Critique the alzheimer’s disease paper in the upload file.

ARTICLES

APP binds DR6 to trigger axon pruning and
neuron death via distinct caspases
Anatoly Nikolaev

1
, Todd McLaughlin

2
, Dennis D. M. O’Leary

2
& Marc Tessier-Lavigne

1

Naturally occurring axonal pruning and neuronal cell death help to sculpt neuronal connections during development, but their
mechanistic basis remains poorly understood. Here we report that b-amyloid precursor protein (APP) and death receptor 6
(DR6, also known as TNFRSF21) activate a widespread caspase-dependent self-destruction program. DR6 is broadly
expressed by developing neurons, and is required for normal cell body death and axonal pruning both in vivo and after
trophic-factor deprivation in vitro. Unlike neuronal cell body apoptosis, which requires caspase 3, we show that axonal
degeneration requires caspase 6, which is activated in a punctate pattern that parallels the pattern of axonal fragmentation.
DR6 is activated locally by an inactive surface ligand(s) that is released in an active form after trophic-factor deprivation, and
we identify APP as a DR6 ligand. Trophic-factor deprivation triggers the shedding of surface APP in a b-secretase
(BACE)-dependent manner. Loss- and gain-of-function studies support a model in which a cleaved amino-terminal fragment
of APP (N-APP) binds DR6 and triggers degeneration. Genetic support is provided by a common neuromuscular junction
phenotype in mutant mice. Our results indicate that APP and DR6 are components of a neuronal self-destruction pathway,
and suggest that an extracellular fragment of APP, acting via DR6 and caspase 6, contributes to Alzheimer’s disease.

The initial formative phase of nervous system development, invol-
ving the generation of neurons and extension of axons, is followed by
a regressive phase in which inappropriate axonal branches are pruned
to refine connections, and many neurons are culled to match the
numbers of neurons and target cells1–3. The loss of neurons and
branches also occurs in the adult after injury, and underlies the
pathophysiology of many neurodegenerative diseases1,4.

Our understanding of regressive events in development remains
fragmentary. Degeneration can result ‘passively’ from the loss of
support from trophic factors such as nerve growth factor (NGF)1–3.

There is also evidence for ‘active’ mechanisms in which extrinsic
signals trigger degeneration by means of pro-apoptotic receptors,
including some members of the tumour necrosis factor (TNF) recep-
tor superfamily such as p75NTR (also known as NGFR), Fas and
TNFRSF1A (previously known as TNFR1) (Fig. 1a)5. However, the
full complement of degeneration triggers remains incompletely
understood.

Our understanding of the intracellular mechanisms of neuronal
dismantling is also incomplete. It is well documented that devel-
opmental neuronal cell body degeneration requires the apoptotic

1Division of Research, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA. 2Molecular Neurobiology Laboratory, The Salk Institute, 10010 North Torrey Pines
Road, La Jolla, California 92037, USA.

0%

1%

a E10.5 E11.5 E12.5

P P
PVC

S

M M
M

V
C

S

V

C

S

b

e d 100
80
60
40
20

NGF-deprived

Sensory axons

NGF-deprived
+ anti-DR6.1

D
e
g

e
n
e
ra

ti
n

g
a

xo
n
b

u
n
d

le
s

(%
)

+ NGF

100
80
60
40
20

TF-deprived

Motor axons

TF-deprived
+ anti-DR6.1

1.5%

+ TFs

100
80
60
40
20

48 h, control

Commissural axons

48 h, + anti-DR6.1

24 h, control

+
N

G
F

N

G
F

-d
e
p

ri
ve

d

Sensory, TuJ1

+
A

n
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-D

R
6
.1

+

I
g

G

+
T

F
s

+
I
g

G

+
A

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ti
-D

R
6
.1

Motor, p75NTR

T
F

-d
e
p

ri
ve

d

f

2
4
h

GFP TUNEL

4
8
h

+

A
n
ti
-D

R
6
.1

+

I
g

G

+
I
g

G

Commissural

c

?
Neuro-
trophins FasL

Fasp75NTRDR6

TNF

TNFRSF1A

0

0

0

Figure 1 | DR6 regulates degeneration of several
neuronal classes. a, Diagram of several TNF
receptor superfamily members possessing death
domains. b, DR6 mRNA is expressed by
differentiating spinal neurons (including motor
(M) and commissural (C)), and by sensory (S)
neurons in DRG at E10.5–E12.5. Expression is
low in neuronal progenitors (P) in the ventricular
zone (V). c–f, Anti-DR6.1 (50 mg ml

21
) reduces

degeneration in vitro. c, Anti-DR6.1 inhibits
commissural axon degeneration (visualized with
green fluorescent protein (GFP), right) and cell
body death (TUNEL labelling, left; dots indicate
explants) seen after 48 h in dorsal spinal cord
cultures. Red arrow indicates degenerating axon.
d, e, Effect on degeneration of sensory (d) or
motor (e) axons triggered by trophic deprivation.
TFs, trophic factors (BDNF and NT3). Axons
were visualized by immunostaining for tubulin
(TuJ1; sensory) or p75NTR (motor). f, The
percentage of degenerating axon bundles in
c–e (mean and s.e.m., n 5 3 replicates). Scale
bars, 110 mm (b, c) and 50 mm (d, e).

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effectors BAX and caspase 3 (refs 6–8); pruning of a particular dend-
rite in Drosophila is also caspase-dependent9,10. Developmental axo-
nal degeneration similarly has many hallmarks of apoptosis—
including blebbing, fragmentation, and phagocytic clearing of debris
by neighbouring cells2,4. However, it has been argued that axonal
degeneration is caspase-independent, because caspase 3 inhibitors
block cell body but not axonal degeneration8 (reflecting higher
activation of caspase 3 in cell bodies compared to axons11), and
because genetic manipulations to inhibit apoptosis did not block
axonal degeneration in some models12,13. These results indicated
the existence of a caspase-independent program of axonal degenera-
tion1,2,4, but its molecular nature has remained elusive.

While studying the expression of all TNF receptor superfamily
members14, we found that DR6—one of eight members possessing a
cytoplasmic death domain (Fig. 1a)—is widely expressed by neurons
as they differentiate and enter a pro-apoptotic state. DR6 is an orphan
receptor15. In transfected cells, it triggers cell death in a Jun N-terminal
kinase-dependent manner16. In vivo, it regulates lymphocyte develop-
ment17,18, but its involvement in neural development is unknown.

Here we show that DR6 links passive and active degeneration
mechanisms. After trophic deprivation, DR6 triggers neuronal cell
body and axon degeneration. Because DR6 signals via BAX and cas-
pase 3 in cell bodies, we revisited the involvement of caspases in
axonal degeneration, and found that axonal degeneration indeed
requires both BAX and a distinct effector, caspase 6. Our results also
indicated that DR6 is activated by a prodegenerative ligand(s) that is
surface-tethered but released in an active form after trophic depriva-
tion. In searching for candidate ligands with these properties, we
considered APP, a transmembrane protein that undergoes regulated
shedding and is causally implicated in Alzheimer’s disease19–22,
because we had previously found it to be highly expressed by devel-
oping neurons and especially axons (see later). Because Alzheimer’s
disease is marked by neuronal and axonal degeneration, we had long
wondered whether APP participates in developmental degeneration.
We show that an extracellular fragment of APP is indeed a ligand for
DR6—as is a fragment of its close relative APLP2—that triggers
degeneration of cell bodies via caspase 3 and axons via caspase 6,
and we propose that this developmental mechanism is hijacked in
Alzheimer’s disease.

DR6 regulates neuronal death

To explore the involvement of the TNF receptor superfamily in
neural development, we screened its 28 members by in situ hybrid-
ization in midgestation mouse embryos. We came to focus on DR6
(Fig. 1a), because its messenger RNA is expressed at low levels in
proliferating progenitors in the spinal cord, but is highly expressed
by differentiating neurons within the spinal cord and adjacent dorsal
root ganglia (DRG) (Fig. 1b).

Because DR6-expressing neurons are becoming dependent for sur-
vival on trophic support at these stages, we examined whether DR6
regulates neuronal death after trophic-factor deprivation in vitro,
focusing on three sets of spinal neurons: commissural, motor and
sensory (Supplementary Fig. 1a). Initially, we found that short inter-
fering RNA (siRNA) knockdown of DR6 protected commissural
neurons from degeneration (Supplementary Fig. 2). This prompted
us to screen monoclonal antibodies to DR6 for their ability to mimic
this protection; we selected antibody 3F4 (anti-DR6.1). When
embryonic day (E)13.5 rat dorsal spinal cord explants are cultured
for 24 h, commissural cell bodies and axons are healthy, but they
degenerate if cultured for 24 h longer 23; anti-DR6.1 inhibited this
degeneration (Fig. 1c, f), mimicking DR6 knockdown. Anti-DR6.1
also protected sensory neurons from E12.5 mouse DRGs cultured for
48 h with NGF, and motor neurons from E12.5 mouse ventral spinal
cord explants cultured for 24 h with brain-derived neurotrophic fac-
tor (BDNF) and neurotrophin 3 (NTF3, also known as NT3): when
these cultures were deprived of trophic factor and cultured for 24 h
longer, they showed massive cell death and axonal degeneration,

which were largely inhibited by anti-DR6.1 (Fig. 1d–f and
Supplementary Fig. 1b). Similar protection was observed when
DRGs or ventral explants from a DR6 null mutant17 were deprived
in the absence of anti-DR6.1 (Supplementary Fig. 4b and data not
shown), confirming that anti-DR6.1 is function-blocking. DR6
inhibition (by antibody, siRNA or genetic deletion) caused a delay
rather than a complete block, because more degeneration was
observed in each case 24–48 h later (Fig. 2b, Supplementary Fig. 4b
and data not shown). Consistent with a delay, there was a higher
motor-neuron number at E14.5 in the DR6 mutant, but this returned
to the wild-type level by E18 (Supplementary Fig. 3), after the cell
death period. Thus, antagonizing DR6 delays the death of several
neuronal populations in vitro and in vivo.

DR6 regulates axonal pruning

DR6 protein is expressed not just by cell bodies (data not shown) but
also by axons (Supplementary Fig. 4a). Protection of axons by DR6

a

b
+ NGF 48 h

C
o

n
tr

o
l I

g
G

A

n
ti
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R
6
.1

NGF-deprived

12 h 24 h

1%

D
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xo
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(%
)

NGF-deprived + control IgG
NGF-deprived + anti-DR6.1

+ NGF

48 h24 h

100

80

60

40

20

c

d

e

f

Scratch in substratum Teflon divider

Neurites NeuritesCell bodies

WT

WT

DR6–/–

DR6–/–

0

Figure 2 | DR6 regulates axon pruning in vitro and in vivo. a, Diagram of
Campenot chamber (adapted from ref. 24). b, Images show the local
degeneration of sensory axons (TuJ1 immunostain) in Campenot chambers
after NGF deprivation from the axonal compartment (top) was delayed by
anti-DR6.1 (50 mg ml

21
) added at the time of deprivation (bottom). The

graph shows the percentage of degenerating bundles at 24 and 48 h (mean
and s.e.m., n 5 3 replicates). c–f, Compromised pruning of retinal axons in
DR62/2 mice.Dorsalviewof (c, e),andvibratomesectionsthrough(d, f),the
superior colliculus of wild-type (WT; c, d) or DR62/2 (e, f) mice at P6. In
wild-type mice (c, d), DiI-labelled temporal RGC axons form a dense
termination zone (TZ) in anterior superior colliculus (arrowheads denote
the anterior border). Few are outside the immediate termination zone area
(arrows). In DR6

2/2
mice (e, f), temporal RGC axons and arbors are present

in areas far from the termination zone (inset, magnified in e, right) and well
posterior to it (arrows). L, lateral; M, medial; P, posterior. Scale bars, 100 mm
(b), 400 mm (c, e, left), 170 mm (e, right) and 250 mm (d, f).

ARTICLES NATURE | Vol 457 | 19 February 2009

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inhibition might therefore reflect a direct role for DR6 in axons. To
explore this, we used compartmented (‘Campenot’) chambers24

(Fig. 2a). Sensory neurons are placed in a central chamber containing
NGF; their axons grow under a partition into NGF-containing side-
chambers. Fluid exchange between the chambers is limited, so NGF
deprivation in a side-chamber elicits local axon degeneration while
sparing cell bodies24. Locally deprived axons degenerate in a stereo-
typical manner with initial signs by 6 h and extensive degeneration by
12–24 h, but when anti-DR6.1 was added to the deprived side-
chamber, degeneration was blocked at 24 h and still largely impaired
at 48 h (Fig. 2b). A similar delay was observed when axons of neurons
from DR6 knockout mice were locally deprived, but in the absence of
anti-DR6.1 (Supplementary Fig. 4b, c). Thus, DR6 function is required
in axons for degeneration.

To determine whether DR6 functions in axonal pruning in vivo, we
studied the well-characterized retino-collicular projection, which
develops from an initially exuberant projection of retinal ganglion
cell (RGC) axons to a focused termination zone in the superior
colliculus. All temporal RGC axons initially extend into posterior
superior colliculus, well past their future termination zone in anterior
superior colliculus (Supplementary Fig. 5a). This diffuse projection is
then refined by selective degeneration of inappropriate axon seg-
ments2, such that by postnatal day (P)6 in wild-type mice few axon
segments persist in areas well beyond the termination zone, as
revealed by focal injection of the lipophilic dye DiI into temporal
retina (Fig. 2c, d and Supplementary Fig. 5a, b). In contrast, in P6
DR6 mutant mice, many more RGC axons and arbors are present in
areas far from the termination zone (Fig. 2e, f and Supplementary Fig.
5c, d): we found an 83% increase in axon-positive domains more
than 400 mm from the termination zone (Supplementary Fig. 5f) in
DR6

2/2
(n 5 7) compared to wild-type mice (n 5 7; P , 0.05,

Student’s t-test). The defect at P6 represents a delay in pruning,
not a complete block, as assessed by examining labelled axons at
P4, P5, P6 and P9: at each age, the mutant has more extraneous axons
than the wild-type, and fewer are observed in both wild-type and
mutant at each age compared to earlier time points, but by P9 the
mutant and wild-type projections are indistinguishable (data not
shown). Thus, blocking DR6 function delays pruning of sensory
axons in vitro and retinocollicular axons in vivo.

Caspase 6 regulates axonal degeneration

Because DR6 regulates both cell body apoptosis and axonal degen-
eration, we revisited whether an apoptotic pathway is also involved in
axons. In support, we found that BAX, an effector in the intrinsic
apoptotic pathway, is required in axons, because local sensory axon
degeneration in Campenot chambers was blocked by the genetic
deletion of Bax (Fig. 3a) or by local addition of a BAX inhibitor
(for example, Supplementary Fig. 10b). Consistent with evidence
that caspase 3 mediates cell body but not axon degeneration8,11, we
found that procaspase 3 is highly enriched in cell bodies, and that
zDEVD-fmk, an inhibitor of effector caspases 3 and 7, blocked cell
body but not axon degeneration (Fig. 3b, c and Supplementary Fig.
6a–c). There is, however, a third effector caspase, caspase 6. We found
that procaspase 6 is expressed in both cell bodies and axons, and that
the caspase 6 inhibitor zVEID-fmk blocked degeneration of sensory,
motor and commissural axons (Fig. 3b, c and Supplementary Fig.
6a–c), suggesting that caspase 6 regulates axonal degeneration. We
verified these results using RNA interference in sensory and commis-
sural neurons: Casp3 knockdown protected cell bodies significantly
but had only a minor protective effect on axons, whereas Casp6
knockdown protected axons significantly with only minor effect on
cell bodies (Fig. 3d, e). Thus, distinct caspases mediate cell body and
axon degeneration.

To visualize caspase activation, we first used the fluorescent repor-
ters FAM-DEVD-fmk (for caspase 3 and 7) and FAM-VEID-fmk (for
caspase 6), which bind covalently to activated target caspases. In
NGF-deprived sensory neurons, the caspase 3/7 reporter labelled cell

bodies but not axons, consistent with a previous study11; in contrast,
caspase 6 reporter labelling was observed in both cell bodies and
axons, and axonal labelling occurred in regularly spaced ‘puncta’,
giving a beads-on-a-string appearance (Supplementary Fig. 6f). To
control for reporter specificity, we used a selective antibody to
cleaved caspase 6 and observed a similar punctate pattern in axons
(Fig. 3f, g), whereas antibodies to cleaved caspase 3 only label cell
bodies11. Caspase 6 activation was confirmed biochemically
(Supplementary Fig. 6e). Interestingly, caspase 6 activation appeared
at sites of microtubule fragmentation (assessed by the loss of tubulin
immunoreactivity) (Fig. 3f, g), suggesting that caspase 6 activation
drives microtubule destabilization. Punctate caspase 6 activation was
markedly reduced by anti-DR6.1 (Fig. 3f) and abolished in Bax

2/2

neurons (not shown), suggesting that caspase 6 acts downstream of
BAX in the pathway triggered by DR6. However, the possibility of
feedback loops in apoptotic pathways makes this interpretation tent-
ative.

Regulated shedding of a DR6 ligand(s)

As DR6 is a receptor-like protein, we addressed whether it is activated
by a ligand(s). If so, the DR6 ectodomain might be capable of binding
the ligand(s) and blocking its action (Fig. 4a). Consistent with this,
the DR6 ectodomain fused to human Fc (DR6–Fc) mimicked anti-
DR6.1 in delaying degeneration (Fig. 4a–c and Supplementary Figs 7a
and 13). To search for DR6 binding sites on axons and in conditioned
medium, we used the DR6 ectodomain fused to alkaline phosphatase
(DR6–AP). Purple alkaline phosphatase reaction product was
observed on sensory and motor axons cultured with trophic factors
when they were pre-incubated with DR6–AP but not with alkaline
phosphatase alone, but binding was markedly reduced after trophic
deprivation (Supplementary Fig. 7b, c). To control for the loss of
axonal membrane, we blocked degeneration using a BAX inhibitor
(data not shown) or using neurons from Bax

2/2
mice (Fig. 4d) and

observed an even greater reduction in DR6–AP binding (residual
binding seen without BAX inhibition might reflect nonspecific bind-
ing to degenerating axons). To determine whether DR6-binding sites
were shed, we collected medium conditioned by sensory axons (in
Campenot chambers) or motor neurons (in explant culture) (a BAX
inhibitor was added to prevent nonspecific release resulting from
degeneration). Proteins were separated on non-reducing gels, blotted
to nitrocellulose, and probed with DR6–AP. Little signal was seen in
medium conditioned by either neuronal type in the presence of
trophic factor. However, 48 h after trophic deprivation, DR6–AP
bound a prominent band around ,35 kDa and a minor band around
,100 kDa in both cultures (Fig. 4e). Together, these results support a
‘ligand activation’ model in which a prodegenerative DR6 ligand(s) is
present on the neuronal surface and inactive, but is shed into med-
ium in an active form after trophic deprivation (Fig. 4f), allowing it to
bind and activate DR6.

N-APP is a regulated DR6 ligand

Several properties of APP made it a candidate for a DR6 ligand: (1) it
is highly expressed by developing spinal and sensory neurons and
their axons (Fig. 4g), (2) its ectodomain can be shed in a regulated
fashion19,20, and (3) it is tied to degeneration through its links to
Alzheimer’s disease19–22. In an initial experiment, we found that
DR6–AP bound APP expressed in COS-1 cells (Supplementary Fig.
8a). This prompted us to test whether the bands detected by DR6–AP
in conditioned medium (Fig. 4e) represent APP ectodomain frag-
ments. APP is cleaved by a- or b-secretases (including, in neurons,
BACE1; ref. 25) at distinct sites in its juxtamembrane region (Fig. 4h)
to release ,100-kDa ectodomain fragments termed sAPPa or sAPPb,
respectively19,20. We probed conditioned medium with a polyclonal
antibody to the APP N terminus (anti-N-APP(poly), which also
binds the APP relative APLP2; see later) and an antibody selective
for the carboxy-terminal epitope of sAPPb exposed by BACE cleav-
age (anti-sAPPb) (Fig. 4h). Notably, anti-N-APP(poly) detected

NATURE | Vol 457 | 19 February 2009 ARTICLES

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similar bands to those detected by DR6–AP: a major band at ,35 kDa
and a minor band at ,100 kDa, both highly enriched after trophic
deprivation (Fig. 4i); anti-sAPPb detected a minor ,100-kDa band
and a major ,55-kDa band (Fig. 4i), also both enriched after trophic
deprivation. These results indicate that trophic deprivation triggers
BACE cleavage of APP to yield the ,100-kDa sAPPb (detected by
both antibodies), which undergoes a further cleavage(s) to yield a
,55-kDa C-terminal fragment (detected by anti-sAPPb) and a ,35-
kDa N-terminal fragment (detected by anti-N-APP(poly)) that we
term N-APP. The site of additional cleavage(s) is unknown, but on
the basis of fragment sizes it is expected to be around the junction
between the APP ‘acidic’ and ‘E2’ domains (amino acid 286); indeed,
recombinant APP(1–286) ran at ,35 kDa and was detected with
anti-N-APP(poly) (Fig. 4j), similar to N-APP.

Supporting cleavage of APP by BACE, we found that APP express-
ion on the surface of cultured sensory and motor axons, as assessed
with anti-N-APP(poly) and with antibody 4G8 to the APP juxta-
membrane region (Fig. 4h), is high in the presence of trophic factor

but lost after trophic deprivation; the surface loss was blocked by
three structurally divergent BACE inhibitors—OM99-2, BACE
inhibitor IV, and the highly selective AZ29 (ref. 26) but not the
a-secretase inhibitor TAPI (Fig. 4k, Supplementary Figs 9a–c and
10a, and data not shown). Interestingly, 4G8 partially inhibited sur-
face loss (Supplementary Fig. 9d), presumably through the steric
hindrance of BACE. Loss of surface APP occurred progressively
and in ‘patches’, with little lost at 3 h, more at 6–12 h, and most lost
by 24 h (Fig. 4k, Supplementary Fig. 10b and data not shown). Total
APP visualized after permeabilization did not change detectably
(Supplementary Fig. 10b). Surface loss was not affected by BAX or
caspase 6 inhibitors, or in neurons from Bax

2/2
mice (Fig. 4k and

Supplementary Figs 9c and 10c).
The marked similarly of bands detected by anti-N-APP(poly) and

DR6–AP suggested that DR6 binds N-APP. Indeed, depletion of
conditioned medium with anti-N-APP(poly) eliminated DR6–AP
binding sites (Fig. 4i), and purified DR6–Fc bound to purified
recombinant APP(1–286) in pull-down (Fig. 4j) and enzyme-linked

a

Cleaved caspase 6 TuJ1/Cleaved caspase 6

+
N

G
F

N

G
F

-d
e
p

ri
ve

d
+

c
tr

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g

G

N
G

F
-d

e
p

ri
ve

d
+

a
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-D

R
6
.1

NGF-deprived, 24 h

+ Caspase 6 inhibitor + Caspase 3 inhibitor

T
u
J1

d

Procaspase 6 TuJ1 Procaspase 3 TuJ1

T
u
J1

/T
U

N
E

L

+ NGF

+ Casp6 siRNA1 + Casp3 siRNA1

NGF-deprived NGF-deprived

D
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g

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xo

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b

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s

(%
)

+ NGF

+ anti-NGF

+ CASP3_si1

+ CASP3_si2

+ CASP3_si3

+ CASP6_si1

+ CASP6_si2

+ CASP6_si3

Casp3
siRNAs

Casp6
siRNAs

Casp3
siRNAs

Casp6
siRNAs

+ NGF
+ Anti-NGF
+ Casp3 siRNA1
+ Casp3 siRNA2
+ Casp3 siRNA3
+ Casp6 siRNA1
+ Casp6 siRNA2
+ Casp6 siRNA3

S
u
rv

iv
in

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ro

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s

(%
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B
ax

+
/+

B
ax

–/
–

+ NGF b

c

f

ge

NGF-deprived

NGF-deprived,
+ caspase 3i

+ NGF

NGF-deprived,
+ caspase 6i

1%

D
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xo
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(%
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100

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NGF-deprived, 12 h, confocal

T
u
J1

/c
le

a
ve

d
c

a
sp

a
se

6

0
10
20
30
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60
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90

100

0
10
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50
60
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80
90

100

Figure 3 | BAX and caspase 6 regulate axonal degeneration. a, Local
sensory axon degeneration (TuJ1 immunostain) 48 h after NGF deprivation
in Campenot chambers was blocked in neurons from Bax

2/2
mice.

b, Dissociated sensory neurons double-labelled for procaspase 3 and TuJ1
(left), or procaspase 6 and TuJ1 (right). Caspase 3 is detected in cell bodies
(arrowheads), whereas caspase 6 is seen in both cell bodies and axons.
c, Local degeneration of sensory axons in Campenot chambers deprived of
NGF for 24 h is inhibited by a caspase 6 inhibitor (zVEID-FMK; caspase 6i),
but not by a caspase 3/7 inhibitor (zDEVD-FMK; caspase 3i). Quantification
is shown to the right (mean and s.e.m., n 5 3 replicates). d, In dissociated
sensory neuron cultures deprived of NGF for 24 h, siRNA knockdown of
Casp3 primarily rescues cell body death (TUNEL label), whereas Casp6
knockdown primarily rescues axonal degeneration. e, Quantification of data

from d. Shown are the percentage of degenerating axon bundles (that is, still
visible bundles that show breakdown) and the percentage of surviving
neurons (that is, TUNEL

2
, TuJ1

1
) (mean 6 s.e.m., n 5 3 replicates). Note

that surviving axons may have TUNEL
1
cell bodies. The extent of inhibition

by individual siRNAs correlates with the degree of target knockdown
(Supplementary Fig. 6d). f, Detection of caspase 6 activation in sensory
axons with a cleaved caspase-6-specific antibody (left; TuJ1 double-label on
right). Punctate activation of caspase 6 after NGF deprivation (16 h, middle
panel) was reduced by anti-DR6.1 (bottom panel). g, Confocal section of a
field from f shows that active caspase 6 puncta correspond to sites of tubulin
loss (fraction non-overlapping: 82 6 3.5%; mean 6 s.e.m., n 5 8 fields).
Scale bars, 75 mm (a, d), 100 mm (b), 50 mm (c, f) and 25 mm (g).

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immunosorbent assay (ELISA) (Supplementary Fig. 8c) assays. The
interaction detected by ELISA is of high affinity (effector concentra-
tion for half-maximum response (EC50) 5 ,4.6 nM). The inter-
action of DR6–AP with full-length APP expressed in COS cells was
also of high affinity (half maximal saturation 5 ,1.3 nM)
(Supplementary Fig. 8a, b). This binding was blocked by anti-N-
APP(poly) (data not shown) and anti-DR6.1 (Supplementary Fig.
8a), consistent with APP being a functional DR6 ligand.

Antibodies 4G8 and anti-sAPPb used earlier are highly specific for
APP. However, like other antibodies to the N terminus of APP27,

anti-APP(poly) also binds the close APP relative APLP2 (data not
shown). We found that a recombinant N-terminal fragment of
APLP2 also binds DR6 (Supplementary Fig. 11a). Thus, APLP2 might
contribute with APP to the bands detected on western by DR6–AP.
Indeed, an antibody selective for the APLP2 N terminus detected a
shed fragment in conditioned medium after trophic deprivation
(Supplementary Fig. 11b). The relative contribution of APP and
APLP2 fragments to DR6–AP binding sites remains to be determined.

To evaluate receptor specificity, we examined by pull-down the
binding of APP(1–286) to ectodomains of the seven other

98

62

38

28

16
14

98

62

38

28

16
14

+ – + – TFs
SA

98

62

38

28

16

DR6–AP
VSC

e f

c d b

T
u
J1

NGF-deprived

Control Fc DR6–Fc
Control Fc

100

80

60

40

20

DR6–Fc

Sensory axons

D
e
g

e
n
e
ra

ti
n
g

a
xo

n
b

u
n
d

le
s

(%
)

+ NGF NGF-deprived

B
ax

-/
–

S
e
n
so

ry
a

xo
n
s

DR6–AP
a

A
P

P

E11.5 E12.5
DREZV

C

S

VLF

S

VLF

V DREZ

SN

g

k

S
u
rf

a
c
e
A

P
P

Control + BACE inhibitor + TAPI

NGF-deprived + BAX inhibitor (12 h) + NGF

h

22C11

Anti-N-APP(poly)

Anti-sAPPβ

4G8
Anti-Aβ
(33-42)

SA CM

NGF

DR6–AP

98

62

38

28

16
14

Anti-APP depletion
Mock depletion

i

IP: Fc (protein A/G)

APP(1–286)–His

Inputs: N-APP

APP(1–286) (~35 kDa)

DR6–Fc

Heparin

Anti-APP

APP(1–286) (~35 kDa)
Anti-APP

DR6–Fc

Fc control

Anti-Fc

j

98

62

38

28

16
14

N-APP

NGF + – + –

sAPPβ

98

62

38

28

16
14

NGF

Ligand?

DR6–Fc

DR6

+ TFs – TFs

DR6

Released
ligand,
active

Surface
ligand,
inactive DR6

?
?

– – –+
– – +–
+ – ––

– – +–
– + ––
– – ++
+ + ++

CuBD

KPI OX2

TM

Aβ

CytoE2/carbohydrate binding

β-secretase γ-secretase

Acidic

APP(1–286)
~35 kDa

Growth
factor-like

N C

0

Figure 4 | The N terminus of APP is a regulated DR6 ligand. a, Diagram of
the hypothesis: if DR6 is ligand-activated, then DR6–Fc might sequester
ligand and inhibit degeneration. b, DR6–Fc inhibits local degeneration of
sensory axons in Campenot chambers 24 h after NGF deprivation.
c, Quantification ofresultsin b (meanands.e.m.,n 5 3replicates).d,e, DR6-
binding sites are lost from axons and released into medium after trophic
deprivation. d, DR6–AP binding (purple) to Bax

2/2
sensory axons (left) is

lost 24 h after NGF deprivation (right). e, Medium conditioned by sensory
axons (SA) (in Campenot chambers) or ventral spinal cord explants (VSC),
maintained with or deprived of trophic factors (TFs) for 24 h (sensory: NGF;
motor: BDNF and NT3; BAX inhibitor present), was resolved under non-
reducing conditions and probed with DR6–AP. The arrow indicates a major
band at ,35 kDa. f, Results in a–e support a ligand activation model in
which an inactive DR6 surface ligand is shed in an active form after tropic
deprivation. g, APP immunostaining on sections of mouse embryos at
indicated ages, showing neuronal and axonal expression. DREZ, dorsal root
entry zone; S, sensory ganglia; SN, spinal nerve; V, ventricular zone; VLF,

ventro-lateral funiculus. h, Domain structure of APP (short form, APP695),
indicating b- and c-secretase cleavage sites and antibody binding sites. KPI
and OX2 denote alternatively spliced domains of the longer form. Adapted
from ref. 20. Ab, amyloid-b peptide; CuBD, copper binding domain; cyto,
cytosolic domain; TM, transmembrane domain. i, DR6 binding sites in
sensory axon conditioned medium (SA CM) include APP ectodomain
fragments. Left, anti-N-APP(poly) detects bands at ,35 kDa (major; filled
arrow) and ,100 kDa (minor; open arrow), enriched after trophic
deprivation. Middle, anti-sAPPb detects bands at ,55 kDa (major; filled
arrow) and ,100 kDa (minor; open arrow). Right, immunodepletion using
anti-N-APP(poly) depletes DR6–AP binding sites. Arrow indicates N-APP
band at ,35 kDa. j, Direct interaction between purified APP(1–286) and
DR6–Fc revealed by pull-down. The effect of heparin (10 mg ml

21
) was also

examined. IP, immunoprecipitates. k, Loss of surface APP in patches from
sensory axons 12 h after NGF deprivation is blocked by the BACE inhibitor
OM99-2 (10 mM) but not by the a-secretase inhibitor TAPI (20 mM). Scale
bars, 75 mm (b, d, k) and 500 mm (g).

NATURE | Vol 457 | 19 February 2009 ARTICLES

985
Macmillan Publishers Limited. All rights reserved©2009

death-domain-containing members of the TNF receptor superfam-
ily, and two orphan members. Only binding to p75NTR was observed
(Supplementary Fig. 8d), suggesting that p75NTR might serve as an
alternative route for APP effects in some settings; however, the affin-
ity was considerably lower (EC50 5 ,300 nM by ELISA;
Supplementary Fig. 8e). Consistent with DR6 being the chief APP
receptor in our systems, a fusion of APP(1–286) and alkaline phos-
phatase bound to sensory axons in culture, but the binding was
significantly reduced by anti-DR6.1 or by using DR6 knockout neu-
rons (Supplementary Fig. 12a, b); residual binding may represent
background or binding to another receptor(s), possibly p75NTR.

Necessity and sufficiency of N-APP

To test whether the N terminus of APP contributes to degeneration,
we performed loss-of-function studies. Degeneration of sensory and
commissural axons in response to trophic deprivation was inhibited
by anti-N-APP(poly) (Fig. 5a, d), which also inhibited the death of
sensory neuron cell bodies (Supplementary Fig. 13a, b), without
affecting the loss of surface APP after trophic deprivation
(Supplementary Fig. 13c). Antibody 22C11 (ref. 28) to the APP N
terminus (Fig. 4h) also inhibited sensory axon degeneration (data not
shown). Because both antibodies also bind APLP2 (ref. 27), we per-
formed a more selective blockade using RNA interference.
Knockdown of App in sensory neurons significantly impaired both
axon degeneration and cell body death after trophic withdrawal
(Fig. 5b). These results support the involvement of an N-terminal
fragment of APP in degeneration. In further support, BACE inhibi-
tors impaired degeneration of sensory axons and cell bodies (Fig. 5c
and Supplementary Figs 13a, b and 14) and of commissural axons
(Fig. 5d) after trophic deprivation. The selective BACE inhibitor
AZ29 blocked degeneration at concentrations consistent with its cel-
lular half-maximal inhibitory concentration (IC50) of 470 nM

26

(Supplementary Fig. 14a).

Importantly, axonal degeneration block by BACE inhibitors could
be reversed by adding purified APP(1–286) to sensory (Fig. 5c) and
commissural (Fig. 5d) neurons, showing that the N terminus of APP
is sufficient to trigger degeneration. This effect was largely blocked by
anti-DR6.1 (Supplementary Fig. 14b), consistent with DR6 being the
most important functional receptor in these cells. Block of sensory
cell body degeneration by BACE inhibitors could similarly be
reversed by the addition of APP(1–286), albeit at higher concentra-
tions (Supplementary Fig. 13a, b). Together, these results support the
model that shed N-APP activates DR6 to trigger degeneration.
Degeneration of sensory axons caused by APP(1–286) in the presence
of BACE inhibitor was blocked if NGF was present (50 ng ml

21
;

Fig. 5c), indicating that trophic factors also inhibit signalling down-
stream of DR6.

Role of amyloid-b in physiological degeneration

BACE cleavage of APP is followed by c-secretase cleavage, yielding
amyloid-b peptides19–22 (Figs 4h and 6c). Because amyloid-b peptides
can be neurotoxic21,22, we examined whether they contribute to
degeneration. The synthetic amyloid-b peptide Ab(1–42) triggered
degeneration in our assays, and an antibody directed at amino acids
33–42 of amyloid-b (anti-Ab(33–42); Fig. 4h) blocked this effect
(Supplementary Fig. 9a, e), but did not block degeneration after
trophic deprivation (Fig. 5e). Conversely, degeneration induced by
synthetic Ab(1–42) was not blocked by the genetic deletion of DR6
(data not shown), indicating that amyloid-b operates by a mode of
action distinct from the physiological degeneration mechanism
studied here.

Antibody 4G8 used earlier, which binds amyloid-b residues 17–24
(Fig. 4h), also blocked the degenerative effect of Ab(1–42)
(Supplementary Fig. 9e), but unlike anti-Ab(33–42) it partially
inhibited degeneration after trophic deprivation (Fig. 5e).
However, as mentioned, 4G8 also partially inhibits the loss of surface

a

c

d

b

+ BACE inhibitor

+ APP(1–286)

Control

+ NGF

e

D
e
g

e
n

e
ra

ti
n

g
a

xo
n

b
u

n
d

le
s

(%
)

Control IgG

+ APP(1–286) + BACEi
+ BACEi

+ APP(1–286) + BACEi + NGF

100

80

60

40

20

Sensory axons

100

80

60

40

20

D
e
g

e
n
e
ra

ti
n
g

a
xo

n
b

u
n
d

le
s

(%
) Control IgG

Anti-N-APP(poly)
BACEi
BACEi + APP(1–286)

100

80

60

40

20

Control IgG

+ Anti-N-APP(poly)

D
e
g

e
n

e
ra

ti
n

g
a

xo
n

b
u

n
d

le
s

(%
)

S
u
rv

iv
in

g
D

R
G

n
e
u

ro
n

s
(%

)

D
e
g

e
n
e
ra

ti
n

g
a

xo
n

b
u

n
d

le
s

(%
)

100

80

60

40

20

+ NGF
NGF-deprived
+ App siRNA1

+ App siRNA2
+ App siRNA3

100

80

60

40

20

100

80

60

40

20

D
e
g

e
n
e
ra

ti
n

g
a

xo
n

b
u
n
d

le
s

(%
)

+ NGF
+ Anti-NGF
+ 4G8
+ Anti-Aβ(33–42)

Sensory axons

Control + Anti-N-APP(poly)

NGF-deprived, 24 h

S
e
n

so
ry

,
T

u
J1

+ NGF

+ Control IgG + Anti-Aβ(33–42)+ 4G8

NGF-deprived, 24 h

T
u
J1

+ BACE inhibitor + Anti-N-APP(poly) Control IgG
+APP(1–286)

C
o

m
m

is
su

ra
l,

4
8
h

S

e
n
so

ry
a

xo
n
s,

T
u

J1

+ NGF NGF-deprived

+ App siRNA1 + App siRNA2

NGF-deprived

S
e
n
so

ry
,
T

u
J1

/T
U

N
E

L

0

0

0

0 0

0

Figure 5 | The APP N terminus regulates
degeneration. a, Local degeneration of sensory
axons in Campenot chambers (NGF deprivation,
24 h) was blocked by anti-N-APP(poly)
(20 mg ml

21
). Quantification is shown to the right

for all panels (a–e). b, In dissociated sensory
neurons, siRNA knockdown of App
(Supplementary Fig. 13d) significantly reduces
axon degeneration 24 h after trophic deprivation,
and partially reduces cell body death. c, Local
degeneration of sensory axons in Campenot
chambers (NGF deprivation, 24 h) was inhibited
by the local addition of BACE inhibitor (BACEi)
OM99-2 (10 mM). Purified APP(1–286) added
locally restored axonal degeneration, an effect
inhibited by 50 ng ml

21
NGF (right).

d, Degeneration of commissural neurons and
axons at 48 h was inhibited by anti-N-APP(poly)
(20 mg ml

21
) or BACE inhibitor OM99-2

(10 mM), but restored by APP(1–286). e, Effect of
amyloid-b antibodies on sensory axon
degeneration (NGF deprivation, 24 h). 4G8
partially inhibited, whereas anti-Ab(33–42) did
not. Scale bars 50 mm (a, c, e), 40 mm (b) and
200 mm (d). All data are mean and s.e.m. for n 5 3
replicates.

ARTICLES NATURE | Vol 457 | 19 February 2009

986
Macmillan Publishers Limited. All rights reserved©2009

APP. In contrast, the APP epitope bound by anti-Ab(33–42) is buried
in the cell membrane, so anti-Ab(33–42) does not bind intact APP
nor inhibit its surface loss (Supplementary Fig. 9a, b, d). Because
anti-Ab(33–42) does not protect, we attribute the partial protective
effect of 4G8 to its ability to inhibit APP shedding, not its ability to
block amyloid-b toxicity. Because 4G8 does not bind APLP2, its
ability to protect also supports the sufficiency of APP in mediating
degeneration.

Evidence for an APP–DR6 interaction in vivo

To seek evidence for an APP–DR6 interaction in vivo, we examined
whether the DR6 knockout exhibits any similar phenotype to those
reported in the App knockout—or, given the potential for redund-
ancy, in compound mutants of App with Aplp2. One phenotype
observed at the neuromuscular junction in the App

2/2
Aplp2

2/2

double knockout is suggestive of a potential pruning defect. In
wild-type animals, motor axons normally terminate at synaptic sites
(Fig. 6a). In App

2/2
Aplp2

2/2
double knockouts, however, there is a

highly penetrant presence of nerve terminals past endplates29.
Notably, a similar phenotype was observed in the DR6 mutant: rather
than terminating at endplates, many terminals were present beyond,
giving characteristic finger-like protrusions (Fig. 6a, b). It is not
known whether this phenotype reflects a failure to retract or excessive
sprouting. Nevertheless, the similarity of phenotypes supports the
view that APP signals via DR6 in regulating axonal behaviour in vivo.
In this system, APP and APLP2 appear redundant because the axonal
phenotype is seen only in App

2/2
Aplp2

2/2
double mutants, not

single mutants29. Whether they are non-redundant in other systems
remains to be determined.

Discussion

Our results reveal a mechanism, the ‘APP–death-receptor’ mech-
anism (Fig. 6c), in which trophic deprivation leads to the cleavage
of surface APP by b-secretase, followed by further cleavage of the
released fragment by an as yet unidentified mechanism (probably
near the junction of APP acidic and E2 domains). This then yields

an N-terminal ,35-kDa fragment (N-APP) which binds DR6, trig-
gering caspase activation and degeneration of both neuronal cell
bodies (via caspase 3) and axons (via caspase 6). Whether the second
cleavage is required for degeneration remains to be determined.
Degeneration induced by added APP(1–286) was blocked when suf-
ficient trophic factor was present, indicating that trophic factor not
only prevents initiation of the APP cleavage cascade, but also blocks
signalling downstream of DR6, providing a fail-safe mechanism to
protect if DR6 is inappropriately activated in an otherwise healthy
neuron.

DR6: an accelerator of self-destruction

In all settings examined, antagonizing DR6 resulted in a delay, rather
than a complete block, in neuronal death and axonal pruning. DR6 is
therefore best thought of as an accelerator of degeneration—neurons
and axons activate it for swift self-destruction when they become
atrophic, but without it they have other, slower, ways of achieving
that end, perhaps involving other pro-apoptotic receptors5 or
intrinsic mechanisms. This function contrasts that of the DR6 rela-
tive p75NTR (which can mediate degeneration when overexpressed11

or when activated by a neurotrophin in neurons lacking the cognate
TRK (also known as NTRK) receptor5). p75NTR is more restricted to
specific neuronal classes than DR6, and its genetic deletion provided
only modest protection of sensory axons in the first 36 h after trophic
deprivation (Supplementary Fig. 15), as reported previously for sym-
pathetic axons30. In sympathetic neurons, p75NTR is thought to
mediate competition for NGF: cells with high NGF/TRKA signalling
upregulate expression of BDNF, which acts via p75NTR to trigger
degeneration of neighbouring neurons with less robust NGF/TRKA
signalling30,31. This mechanism shares with ours the expression of a
prodegenerative ligand(s) by the neurons themselves. However, the
DR6 ligand APP is activated by trophic-factor deprivation, whereas
p75NTR ligand expression is increased by trophic-factor stimu-
lation31. Thus, p75NTR ligands are released by ‘strong’ neurons to
kill ‘weak’ neurons (a paracrine prodegenerative effect)31, whereas
APP gets activated within weak neurons to accelerate self-destruction

APP–death-receptor mechanism c

DR6+/– DR6–/–
N

F
+

S
Y

P
/B

T
X

DR6+/–, high mag DR6–/–, high mag

B
T

X

a

N
e
rv

e
e

n
d

t
e
rm

in
a
l s

p
ro

u
ts

>
5

0
µ

m
(
%

)

DR6–/–
DR6+/–

P0 NMJs

9

7

5

3

1

b

DR6 APP

ICD

AICD

Caspase

Degeneration

TF
deprivation

A
β

A
β

A
β

γ-secretase

β-secretase

?

Figure 6 | APP and DR6 signalling: in vivo
evidence, and model. a, In control (DR61/2) P0
diaphragm muscle, few axons (green,
neurofilament (NF) and synaptophysin (SYP)
stain) overshoot endplates (red, fluorescent
a-bungarotoxin (BTX) stain), and those that do
are short, but in DR6 mutants more overshoot
and many are long (arrowheads). Scale bar, 60
mm (left four panels) and 15 mm (right four
panels). b, The number of axons overshooting
by .50 mm (mean and s.e.m., n 5 3 wild-type, 4
mutants); this underestimates the effect, because
overshooting axons are longer in mutants. NMJ,
neuromuscular junction. c, The APP–death-
receptor mechanism is shown. Trophic factor
(TF)-deprivation triggers the cleavage of surface
APP by b-secretase, releasing sAPPb, which is
further cleaved by an unknown mechanism (‘?’)
to release N-APP. This then binds DR6 to trigger
degeneration through caspase 6 in axons and
caspase 3 in cell bodies. Also illustrated is
cleavage by c-secretase to release amyloid-b (Ab)
and the APP intracellular domain (AICD).

NATURE | Vol 457 | 19 February 2009 ARTICLES

987
Macmillan Publishers Limited. All rights reserved©2009

triggered by trophic deprivation or perhaps other insults (an auto-
crine prodegenerative effect).

Caspase 6 mediates axonal degeneration

The intracellular mechanisms of axonal degeneration and their rela-
tion to apoptosis have been unclear. Our results indicate that devel-
opmental axonal degeneration does involve an apoptotic pathway,
but with a non-classical effector, caspase 6. Epistatis analysis supports
a linear activation model from DR6 to BAX to caspase 6, but does not
exclude the possibility that active caspase 6 might feedback, for
example, to accelerate the process; in this context, it is intriguing that
the APP cytoplasmic domain is a caspase 6 substrate32. Activation of
caspase 6 by trophic deprivation occurs in a punctate pattern in
axons, leading to a beads-on-a-string appearance, and sites of punct-
ate caspase 6 activation correspond to sites of microtubule frag-
mentation. Caspase 6 might trigger microtubule destabilization by
cleaving microtubule associated proteins such as TAU (also known as
MAPT), a documented target of caspase 6 (refs 33, 34); in a recent
proteomic analysis, almost half the identified caspase 6 targets were
cytoskeleton-associated35.

Ligands, receptors for self-destruction

Although p75NTR also binds APP(1–286), DR6 binds with a much
higher affinity, and blocking DR6 function largely blocks both
APP(1–286) binding to sensory axons and degeneration triggered
by APP(1–286). Thus, DR6 seems to be the major functional APP
receptor in these neurons, although p75NTR might contribute in
other contexts. Conversely, APP may not be the only DR6 ligand:
APLP2, which is coexpressed with APP in many neurons27, may also
contribute to degeneration, because an N-terminal fragment is shed
in response to trophic deprivation, can bind DR6, and can trigger
degeneration when added exogenously (Supplementary Fig. 16).
Future studies will define the relative contributions of APP and
APLP2 in different neuronal populations.

The finding of similar neuromuscular junction phenotypes in DR6
and App

2/2
Aplp2

2/2
mutants supports a ligand–receptor inter-

action, and indicates that APP and APLP2 both contribute in this
system. The aberrant axonal extensions seen could reflect an impair-
ment of pruning, or, alternatively, a failure of axons to stop; of note,
the APP ectodomain has been implicated in neurite growth inhibi-
tion36. Previous studies have not reported changes in neuronal cell
death in vivo in App mutants, either singly or in combination with
Aplp1 and/or Aplp2 mutations37,38. However, such studies did not
examine spinal cord or sensory ganglia, nor involve time-course
analysis to evaluate possible delays in degeneration. In vitro analysis
of cortical neurons from mutants has given divergent results about
their basal survival rates and susceptibility to glutamate excitotoxi-
city37–39, but their response to trophic deprivation has not been
reported.

In recent findings paralleling ours, trophic deprivation was found
to trigger BACE cleavage of APP in PC12 cell-derived neurons and
primary hippocampal neurons, and degeneration was reduced by
App knockdown (in PC12 cells) and BACE inhibition40,41. The pro-
degenerative function of APP was, however, attributed to amyloid-b,
because antibodies to amyloid-b inhibited degeneration40,41. We too
observed protection by antibody 4G8, but attribute this to the ability
of 4G8 to bind full-length APP and inhibit cleavage, because a dif-
ferent anti-amyloid-b antibody that does not bind native APP inhib-
ited neither shedding nor physiological degeneration, but blocked
the toxic action of added amyloid-b. Conversely, the toxic effect of
amyloid-b was not blocked by DR6 inhibition. It was also found that
a c-secretase inhibitor, which reduced amyloid-b production after
BACE cleavage, inhibited degeneration40,41. We too found that
c-secretase inhibitors can partially inhibit degeneration of commis-
sural and sensory axons (Supplementary Fig. 17), but c-secretase has
many substrates, and it is possible that the efficient activation of DR6
signalling requires a distinct c-secretase-dependent process. Thus,

our results argue against the involvement of amyloid-b in initiating
DR6-dependent degeneration in the neurons studied here, but this
does not exclude its possible involvement in other neurons, or at later
times in these neurons to augment the effects of APP–DR6 signalling.

APP–DR6 signalling and neurodegeneration

APP is expressed in adult brain and upregulated in damaged axons42.
DR6 is also highly expressed in adult brain (Supplementary Fig. 18).
Given our findings, it is reasonable to assume that the APP–death-
receptor mechanism might contribute to adult plasticity, or to neu-
rodegeneration after injury or in disease. Interestingly, DR6 is upre-
gulated in injured neurons43, raising the question as to whether
overexpressed DR6 in neurons can trigger ligand-independent
degeneration, as reported for p75NTR11.

Given the genetic evidence linking APP and its cleavage to
Alzheimer’s disease, we propose that signalling of APP via DR6
(and possibly p75NTR) may in particular contribute to the initiation
or progression of Alzheimer’s disease, either alone or in combination
with other proposed APP-dependent mechanisms, such as amyloid-
b toxicity21,22 or effects of the APP intracellular domain44. Of note,
previous studies showed immunoreactivity for the APP N terminus
associated with Alzheimer’s plaques45,46, DR6 maps to chromosome
6p12.2-21.1, near a putative Alzheimer’s disease susceptibility
locus47, and sites of DR6 mRNA expression in adult brain correlate
in an intriguing way with known sites of dysfunction in Alzheimer’s:
very high in hippocampus, high in cortex, but low in striatum
(Supplementary Fig. 18), and high in forebrain cholinergic neu-
rons48, for instance. In addition, activated caspase 6, a downstream
DR6 effector, is associated with plaques and tangles in Alzheimer’s
disease, and with mild cognitive impairment34,49, consistent with the
possible activation of caspase 6 in neuritic processes by the APP–
death-receptor mechanism (caspase 6 is also implicated in
Huntington’s disease50). Although these results are compatible with
the involvement of APP–DR6 signalling in Alzheimer’s, it is less clear
how the mechanism fits with genetic evidence implicating altered
c-secretase processing in this disease19–22. However, the fact that
c-secretase inhibitors antagonize DR6-dependent degeneration hints
at a possible relationship.

Thus, further study is required to determine the full implications
of the APP–death-receptor mechanism in development, adult physi-
ology and disease. Nonetheless, our results already tie APP to a new
mechanism for neuronal self-destruction in development, and sug-
gest that the APP ectodomain, acting via DR6 and caspase 6, con-
tributes to the pathophysiology of Alzheimer’s disease.

METHODS SUMMARY

Antibodies to the following targets were used: procaspase 3 (1:200, Upstate),

active capsase 3 (1:200, R&D), procaspase 6 (1:200, Stratagene), active caspase 6

(1:100, BioVision), Tuj1 (1:500, Covance), p75NTR (Chemicon), 2H3 (1:200,

DSHB), Islet1/2 (1:100, Santa Cruz Biotech), N-APP (polyclonal, 1:100, Thermo

Fisher Scientific; monoclonal 22C11, Calbiochem), DR6 (R&D), NGF (Abcam

and Genentech), BDNF (Calbiochem), NT3 (Genentech), amyloid-b (4G8,
Covance), the C-terminal cleavage-specific anti-amyloid-b antibody (anti-
Ab(33–42), Sigma), and the C-terminal cleavage site of sAPPb (Covance).
Monoclonal antibodies to human DR6 ectodomain fused with human Fc

(A.N., K. Dodge, V. Dixit and M.T.L., manuscript in preparation) were screened

for binding to murine DR6 and block of commissural neuron degeneration.

Proteins used were: netrin-1 (R&D), NGF (Roche), BDNF and NT3

(Calbiochem), and control IgG (R&D). Transiently expressed murine DR6 ecto-

domain (amino acids 1–349) fused to human Fc, His-tagged human APP(1–

286), and APP(1–286) and APLP2(1–300) fused to human Fc, were affinity-

purified from CHO cell supernatants (similar results were obtained using

APP(1–286) and APP(1–306), Novus Biologicals). Inhibitors were used against:

caspase 3 (Z-DEVD-FMK, Calbiochem), caspase 6 (Z-VEID-FMK, BD

Pharmingen), BACE (OM99-2, Calbiochem; BACE inhibitor IV, Calbiochem;

AZ29, Genentech), c-secretase (DAPT, Calbiochem).

See Methods for details of in situ hybridization and immunohistochemistry,

explant, dissociated, and Campenot chamber cultures, siRNA transfection,

ARTICLES NATURE | Vol 457 | 19 February 2009

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tracing and quantification of retinotectal projections, alkaline-phosphatase-
binding assays and pull-down assays.

Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.

Received 24 May; accepted 31 December 2008.

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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.

Acknowledgements We thank R. Axel, C. Bargmann, B. de Strooper, V. Dixit,
C. Henderson, J. Lewcock, R. Scheller, R. Vassar, R. Watts, and members of the
M.T.-L. laboratory for helpful discussions and suggestions, and critical reading of
the manuscript, and A. Bruce for making the diagrams. We thank P. Hass and
members of his laboratory (Genentech) for generation and purification of the DR6
ectodomain and APP(1–286), and W.-C. Liang and Y. Wu (Genentech) for binding
experiments with purified proteins. Supported by Genentech (A.N. and M.T.-L.)
and National Eye Institute grant R01 EY07025 (D.D.M.O.’L.).

Author Contributions A.N. performed most of the experiments, with the exception
of the analysis of retinal projections and the experiments listed in the
Acknowledgements, and co-wrote the paper. The retinotectal analysis was
performed by T.M. and supervised by D.D.M.O.’L. M.T.-L. supervised or
co-supervised all experiments, and co-wrote the paper.

Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare competing financial interests:
details accompany the full-text HTML version of the paper at www.nature.com/
nature. Correspondence and requests for materials should be addressed to M.T.-L.
([email protected]).

NATURE | Vol 457 | 19 February 2009 ARTICLES

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METHODS
In situ hybridization and immunochemistry. Radioactively labelled

35
S in situ

mRNA hybridization was as described51, using the mRNA locator kit (Ambion).

Radiolabelled mRNA probes to antisense sequences of mouse TNF receptor

superfamily member 39 untranslated regions were generated using the

MAXIscript kit (Ambion). Immunochemistry was as described on sections51

or cultured cells52. Surface labelling was done without detergent. Double label-

ling was performed using Zenon Technology (Invitrogen). Fluorescent caspase
reporter assays were as recommended (MP Biologicals). TUNEL assays was as

recommended (in situ cell death detection kit, Roche).

Quantification of axon degeneration. To measure the percentage of degenerat-
ing axon bundles, the number of still visible bundles that showed breakdown was

counted.

Quantification on sections. Twenty-micrometre serial cryosections were taken
from axially matched cervical (C1–C3) levels of DR6

2/2
embryos and hetero-

zygous littermates. Motor neurons were counted in all sections at E14.5 (large

Islet1/2-positive ventral neurons; 4 mutants, 3 controls) or E18 (large H&E-

stained ventral neurons; 7 mutants, 5 controls).

Neuronal cultures. E13 rat dorsal spinal cord was dissected after the introduc-
tion of plasmids and siRNAs by electroporation53; the dorsal explant survival

assay was as described23. DR6 siRNA1 and siRNA2 (sense) were 59-CAAU-

AGGUCAGGAAGAUGGCU-39 and 59-AAUCUGUUGAGUUCAUGCCUU-39,

respectively. The mismatch sequence complementary to siRNA1 was 59-
GGACTCTGTGTACAGTCACCTCCCAGATCTGTTATAG-39. Mouse sensory

and motor neuron explants or dissociated cells were cultured on laminin-coated

35-mm tissue culture dishes in culture medium (Neurobasal medium with B27

supplement) with appropriate trophic factor (sensory: NGF, 50 mg ml
21
; motor:

BDNF and NT3, 10 mg ml
21
). Trophic deprivation was achieved by removing

growth factor and adding appropriate antibodies (sensory: anti-NGF,

50 mg ml
21
; motor: anti-BDNF and anti-NT3, 50 mg ml

21
). The introduction

of siRNAs into dissociated sensory neuron cultures was performed as

described54.

Campenot chamber assay. The Campenot chamber assay was carried out as
described24 with minor modifications. In brief, 35-mm tissue culture dishes were

coated with poly-D-lysine and laminin and scratched with a pin rake (Tyler

Research) to generate tracks, as illustrated in Fig. 2a. A drop of culture medium

containing 4 mg ml
21

methylcellulose was placed on the scratched substratum. A

teflon divider (Tyler Research) was seated on silicone grease and a dab of silicone

grease was placed at the mouth of the centre slot. Dissociated sensory neurons

from E12.5 mouse DRGs were suspended in methylcellulose-containing med-
ium, loaded into a disposable sterile syringe fitted with a 22-gauge needle,

injected into the centre slot under a dissecting microscope, and allowed to settle

overnight. The outer perimeter of the dish (the cell body compartment) and the

inner axonal compartments were filled with methylcellulose-containing med-

ium. Within 3–5 days in vitro, axons begin to emerge into left and right compart-

ments. To trigger local axonal degeneration, NGF-containing medium from

axonal compartments was replaced with neurobasal medium containing anti-

NGF. Where indicated, anti-DR6.1 or control IgG were added (50 mg ml
21

final

concentration) at the time of NGF deprivation. Cultures were fixed at different

times after deprivation with 4% paraformaldehyde for 30 min at room temper-

ature and processed for TuJ1 immunofluorescence.

Tracing of RGC axons. Injections of DiI into temporal retina and subsequent
analyses were performed essentially as previously described55. The centre of the

termination zone was determined to be the centre of a circumscribed circle.

Injection size, termination zone size and the efficiency of axon labelling were

not different between wild-type and mutant. The termination zone position was

also unchanged (average termination zone centre: 50.3% for controls and 49.8%
for mutants (P . 0.9), from the medial edge). The retina in mutants appeared

morphologically normal, with all retinal layers present in similar proportions to

wild-type.

For quantification, the presence of axons was determined in 100-mm vibra-
tome sections and transposed onto the superior colliculus. Sections were photo-

graphed and axon presence was recorded in 100-mm segments from the anterior
border. Using landmarks such as the termination zone, unique arbors, or the

edge of the superior colliculus, these data were transposed from photos of sec-

tions to photos of the wholemount superior colliculuses in dorsal view, resulting

in a grid of 100-mm squares covering each superior colliculus. The termination
zones and grids were aligned for the analyses. All analyses were performed

blinded to genotype.

Alkaline phosphatase binding assays. Alkaline phosphatase fused to the DR6
ectodomain (DR6–AP) and to APP(1–286) (APP–AP) were transiently

expressed in COS-1 cells. Medium was changed after 12 h to OPTI-MEM
(Invitrogen), and conditioned medium was collected 36 h later and filtered.

The DR6–AP blot assay on conditioned medium was performed as

described56. In brief, conditioned medium derived from sensory axons main-

tained in Campenot chambers or ventral spinal cord explants in explant culture

(with or without trophic deprivation) was concentrated tenfold using centriprep

centrifugal filters (Millipore), resolved in 4–20% gels under non-reducing con-

ditions, and blotted with DR6–AP in alkaline phosphatase binding buffer.

For in situ sensory axon binding assays, wild-type or Bax
2/2

sensory explants

were cultured, deprived of NGF for 12 h (with or without BAX inhibitor, as

indicated), then washed twice with the alkaline phosphatase binding buffer

(HBSS, Gibco, with 0.2% BSA, 0.1% NaN3, 5 mM CaCl2, 1 mM MgCl2,

20 mM HEPES, pH 7.0). The alkaline phosphatase binding assay was carried

out by making a 1:1 mixture of binding buffer and conditioned medium contain-

ing DR6–AP, APP–AP, or control alkaline phosphatase, applied to DRG explants

in 8-well culture slides and incubated for 90 min at room temperature. Explants

were rinsed five times with binding buffer, fixed with formaldehyde (3.7% in

PBS) for 12 min at room temperature, and rinsed three times with HBS (20 mM

HEPES, pH 7.0, 150 mM NaCl). Endogenous alkaline phosphatase activity was
blocked by heat inactivation at 65 uC in HBS for 30 min. After rinsing three times
in alkaline phosphatase reaction buffer (100 mM Tris, pH 9.5, 100 mM NaCl,

50 mM MgCl2), bound alkaline phosphatase fusion was visualized by developing

colour stain in alkaline phosphatase reaction buffer with 1/50 (by volume) of

NBT/BCIP stock solution (Roche) overnight at room temperature.

The in situ binding of DR6–AP to COS cells transiently expressing APP695 was

performed in the same way, but with heparin (2–10 mg ml
21
) added to reduce

nonspecific binding. DR6–AP did not bind to controls (p75NTR or DR6

expressed in COS-1 cells) under these conditions. For quantitative analysis,

the amount of DR6–AP protein in medium was quantified as described57. In

brief, 100 ml of 23 alkaline phosphatase buffer (prepared by adding 100 mg para-
nitrophenyl phosphate (Sigma) and 15 ml of 1 M MgCl2 to 15 ml 2 M diethano-

lamine, pH 9.8) was mixed with conditioned medium containing DR6–AP or

control alkaline phosphatase. The reaction was developed over 5–15 min, with

the absorbance being in the linear range (0.1–1). The volume of reaction was

adjusted by adding 800 ml of distilled water, and absorbance was measured at
405 nm. Concentration in nM was calculated according to the formula (for

100ml): C (nM)5A31003(60/developing time)/30. Saturation binding analysis
was performed as described57. Prizm 4 software (GraphPad) was used to generate

saturation binding curves and to determine half-maximal saturation value.

APP pull-down assays. Soluble ectodomains of TNF receptor superfamily mem-
bers fused to human Fc were expressed in CHO cells and affinity purified. They

were incubated at 5 mg ml
21

in binding buffer (HBSS, with 0.2% BSA, 0.1%

NaN3, 5 mM CaCl2, 1 mM MgCl2, 20 mM HEPES, pH 7.0) with 1 mg ml
21

of

His-tagged APP(1–286) and protein A/G beads (Santa Cruz Bio) at 4 uC over-
night. Beads were washed five times with binding buffer. Bound complexes were

eluted from beads with SDS loading buffer, resolved in 4–20% SDS PAGE gels,

and blotted for APP (with anti-NAPP(poly)) and for TNF receptor family mem-

bers (with anti-human Fc).

Mice. The following mutant mice were used: DR6 knockout17 (gift from V.
Dixit), Bax knockout58 (gift from S. Korsmeyer) and p75NTR knockout59

(Jackson laboratory).

51. Sabatier, C. et al. The divergent Robo family protein rig-1/Robo3 is a negative
regulator of slit responsiveness required for midline crossing by commissural
axons. Cell 117, 157–169 (2004).

52. Atwal, J. K. et al. PirB is a functional receptor for myelin inhibitors of axonal
regeneration. Science 322, 967–970 (2008).

53. Chen, Z. et al. Alternative splicing of the Robo3 axon guidance Receptor governs
the midline switch. Neuron 58, 325–332 (2008).

54. Higuchi, H., Yamashita, T., Yoshikawa, H. & Tohyama, M. Functional inhibition of
the p75 receptor using a small interfering RNA. Biochem. Biophys. Res. Commun.
301, 804–809 (2003).

55. McLaughlin, T., Torborg, C. L., Feller, M. B. & O’Leary, D. D. Retinotopic map
refinement requires spontaneous retinal waves during a brief critical period of
development. Neuron 40, 1147–1160 (2003).

56. Pettmann, B. et al. Biological activities of nerve growth factor bound to
nitrocellulose paper by western blotting. J. Neurosci. 8, 3624–3632 (1988).

57. Okada, A. et al. Boc is a receptor for sonic hedgehog in the guidance of
commissural axons. Nature 444, 369–373 (2006).

58. Knudson, C. M. et al. Bax-deficient mice with lymphoid hyperplasia and male germ
cell death. Science 270, 96–99 (1995).

59. Lee, K. F. et al. Targeted mutation of the gene encoding the low affinity NGF
receptor p75 leads to deficits in the peripheral sensory nervous system. Cell 69,
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doi:10.1038/nature07767

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