane cleavage that leads to the

Received February 11, 2002; revised manuscript received April 3, 2002;

accepted April 4, 2002.

Address correspondence and reprint requests to Dale E. Bredesen,

Buck Institute for Age Research, 8001 Redwood Boulevard, Novato,

CA 94945–1400, Tel. 415-209-2090, Fax: 415-209-2230,

E-mail: [email protected]

Abbreviations used: Ab, amyloid b-peptide; AD, Alzheimer’s disease;APLPa, AAP-like protein 1; APP, amyloid precursor protein; AraC,

cytosine arabinoside; BAF, Boc-aspartyl-fluoromethylketone; DMSO,

dimethylsulfoxide; DP, delivery peptide; EthD-1, ethidium homodimer-

1; FITC, fluorescein isothiocyanate; GFAP, glial fibrillary acidic protein;

MTT, 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyltetrazolium bromide;

PBS, phosphate-buffered saline; RT, room temperature; TBS, Tris-

buffered saline.

Journal of Neurochemistry, 2002, 82, 283–294

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 283–294 283

generation of Ab. This hypothesis, however, has not beenverified in other more recent studies (Soriano et al. 2001).

Independent from its effect on the generation of Ab, recentwork has demonstrated that the C-terminal fragment released

by intracellular caspase cleavage of APP (APPC31) is

cytotoxic (Lu et al. 2000; Dumanchin-Njock et al. 2001).

Evidence for cleavage of APP at the C-terminal caspase site,

D664, was obtained from brains of patients with AD, but not

control patients (Lu et al. 2000). Taken together, these data

suggest that the cleavage of the C-terminal portion of APP

may play an important role in the neural toxicity observed in

AD pathogenesis by generating a pro-apoptotic C-terminal

fragment, and possibly by increasing the production of the

toxic Ab peptide. If this is indeed the case, then

the accumulation of Ab at neuronal terminals could providethe trigger for a feedback loop of toxicity by inducing the

initial activation of caspases. It is possible that even low

levels of activated caspases may suffice to cleave APP

molecules intracellularly at the C-terminal caspase site.

Cytotoxic peptides would then be released as a consequence

of C-terminal cleavage of APP, further amplifying the

activation of the caspase cascade. Consistent with this idea,

mice expressing an APP transgene carrying two point

mutations linked to autosomal forms of familial AD develop

neurological symptoms and synapse loss in the absence of

significant Ab accumulation or amyloid plaque formation

(Mucke et al. 2000).

Here we extend our original studies on APPC31: first,

because the initial studies documented toxicity in cell lines

but did not evaluate toxicity in primary neuronal cultures, we

assessed such neurotoxicity. Second, we addressed the

possibility that APP-like protein 1 (APLP1) and APP-like

protein 2 (APLP2) may undergo cleavage events similar to

that which generates APPC31.

APLP1 and APLP2 are members of the APP family of

proteins. However, the sites required for c and b-secretasecleavage of APP are not conserved in either APLP1 or

APLP2. These molecules therefore do not have the capacity

to generate b-amyloid-like peptides. However, the C-terminalcaspase cleavage site that allows for the generation of

APPC31 is conserved in both APLP1 and APLP2. This

observation hinted at the possibility that either one or both of

the APP family members are cleaved by caspases to generate

potentially toxic peptides.

We provide evidence here that APPC31 induces apoptosis

in primary neuronal cultures and that, like APP, APLP1 and

APLP2 can be cleaved by caspases at their C-termini. Even

though the homology between the peptide released by

caspase cleavage of APLP1 (APLP1C31) and APPC31 is

relatively low and is mainly restricted to a C-terminal

YENPTY motif, APLP1C31 induces apoptosis in primary

neuronal cultures with an LC50 similar to that of APPC31.

The present work supports the notion that, in addition to

the well-established role of APP in the pathogenesis of AD,

APLP1 and possibly APLP2 may release C-terminal toxic

peptides and thus play a role in the neurotoxicity associated

with AD.

Materials and methods

Cells and reagents

Hippocampal or cortical neurons derived from 17-day-old rat

embryos were plated in modified minimum essential media (MEM-

PAK) supplemented with 5% horse serum. Three days later, the

cultures were treated with 10 lM cytosine arabinoside (AraC).

Twenty-four hours later the cells were treated with peptide

conjugates as indicated in the figures and incubated for an additional

24 or 48 h. Where indicated, cells were incubated in the presence of

50 lM of the general caspase inhibitor BOC-Asp(Ome)-FMK (BAF)for 30 min prior to addition of peptides or with an equivalent

volume of dimethylsulfoxide (DMSO) and then maintained in the

presence of the same concentration of the inhibitor for the duration

of the experiment by adding fresh BAF at 12-h intervals.

All peptide conjugates were prepared as 1 mM and 10 mM stock

solutions in water, aliquoted and frozen at ) 80�C. Aliquots werethawed in ice and used promptly.

Peptide delivery into cells

Peptides were synthesized and purified at the Stanford University

Protein and Nucleic Acid (PAN) Facility. All peptide stocks were

solubilized in water at 1 or 10 mM concentration. The delivery

peptide derived from the Drosophila Antennapedia homeodomain

(C-RQIKIWFQNRRMKWKK; Dorn et al. 1999), also called

penetratin (Nakagawa et al. 2000), was cross-linked via an

N-terminal Cys–Cys bond to the 31 amino acid peptide generated

by caspase cleavage of APP (DP–APPC31) or APLP1 (DP–

APLP1C31), to fluorescein isothiocyanate (DP–FITC) or to itself

(DP) at the Stanford University PAN facility. Cargo peptides are

released from the carrier by reduction of the disulfide bond in the

intracellular environment. We have observed no toxicity in

transduction experiments using the conjugate of the DP to itself at

the concentrations assayed. Additional controls are indicated in the

figures and text.

Cell death assessment

Primary hippocampal or cortical neuronal cultures or 293 cells

transduced with the different delivery peptide–peptide conjugates

were assayed for viability at 24 and 48 h after transduction by

trypan blue exclusion as described (Lu et al. 2000) or by conversion

of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide;

thiazolyl blue (MTT, Sigma, St Louis, MO, USA) to insoluble

formazan in metabolically active cells and by the LIVE/DEAD

assay (Molecular Probes, Eugene, OR, USA). This assay distingui-

shes live cells by the presence of intracellular esterase activity,

which results in the conversion of the non-fluorescent cell permeant

calcein–AM to the intensely green fluorescent calcein. Calcein is

retained within live cells. Ethidium homodimer-1 (EthD-1) enters

cells with damaged membranes and becomes intensely fluorescent

when bonding to nucleic acids. EthD-1 is excluded by the intact

plasma membrane of live cells. Media were removed and replaced

by 4 lM EthD-1 and 2 lM calcein in phosphate-buffered saline

284 V. Galvan et al.

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 283–294

(PBS). Images were taken 30 min after treatment. The morphology

of nuclei in the cultures was examined by staining with 0.1 lg/mLHoechst 33342. LC50 values were calculated as 100*[(T)T0)/T0] ¼ )50 and by straight line interpolation.

In vitro protein synthesis and caspase cleavage

In vitro transcription and translation used the Promega Coupled kit

(Promega, Madison, WI, USA). The constructs encoding wild-type

APP or the APP D664A mutant (pC-FL-APP and pC-APPD664A),

APP truncated at D664 (APP C31), pC-APLP1 and pC-APLP2 were

translated and the protein products were used to assess caspase

cleavage. Cleavage with caspases )3, ) 6, ) 7 and ) 8 was done andassessed as described (Ellerby et al. 1999).

Immunostaining and image analysis

Hippocampal cultures were fixed in 4% paraformaldehyde in 1 ·PBS for 20 min at room temperature (RT). Cells were then rinsed in

1 · PBS and then washed once in 1· Tris-buffered saline (TBS)followed by blocking in 10% donkey serum (Jackson Immuno-

Research Laboratories, West Grove, PA, USA) with 0.1% Triton

X-100 in 1 · TBS for 1 h at RT. Cultures were incubated overnightin the presence of rabbit anti-GFAP (Sigma) at 1 : 800 dilution and

mouse anti-NeuN (Chemicon, Temecula, CA, USA) at 1 : 100 at

4�C. Negative controls were incubated in 2 mg/mL rabbit and

mouse pre-immune IgGs (Sigma). All primary antibodies were

diluted in 1 · TBS containing 10% donkey serum. Cultures were

washed for 90 min in four changes of 1 · TBS and incubated in thepresence of donkey anti-rabbit IgG conjugated to Cy3 and donkey

anti-mouse IgG conjugated to FITC (Jackson ImmunoResearch

Laboratories, Inc., West Grove, PA, USA), at 1 : 250 and 1 : 400,

respectively, in 1 · TBS containing 1% donkey serum for 1 h at RT.Cells were washed for 90 min in four changes of 1 · TBS andmounted in VectaShield-DAPI mounting medium (Vector Laborat-

ories, Burlingame, CA, USA). Low magnification images were

acquired using Nikon Eclipse-800 microscope and Optronics

MagnaFire camera and software, and analyzed using Compix

Simple PCI software. The total surface area corresponding to red

and green fluorescence in each confocal image was determined by

image analysis using Simple PCI software (Compix, Inc., Philadel-

phia, PA, USA). In each experiment, areas of positive immunore-

activity were identified in the control sample and used to define

representative ranges of values for red and green pixels. Using these

defined parameters, randomly chosen fields from each sample in a

given experiment were collected and the images were analyzed by

the PCI image analysis software, which calculated the total area

covered by pixels matching the defined intensities for each color

component. The values corresponding to total green and red

fluorescent areas were averaged.

Generation of the APP-Neo antibody

An antibody that recognizes specifically the epitope generated by

cleavage of APP at D664 by caspases was generated at ResGen

(Invitrogen Corp., Carlsbad, CA, USA). Briefly, rabbits were

immunized with the peptide 657CIHHGVVEVD664, which includes

the nine amino acids immediately preceding the caspase cleavage

site at position 664 in APP695, coupled to KLH. Antisera from three

bleeds over a 10-week period were pooled and affinity-purified in

three successive steps:

1. Peptide antigen was immobilized on an activated support.

Antisera were passed through the column and then washed. After

washing, the bound antibodies were eluted by a pH gradient.

2. The eluate from (1) was depleted of immunoglobulins that

recognize the intact APP molecule by adsorption to a bridging

peptide that encompasses the caspase cleavage site (TSIHHGVVE-

VDAAVTPEE).

3. The flowthrough from (2) was affinity-purified on the

immobilized immunogenic peptide. After washing, specific anti-

bodies were eluted by a pH gradient, collected and stored in borate

buffer.

The ELISA titer for this preparation was < 1 : 142 000

(< 5 ng/mL) against the immunizing peptide (corresponding to the

�novel� C-terminus of APP, an epitope that is generated only aftercaspase cleavage) versus > 1 : 70 (> 10 mg/mL) against the

bridging peptide that corresponds to the intact APP sequence across

the caspase cleavage site at D664.

Immunohistochemistry

Human hippocampi obtained from AD or age-matched control

patients (Harvard Brain Tissue Resource Center, Belmont, MA,

USA) fixed with 4% paraformaldehyde were embedded in paraffin.

Seven-micrometer microtome sections were deparaffinized in

xylene, rehydrated in 100, 95, 80 and 70% ethanol, and washed

in 1 · TBS for 15 min at room temperature. A 3% H2O2 solution in

methanol was used to neutralize endogenous peroxidase-like

activity. Microwave antigen retrieval was performed in 10 mM

citrate buffer (pH 6.0) for 5 min at 440 Watts. Slides were allowed

to cool to room temperature and were washed in 1 · TBS for

15 min. Samples were blocked in 10% normal horse serum in

1 · TBS for 1 h at room temperature. Primary rabbit IgG to APP-

Neo was applied at a dilution of 1 : 10000 in 1% bovine serum

albumin (BSA) in 1 · TBS; sections were incubated overnight at4�C. Rabbit pre-immune IgG (Sigma) diluted to 1 lg/mL in the 1%BSA in 1 · TBS were used as a negative control. Sections werewashed for 30 min in three changes of 1 · TBS; biotinylated horseanti-rabbit IgG (Vector Laboratories) was applied at a dilution of

1 : 250 for 1 h at room temperature. Peroxidase-based ABC Elite kit

(Vector Laboratories) was used according to the manufacturer’s

instructions followed by a 30-min wash in three changes of

1 · TBS. A liquid DAB kit (Vector Laboratories) was used for thedetection; color development was monitored under the microscope.

Sections were washed in 1XTBS, briefly counterstained in aqueous

hematoxylin, dehydrated, cleared, and mounted in Permount (Fisher

Scientific, Pittsburgh, PA, USA). Images were acquired using Nikon

Eclipse-800 microscope and the Optronics MagnaFire camera and

software. Low magnification images were acquired using Nikon

SMZ-U dissecting microscope and the CoolSnap camera and

software.

Results

C31 induces death of rat hippocampal neurons

in primary culture

Previous work demonstrated that the C-terminal fragment

released by caspase cleavage of APP is toxic in transformed

Caspase cleavage of APP family of proteins 285


cell lines (Lu et al. 2000). To assess the cytotoxicity of

APPC31 in a system more closely related to the mammalian

brain, we performed experiments to determine whether

APPC31 induced apoptosis in primary cultures of hippo-

campal neurons. Given that transfection efficiencies in

neuronal primary cultures are relatively low, we used a

protein transduction method. This approach allows for the

introduction of polypeptides into cells with an efficiency

close to 100%, and utilizes relatively stress-free conditions

(Schwarze et al. 2000). We chose to use the Drosophila

melanogaster Antennapedia homeodomain-derived DP, also

called penetratin, linked by an N-terminal disulfide bond to

the APP-derived C31 peptide or to control peptides. This

delivery peptide has been used successfully in similar

experiments in the past (Dorn et al. 1999; Gallouzi and

Steitz 2001). Disulfide linkage was chosen over other types

of covalent bond in order to allow the APPC31 peptide to be

released inside the cells, in association with reduction of the

S–S bond in the intracellular environment.

To assess the efficiency of transduction, primary neuronal

cultures were transduced with penetratin conjugated to FITC

(DP–FITC) and analyzed by confocal microscopy. The DP–

FITC peptide was internalized in > 95% of the cells in the

culture (Fig. 1a). We then transduced hippocampal neuronal

cultures derived from 17-day-old rat embryos with 10 lMDP–APPC31 peptide or DP control. A marked decrease in

viability in the DP–APPC31-transduced cultures, but not in

the control cultures, was observed 24 h after transduction.

The cells treated with the DP–APPC31 peptide showed

prominent cytoplasmic shrinkage and an almost complete

disappearance of the neuritic network (not shown). Fluores-

cence microscopic examination of the same cultures showed

a profound reduction in the number of viable cells (cells

capable of calcein retention in their cytoplasm), and a

proportional increase in the number of cells with damaged

membranes permeable to ethidium homodimer (EthD-1)

(Fig. 1b). Essentially identical results were obtained for

cortical neuronal cultures (not shown). Also, incubation of

primary hippocampal neurons in the presence of increasing

concentrations of DP–APPC31, but not DP alone, signifi-

cantly reduced the number of viable cells (capable of

converting MTT into insoluble formazan) present in the

cultures (Fig. 1c).

Immunocytochemical examination of hippocampal cul-

tures using antibodies specific for a neuronal marker, neuron-

specific nuclear protein (NeuN), and a glial marker, glial

fibrillary acidic protein (GFAP), revealed a marked decrease

in the number of NeuN-immunoreactive cells present in the

cultures that had been treated with 10 lM DP–APPC31

compared to cultures treated with vehicle (Fig. 2a versus

Fig. 2b). A slight reduction in the number of NeuN-immuno-

reactive cells was seen in cultures treated with delivery

peptide alone (Fig. 2c). The decrease in the number of

NeuN-immunoreactive cells present in cultures treated with

10 lM DP–APPC31 was prevented by the addition of the

broad-spectrum caspase inhibitor BAF (Fig. 2d). To deter-

mine precisely the extent of cell death induced by APP-C31

and to evaluate the possible cell-type specifity of the

peptide’s toxicity we performed experiments similar to those

shown in Fig. 2 and quantitated the total area of NeuN and

GFAP immunoreactivity of representative fields using con-

focal microscopy and digital image analysis.

C31 induces programmed cell death in both neuronal

and glial cells

The morphology of the cells that survived transduction with

DP was suggestive of glial origin (Fig. 2). To investigate

whether this was due to a greater sensitivity of neurons than

glial cells to DP–APPC31-induced death, 3-day-old rat

hippocampal cultures were exposed to increasing concen-

trations of DP–APPC31 or control DP peptide and fixed

48 h after transduction. The fixed cultures were then

immunostained with antibodies specific for GFAP (red) and

NeuN (green). A quantitative assessment of the total area of

red and green fluorescence present in low-magnification

confocal images of representative fields obtained from three

independent experiments was performed using a digital

image analysis system (SimplePCI, Compix, Inc, Philadel-

phia, PA, USA). We found that both the neuronal and the

glial population were significantly reduced in cultures treated

with 10 lM DP–APPC31 when compared to untreated or

control peptide-treated cultures (Fig. 3). Transduction with

higher concentrations of DP–APPC31 (25 lM) were requiredto reduce the viability of the neuronal population further,

while no further toxicity was observed for glial cells at the

concentrations assayed. Incubation in the presence of the

broad caspase inhibitor, Boc-aspartyl-fluoromethylketone

(BAF), delayed the toxicity resulting from transduction with

DP–APPC31, arguing that C31-induced neuronal death in

primary cultures is caspase-mediated.

To resolve the discrepancy in the LC50 values obtained by

the MTT assay for cell viability (Fig. 1c, 7.2 lM) and byquantitative image analysis (Fig. 3, 3.75 lM), the extentof cell death induced by transduction of DP–APPC31 in

neuronal cultures was further examined by the trypan blue

exclusion method. The LC50 value obtained by trypan blue

exclusion for neuronal cultures transduced with DP–APPC31

was 3.75 lM, in agreement with the value obtained by

quantitative image analysis (Fig. 4a). Given that 3-day-old

cultures of primary neurons were used in all experiments, it is

conceivable that the higher LC50 value obtained using the

MTT assay was due to variability in the proportion of glial

cells present in different batches of primary neurons at the

time of plating.

Finally, we quantitated the number of apoptotic nuclei in

cultures transduced with different concentrations of

DP–APPC31 by Hoechst 33342 staining. An increase in

the percentage of condensed, fragmented nuclei present in



neuronal cultures was observed when increasingly high

concentrations of DP–APPC31 were used for transduction

(Fig. 4b). This observation, together with the finding that

APPC31 toxicity was delayed by caspase inhibitors, suggests

that the cellular death induced by the C31 peptide was

apoptotic in nature.

Caspase-cleaved APP in the brains of patients

with AD and control, non-AD patients

To document the generation of C31 peptides in cultured cells

and tissues, we generated an antibody capable of recognizing

exclusively the novel epitope that arises by caspase cleavage

Fig. 3 Neuronal and glial cells are comparably susceptible to DP–

APPC31 toxicity. Confocal images of primary hippocampal cultures

transduced with (a), 0; (b), 2.5 lM ; (c) 10 lM; (d), 25 lM DP–APPC31,

(e) with DP alone, or (f) with 10 lM DP–APPC31 in the presence of

50 lM BAF. Cultures were fixed and immunostained with anti-NeuN

and anti-GFAP 30 h after treatment. A representative experiment is

shown. (g) Total green (NeuN) and red (GFAP) fluorescent areas in

primary hippocampal cultures treated as in (a–f) were determined by

image analysis as described in Materials and Methods using the PCI

image analysis software (Compix). Values are shown as mean ± SD

(n ¼ 3).

Fig. 2 DP–APP31 toxicity in primary neuronal cultures is dependent

on the activation of caspases. Confocal images of primary hippo-

campal cultures pre-incubated in 50 lM BAF or in an identical volume

of DMSO and then treated with: (a) water; (b) 10 lM DP–APPC31;

(Cc) 10 lM DP; (d) 10 lM DP–APPC31 in the presence of 50 lM BAF.

Twenty-four hours later, the cultures were fixed and immunostained

with antibodies specific for NeuN and GFAP to identify neuronal and

glial populations, respectively.

Fig. 1 DP–APPC31 decreases the number of viable cells in primary

neuronal cultures. (a) Left panels: Confocal images of HEK293 cells

transduced with DP–APPC31 or with DP alone, fixed and stained with

an anti-APPC31 antibody followed by anti-rabbit FITC. Right panels:

Confocal images of HEK293 cells transduced with unlabelled or FITC-

labeled DP. (b) Primary cultures of hippocampal neurons transduced

with 10 lM of the indicated peptides were subjected to the LIVE/DEAD

assay 24 h later as described in Materials and Methods. Green-

fluorescent calcein is retained within living cells, while EthD-1

becomes red fluorescent when bound to nucleic acids of cells with

damaged membranes. (c) Primary cultures of hippocampal neurons

transduced with the indicated concentrations of DP–C31 or DP alone

were assayed for viability 36 h later by the MTT assay.



of APP at its C terminus (APP-Neo). The method utilized for

the generation of this antibody has been described previously

(Gervais et al. 1999). We examined sections from hippo-

campi obtained from AD or age-matched control subjects by

immunohistochemistry using the APP-Neo antibody. Hippo-

campal sections from AD brains showed that APP-Neo

immunoreactivity, indicative of cleavage of APP at its

C-terminus, is intense anteriorly in the polymorphic layer,

reduced in the stratum granulosum, decreased in CA4-CA2

and absent from the stratum moleculare (Fig. 5a). APP-Neo

staining was less intense at more posterior levels (Fig. 5b),

but could be detected as dense deposits and in efferent fibers

near CA3. Staining was abolished if the primary antibody

was pre-adsorbed with the immunizing peptide (Fig. 5c) but

not if it was pre-adsorbed with a peptide that encompasses

the immunizing peptide sequences and the first five

N-terminal amino acids of the C31 peptide, past the caspase

cleavage site (bridge peptide; Fig. 5d). We observed that

specific APP-Neo immunostaining occurred in the hippo-

campus of a 90-year-old without AD as well (i.e. control

brain), but to a lesser degree, staining was low to moderate in

cells and fibers of the polymorphic layer and stratum

granulosum, declining in CA4–CA2 and absent from the

stratum moleculare (Fig. 5e). In contrast to the AD brains, no

APP-Neo staining could be detected at more posterior levels

in the hippocampus (Fig. 5f). Staining was abolished by pre-

adsorption with the immunogenic peptide (Fig. 5g) but not

by preadsorption with bridge peptide (Fig. 5h).

Closer examination revealed that the pattern of staining

intensity reflected uneven distribution of APP-Neo immuno-

reactivity. In the AD hippocampus, cytoplasmic staining was

seen in occasional granular layer neurons and most poly-

morphic neuronal cell bodies. Extracellular deposits of APP-

Neo surrounded immunoreactive neurons, neuronal proces-

ses, and vacant areas which may have formerly held neurons

(Fig. 5i). Immunostaining in hippocami of normal controls

was similar but less intense. APP-Neo staining occurred in

isolated granule cells, polymorphic layer neurons, and small

foci along cellular processes (Fig. 5j). Extracellular deposits

Fig. 5 Truncation of APP at the C-terminal caspase site is increased

in human AD brain. An antibody that specifically recognizes the novel

C-terminus of APP generated by caspase cleavage (APP-Neo) but not

the intact APP molecule (see Materials and methods) was used to

stain hippocampal sections of (a–d and i) a 77-year-old with Alzhei-

mer’s disease and (e–h and j) a normal 90-year-old control patient. (a)

In the anterior hippocampus of the 77-year-old AD patient, APP-Neo

staining is intense in the polymorphic layer (P), decreased in CA4,

further reduced in stratum granulosum (G) and absent in stratum

moleculare (M). (b) At more posterior levels, less intense APP-Neo

staining is seen in efferent fibers near CA3 (clear arrow) and some-

times in dense accumulations (black arrow). (c) DAB staining is

abolished if the APP-Neo antiserum is pre-absorbed with the immu-

nogen (shown), with immunogen plus a peptide encompassing the

cleavage site and the first four amino acids of APPC31 (bridging

peptide), or is replaced by buffer (not shown). (d) Immunostaining is

unaffected by pre-absorbing the APP-Neo antiserum with the bridging

peptide alone. (e) In the hippocampus of a normal 90 y.o., APP-Neo

staining intensity is low to moderate in the polymorphic layer (P), low in

CA4-CA3, even less in stratum granulosum (G), and absent in stratum

moleculare (M). (f) No DAB immunostaining is seen when the APP-

Neo antiserum has been preabsorbed with immunogen, or (g) with

immunogen plus bridging peptide. (h) APP-Neo immunostaining is

unaffected when the primary antiserum is preabsorbed with bridging

peptide alone. Magnification (a–d) · 7; (e-h) · 6. Hematoxylin count-

erstain. Higher magnification of the 77-year-old AD hippocampus (i)

shows substantial APP-Neo staining in individual cells (clear arrows)

of the granular layer (G) and in most neurons (large black arrows) in

the polymorphic layer (P). Extracellular deposits of APP-Neo surround

these neurons, former processes (thin arrows), and vacant areas

(stars), which may have formerly held neurons. The normal 90-year-

old hippocampus is shown without hematoxylin counterstain (j) to

reveal light APP-Neo staining in isolated granular layer cells (clear

arrow), polymorphic layer neurons (small black arrow), and extracel-

lular deposits along processes (thin arrows). Use of immunogen-

absorbed APP-Neo antibody in controls eliminated DAB staining, but

not the lipofuscin pigment in many neurons of this elderly individual

(large black arrows). Magnification (i and j) · 200.

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nuc

lei

(a)

(b)

Fig. 4 DP–APPC31-induced death is apoptotic in nature. (a) Primary

hippocampal cultures were transduced with the indicated concentra-

tions of DP–APPC31 or DP alone. Thirty hours later, the viability of the

cultures was determined by the trypan blue exclusion method. Values

are shown as mean ± SD. (b) Experiments similar to those in (a) were

performed using the indicated concentrations of DP–APPC31 or DP

alone. Cultures were stained with 0.1 lg/mL Hoechst 33342 30 h after

transduction and the percentage of condensed, fragmented nuclei

were determined for five randomly chosen fields in each sample.

Values are shown as mean ± SD (n ¼ 3).



were rarely clustered around polymorphic layer neurons or

open areas of tissue. Control sections incubated in immuno-

gen-absorbed APP-Neo antibody helped identify and confirm

specific staining, and to differentiate it from lipofuscin

pigment in older individuals.

APLP1 and APLP2 may be cleaved by caspases

Three members of the APP family of proteins exist: APP,

APLP1, and APLP2. Even though the overall similarity of

the APP family C-termini is not high, the caspase cleavage

site that is required for the generation of APPC31 is

completely conserved in all three members. If the DEVD

sequences in APLP1 and APLP2 can function as caspase

cleavage sites, both proteins could potentially generate

C-terminal peptides. It should be noted, however, that the

P1¢ position in APP is Ala (VEVDA), whereas the P1¢position in APLP1 is Pro (VEVDP) as it is in APLP2.

Caspases tend to prefer less bulky residues such as Gly, Ala,

(a) (b)

(c) (d)

(e) (f)

(g) (h)

(i) (j)



or Ser, in the P1¢ position, rather than more bulky residuessuch as Pro (Stennicke et al. 2000). Therefore, at least in

theory, the VEVD site in APP should be more readily

cleaved by caspases than the sites in APLP1 and APLP2. To

determine whether APLP1 and APLP2 can be cleaved by

caspases, we assayed a panel of recombinant caspases for

their abilities to cleave 35S-labelled, in vitro transcribed/

translated APP, APLP1 and APLP2. The results shown in

Fig. 6(a), indicate that APP can be cleaved by caspases-3 and

-6, but not by caspase-8. APLP1, on the other hand, was

cleaved in vitro only by caspase-3 (Fig. 6b), not by caspase-6

or -8. Like APLP1, APLP2 may be cleaved in vitro by

caspase-3 only, but with very low efficiency, if at all

(Fig. 6b). The 35S-Met-labelled C31 peptide product of the

cleavage of APP by caspase-3 and -6 (Fig. 6a) was detected

as a � 4 kDa band. However, we could not detect the

homologous peptide generated by caspase-3 cleavage of

APLP1. Our inability to detect APLP1C31 could be due to

the fact that only one methionine (of a total of two in

APPC31) is conserved in APLP1C31.

To determine whether cleavage of APLP1 and APLP2 can

occur in cultured cells, we transfected CMV-driven con-

structs expressing N-terminally FLAG-tagged APLP1 and

APLP2 or a full-length APP construct in 293 cells and

activated the caspase cascade by treatment with staurospo-

rine. Both APP and APLP1 were cleaved in staurosporine-

treated 293 cells (Figs 6c and d) and in both cases, cleavage

was prevented by incubation of the cells in the presence of

BAF. We were able to detect both the full-length and the

truncated forms of APP (Fig. 6c, arrows). Full-length APLP1

appeared to be completely degraded in 293 cells treated with

staurosporine, but not when BAF was present (Fig. 6d). No

cleavage products of FLAG-APLP1 could be detected in

these cultures. Both in vitro and in transfected 293 cells, we

found that caspases could cleave APP and APLP1 at more

than one site. No evidence was found for the cleavage of

FLAG-APLP2 in transfected 293 cells (Fig. 6d).

To determine whether APLP1 is effectively cleaved at

position 664, we took advantage of the selective reactivity of

the APP-Neo antibody. Given that the five amino acids that

constitute the novel C-termini in cleaved APLP1 and APLP2

are relatively conserved, epitopes could be generated that

might be recognized selectively by APP-Neo after caspase

cleavage. To determine whether APP-Neo-immunoreactive

epitopes are generated by caspases in APP, APLP1 and

APLP2, we incubated unlabelled, in vitro transcribed/trans-

lated full-length APP (APP695), APPD664A (a mutant of APP

in which the D residue at position 664 has been replaced by

Fig. 6 APLP1 and APLP2 are cleaved by caspases in vitro. (a) Full-

length APP and APPDC31 were in vitro-transcribed and translated in

the presence of 35S-Met. The reactions were then subjected to clea-

vage by the indicated caspases in vitro. Arrows indicate cleavage

products. (b) Full-length APLP1 and APLP2 were in vitro-transcribed

and translated in the presence of 35S-Met. The reactions were then

subjected to caspase cleavage as in (a). Untreated full-length APP

and APPDC31 were included as references. (c) FL-APP or APPDC31

were expressed from the CMV promoter in HEK293 cells untreated or

treated with staurosporine in the presence or absence of BAF. Lysates

were electrophoresed, blotted and reacted with an antibody specific for

APP (5A3). Arrows indicate full-length and cleaved forms of APP. (d)

APLP1-FLAG and APLP2-FLAG were expressed from the CMV pro-

moter in HEK293 cells treated and processed as in (b). Blots were

reacted with anti-FLAG antibody (M2, Sigma).



A), and full-length APLP1 and APLP2 in the presence of

recombinant caspases. The products of the reactions were

separated on polyacrylamide gels and immunoblotted with

APP-Neo antibody. As a control, we performed immunoblots

using lysates from 293 cells transfected with full-length

APP695, with an APP construct lacking the APP C-terminal

31 amino acids (APPDC31), or with APP695 and treated with10 lM staurosporine, in the presence or absence of 50 lMBAF. As expected, APP-Neo-immunoreactive bands were

detected only in lysates from 293 cells expressing APPDC31and in lysates from cells expressing APP695 and treated with

staurosporine in the absence of BAF (Fig. 7a). Also, an APP-

Neo-immunoreactive epitope was detected in immunoblots

of in vitro transcribed/translated full-length APP that had

been incubated in the presence of recombinant caspase-3

(Fig. 7a), but not caspase-7 or -8 (Fig. 7a). Likewise, in vitro

transcribed/translated APLP1 and APLP2 yielded APP-Neo-

immunoreactive cleavage products only when incubated in

the presence of recombinant caspase-3 (Fig. 7b). Even

though the caspase-cleaved APLP2 species was detected as

a very faint 35S-labeled band in in vitro cleavage assays, the

higher sensitivity of the APP-Neo antibody enabled us to

detect the low-abundance, cleaved species in western blot

assays. Providing a control for the specificity of the reaction,

a mutant form of APP that cannot be cleaved by caspases,

APPD664A, did not yield detectable APP-Neo-immunoreac-

tive products after incubation with recombinant caspase-3, -7

or -8.

APLP1C31 induces death in primary hippocampal

cultures

The results shown above suggest that APLP1 can be cleaved

by caspase-3 at the aspartic acid residue at position 620. If this

event occurs in vivo, APLP1 would have the potential to

generate a pro-apoptotic C-terminal peptide homologous to

APPC31. To determine whether the peptide generated by

caspase cleavage of APLP1 is toxic, we generated a fusion of

APLP1C31 to the Antennapedia delivery peptide (DP–

APLP1C31) and assayed it in protein transduction experi-

ments similar to those presented in Figs 1–4 using rat

embryonic hippocampal cultures. Three-day-old rat hippo-

campal cultures were exposed to increasing concentrations of

DP–APLP1C31 or control peptide, fixed 36 h after transduc-

tion and immunostained with antibodies specific for GFAP

and NeuN. A quantitative assessment of the relative areas of

red (GFAP) and green (NeuN) fluorescence present in low-

magnification confocal images was performed using a digital

image analysis system (SimplePCI, Compix, Inc.). We found

that the NeuN-immunoreactive population was markedly

reduced in cultures treated with 10 lM DP–APLP1C31

(Fig. 8a). At higher concentrations of DP–APLP1C31

(25 lM), the viability of the neuronal population was reducedfurther. Surprisingly, we saw only a modest decline in the

viability of glial cells, which may have been due to a relatively

higher sensitivity of neurons to APLP1C31 toxicity. Incuba-

tion in the presence of the broad caspase inhibitor, BAF,

delayed DP–APLP1C31 toxicity, arguing that cell death

induced by APLP1C31 depends on caspase activity.

The extent of cell death induced by transduction of DP–

APPC31 in neuronal cultures was further examined by

the trypan blue exclusion method. As shown in Fig. 8(b), a

dose-dependent reduction in the viability of the cultures was

observed at increasing concentrations of transduced DP–

APLP1C31 but not of control DP peptide. The LC50 value

obtained for neuronal cultures transduced with DP–APPC31

was 4 lM, while the value obtained by image analysis wasapproximately 5 lM (Fig. 8a).

Discussion

Our data indicate that the APP fragment that is generated by

caspase cleavage of the APP C-terminus at Asp664 is toxic

to hippocampal and cortical neurons in primary culture. The

presence of this peptide in primary neuronal cultures triggers

the activation of programmed cell death, as demonstrated by

the condensation and fragmentation of nuclei in transduced

Fig. 7 Caspase cleavage of APP, APLP1 and APLP2 generates an

APP-Neo-immunoreactive epitope. (a) Left panel: Full-length APP or

APPDC31 were expressed from the CMV promoter in HEK293 cells

untreated (mock) or treated with staurosporine in the presence or

absence of BAF. Lysates were subjected to immunoblotting using the

APP-Neo antibody as described in the text. Right panel: In vitro

transcribed/translated 35S-Met labeled full-length APP was subject to

cleavage by caspases as indicated and immunoblotted using APP-

Neo. (b) In vitro-transcribed/translated 35S-labelled APLP1 (left panel)

and APLP2 (right panel) were subjected to cleavage with the indicated

caspases in vitro, blotted and reacted with APP-Neo.



cells and by the ability of the general caspase inhibitor BAF

to delay the death process. This finding is compatible with

our earlier finding that caspase-8 and caspase-9, but not

caspase-3, were required for C31-induced cell death (Lu

et al., submitted for publication). The biochemical path-

way(s) leading from C31 to caspase-8 and -9 and apoptosis

activation is not yet known. However, it is compatible with

the previous finding of caspase-9 activation in synaptosomal

preparations from the brains of patients with AD, but not

from control patients (Lu et al. 2000). It is important to add,

however, that there is no evidence that apoptosis, as

classically defined (Kerr et al. 1972), represents the predom-

inant mode of cell death in neurons in AD. One possible

explanation for this apparent discrepancy is that, whereas

immature neurons may be induced to undergo apoptosis

readily, more mature neurons are more resistant to at least

some pro-apoptotic insults (Yakovlev et al. 2001). Thus,

insults demonstrated to be pro-apoptotic in neurons in

primary culture, such as Ab, APP-C31 and APLP1-C31,may turn out to induce non-apoptotic cell death in mature

neurons in vivo.

To document the caspase-mediated cleavage of APP,

which provides indirect evidence of the release of the toxic

C31 peptide, we used an antibody that selectively recognizes

the neo-C-terminus generated by cleavage of APP at position

664 (APP-Neo). Immunohistochemical examination of brain

tissues from AD patients and age-matched controls revealed

a pronounced accumulation of the caspase-cleaved form of

APP in aggregates adjacent to neuronal cell bodies and in the

perikaryal cytoplasm of some neurons in AD, but to a lesser

extent in the normal, age-matched control sections.

The evidence, taken as a whole, suggests that APP is

cleaved both in cultured cells and in vivo, releasing not only

the Ab peptides, but also APPC31, a neurotoxic peptide.

Thus, the C31 peptide is a good candidate to play a role in

the death of neurons associated with AD. It should be added

that recent work from the d’Adamio Laboratory has shown

that the APPC57 peptide, which results from c-secretasecleavage, may also be cytotoxic (Passer et al. 2000).

However, it is not yet clear whether generation of C31 is

required for C57 toxicity, as was previously demonstrated for

C100 (Lu et al. 2000).

The APP-related gene products, APLP1 and APLP2,

demonstrate putative caspase cleavage sites that liberate C31

fragments: for APLP1, the P4-P1¢ positions are VEVDP, andfor APLP2, the P4-P1¢ positions are VEVDP, while in APP,the P4-P1¢ positions are VEVDA. These sequences, likethose in APP, fit well with previously described caspase

cleavage sites for the initiator/apical caspases such as

caspase-8 and caspase-9, except at the P1¢ position. Wehave shown here that APLP1, and to a lesser extent APLP2,

may be cleaved by caspases. The APLP2 cleavage efficiency

is likely to be lower than that for APLP1, given that caspase

cleavage products were only detected by immunoblot with a

cleavage-specific antibody. The observation that caspase-

cleaved APLP1 and APLP2 were recognized by APP-Neo in

immunoblots raises the intriguing possibility that potential

neo-epitopes derived from APLP1 and APLP2, in addition to

APP, could be generated in brain tissues and detected by

APP-Neo (Fig. 5). The APLP1C31 peptide is 52% identical

and 82% similar to APPC31, while the predicted APLP2C31

is 71% identical and 83% similar to APPC31. In the case of

APLP1C31, however, even though identity is mainly

restricted to the YENPTY motif, the peptide released by

cleavage of APLP1 C-terminus shows a level of toxicity in

primary neuronal cultures comparable to that of APPC31.

This observation hints at the possibility that toxicity may be

mediated by interference, or at least modulation, of the

endocytic signal that regulates APP turnover and Absecretion (Perez et al. 1999).

Fig. 8 Transduction of DP–APLP1C31 induces death in primary hip-

pocampal cultures. (a) Primary hippocampal cultures preincubated in

50 lM BAF or in an identical volume of DMSO were treated with

increasing concentrations of the indicated peptides in the presence or

in the absence of BAF. Twenty-four hours later, the cultures were fixed

and immunostained with antibodies specific for NeuN and GFAP to

identify neuronal and glial populations, respectively. Total green

(NeuN) and red (GFAP) fluorescent areas in the cultures were

determined by image analysis as described in Materials and Methods

using the PCI image analysis software (Compix). The values shown

are the average of two independent experiments. (b) Primary hippo-

campal cultures were transduced with the indicated concentrations of

DP–APLP1C31 or DP alone. Thirty hours later, the viability of the

cultures was determined by the trypan blue exclusion method. Values

are shown as mean ± SD (n ¼ 5).



We propose that low levels of caspase activation may

occur in neurons, particularly in neuronal terminals

exposed to mild stress, such as the accumulation of

insoluble Ab peptides. These low levels of caspase

activation may be sufficient to cleave APP family mem-

bers present in synaptic terminals but not be sustained or

high enough to propagate the apoptotic signal to the soma.

A considerable number of experiments reported in the

literature seem to support this idea (Mattson et al. 1998;

Chan and Mattson 1999) and also suggest that apoptotic

signals in neurons may be continuously �dampened� bypro-survival signals that protect them from stress-induced

death (Bergmann et al. 2002; Mattson 2000). Locally,

transiently activated caspases could thus support the

generation of toxic C-terminal peptides. Even though the

mechanism underlying the toxicity of these C-terminal

peptides is still unknown, recent work has suggested that

they may be involved in the modulation of transcriptional

activity of APP-binding proteins (Cao and Sudhof 2001;

Cupers et al. 2001; Kimberly et al. 2001). Our results

indicate that the toxicity of C-terminal peptides derived

from members of the APP family is dependent on the

activity of caspases, which suggests that a �feedback�mechanism of caspase activation may exist. We hypothes-

ize that strong pro-survival signals operate in neurons that

inhibit this process for prolonged periods of time but that

may be eventually overridden by the sustained activation

of caspases, eventually leading to neuronal death. It is also

possible that the classic apoptotic pathway is not comple-

ted in neurons in vivo, and that the mechanisms respon-

sible for the eventual demise of the cell may involve

stress-response-related, non-apoptotic pathways.

Acknowledgements

Supported by the National Institute of Neurological Disorders and

Stroke (NS35155 and NS33376 to D.E.B); by the National Institute

on Aging (AG05131 to E.H.K); by the UCSD Alzheimer’s Disease

Research Center and the Joseph Drown Foundation.

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Caspase cleavage of members of the amyloid precursor family of proteins